Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer

Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer Qiuyu Gong, Zhang Guangjian, Bohao Liu, Deqian Qiao, Xingzhuo Zhu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5702716/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Molecular targeted therapy (MTT) for non-small cell lung cancer (NSCLC) has been a central issue for a long time. However, drug resistance and extra toxicity have limited its further clinical applications. Herein, taking advantages of the proteolysis-targeting chimeras (PROTACs), a series of PROTAC degraders ( P4-1 to 4 ) targeting cell-surface CD26 (a potential target for NSCLC) have been developed for MTT of NSCLC. To achieve the efficient degradation of cell surface proteins, which is a huge challenge, the molecular structures of degraders were rational designed and optimized. Remarkably, CD26 can be degraded by P4-3 evidently at low dose (~ 500 nM) without degrading CD26 isoenzymes, which was independent of autophagy pathway. Surprisingly, the proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299 cells) and tumors were significantly inhibited by P4-3 , and no toxicity of P4-3 for BEAS-2B cells (human lung normal epithelial cells) were obtained. More interestingly, the powerful proliferation inhibition capabilities of P4-3 for organoids were observed. Moreover, a mechanism of P4-3 for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC. Biological sciences/Cancer/Cancer therapy/Targeted therapies Biological sciences/Chemical biology/Small molecules CD26 non-small cell lung cancer proteolysis-targeting chimeras molecular targeted therapy organoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Lung cancer is a very aggressive and highly prevalent disease worldwide, with the leading cause of death among male malignant tumors, and its mortality rate among female malignant tumors ranks second only to breast cancer 1 , 2 . It is reported that lung cancer was one of the most frequently diagnosed cancers (about 2.5 million new cases worldwide) in 2022 3 , which brings great challenges to global health issues. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which accounts for approximately 85% of all lung cancer cases 4 . The treatment of NSCLC is therefore extremely urgent. The most traditional NSCLC therapy method is chemotherapy and the representative drugs include cisplatin, carboplatin, taxanes and gemcitabine 5 – 8 . Besides, radiotherapy is a standard means for NSCLC treatment, which could improve overall survival rate of patients 9 . However, the drug resistance of chemotherapy and side effects of radiotherapy may cause pains to patients, restricting their further applications. Molecular targeted therapy (MTT) is another ideal way for NSCLC treatment, and several representative drugs, such as epidermal growth factor receptor (EGFR) inhibitors (gefitinib, erlotinib, dacomitinib, osimertinib) 10 – 13 and anaplastic lymphoma kinase (ALK) inhibitors (crizotinib, alectinib, and brigatinib) 14 – 16 , have been used successfully. In recent years, pembrolizumab 17 and atezolizumab 18 targeting PD-L1 have been developed for immunotherapy for NSCLC, which brings hope to NSCLC treatment. Unfortunately, drug resistance still remains an obstacle to overcome in MTT and immunotherapy, which makes the development of NSCLC treatment strategies with low drug resistance imperative. Proteolysis-targeting chimeras (PROTACs) with the advantages of lower drug-resistance, better selectivity and catalytic dosage compared to classic inhibitors, has emerged as a promising approach for MTT of cancers 19 – 23 . For NSCLC, several PROTAC degraders have been developed. For example, some degraders targeting kinases (EGFR tyrosine kinase, ALK and Kirsten rat sarcoma viral oncogene homolog (KRAS)) were utilized for NSCLC cells inhibition, which exhibits a great potential for NSCLC treatment 24 – 27 . Unfortunately, the developments kinase-based NSCLC degraders remain huge challenges due to the features of high structural conservation of kinase ATP binding sites. Therefore, developing other cell surface proteases (low conserved binding sites) PROTAC degraders attracts our great interest. CD26 is a 110 kDa cell surface glycoprotein with an extracellular domain with dipeptidyl peptidase 4 (DPP4) activities, which owns diverse biological functions 28 . CD26 has been considered as potential biomarkers and targets for several cancers, including NSCLC 29 . However, Wesley et al. proposed that CD26 acted as the tumor suppressor in NSCLC cells 30 , which is contrary to the above-mentioned point. The reason for this inconsistency may be that powerful molecular probe tools have not yet been reported. So developing a degrader based on CD26 PROTAC may help to eliminate our confusion. Herein, a series of PROTAC degraders ( P4-1 to 4 ) targeting cell-surface CD26 have been developed. To achieve the efficient degradation of cell surface proteins, which is a huge challenge, the molecular structures of degraders were rational designed and optimized. Remarkably, CD26 can be degraded by P4-3 obviously at low dose (~ 500 nM) without degrading CD26 isoenzymes, which was independent of autophagy pathway. Surprisingly, the proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299) and tumors were significantly inhibited by P4-3 , and no toxicity of P4-3 for BEAS-2B cells (human lung normal epithelial cells) were obtained. More interestingly, the powerful proliferation inhibition capabilities of P4-3 for organoids were observed. In addition, a mechanism of P4-3 for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC. Result and Discussion Screening of CD26 degraders The CD26 PROTAC-based degraders are designed according to the previous strategies. The candidate CD26 degraders (named P4-1 to P4-4 , von Hippel-Lindau-P1: VHL-P1 to VHL-P4 ) contain three parts: protein-of-interest (POI) ligand (Alogliptin were used here due to its superior binding capability 31 , 32 ), a linker and E3 ligase ligand (Lenalidomide or VHL ligand here, Fig. 1 a). The linker structure is optimized to obtain the molucule with the best degradation performance. As shown in Fig. 1 b, the bind energies of all VHL ligands-based degraders are higher than the Lenalidomide-based degraders, suggesting the degraders P4-1 to P4-4 are more suitable here. The detailed syntheses routes are described in Fig. S1 . The structures of P4-1 to P4-4 and related products are well characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (Figs. S2-S17). To evaluate the binding capabilities of candidate degraders, the half-maximal effect concentration (EC 50 ) were performed. As shown in Fig. S1 8, the EC 50 values of P4-3 and P4-4 are 86.95 nM and 10.32 nM, respectively. These results suggest that P4-3 may be a favorite degrader. Next, the degradation abilities of all candidates’ degraders are explored. The distribution of CD26 was first studied, as shown in Fig. S1 9, CD26 is mainly exists in cell membranes (or cell surfaces), and this trend is unaffected by the use of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). So unless otherwise mentioned, the western blot results of CD26 later refer to the expressions of cell membrane CD26. Then, NCl-H460, NCl-H1299 (two representative NSCLC cells) and BEAS-2B cells (human lung normal epithelial cells) were selected as models. As shown in Fig. 1 c, with concentration increases of degraders, CD26 from H460 cells are degraded and it is found that P4-3 has the best degradation capility (effective concentration is ~ 500 nM). At the same time, it is found that CD26 can be degraded by P4-3 with 9 hours (Fig. S20). Furthermore, CD26 from H1299 and BEAS-2B cells are also degraded by P4-3 as well (Fig. S21). Undoubtedly, the selectivity of P4-3 is another important issue, so the degradations of several isoenzymes by P4-3 were studied. As shown in Fig. 1 d and S21, DPP7, DPP8, DPP9 and fibroblast activation protein (FAP) from H460, H1299 and BEAS-2B cells are almost unaffected by P4-3 , which means P4-3 has extremely excellent selectivity toward CD26. Moreover, the specificity of P4-3 was confirmed by proteomics. As shown in Fig. 1 e, the changed proteins (Fold change > 1.5) from H460 cells occupy a little (< 1%). Meanwhile, the relative expressions of DPP3, DPP7, DPP8, DPP9, DPP10 and FAP from cells treated with P4-3 are almost unchanged compared with DMSO group (Fig. 1 f). Interestingly, CD26 was not detected in our mass spectrometry-based proteomic analysis. This observation may be attributed to the inherent limitations of mass spectrometry, such as the instrument’s detection threshold and dynamic range constraints, which can impact the detection of proteins with lower abundance, indicating that the selectivity of P4-3 is not affected by the high expressions of DPP3, DPP7, DPP8, DPP9, DPP10 and FAP. And these results further confirms that CD26 degrader with superior capacity is successfully constructed and the good performances of P4-3 can be attributed the rational design and optimization of molecular structures. In addition, the possible degradation pathways of P4-3 are investigated. As shown in Fig. S22, the addition of chloroquine (one kind of autophagy inhibitor) has no effect on the degradation results, suggesting CD26 degradation is independent of autophagy pathway. Furthermore, it can be seen that the degradation capacity of P4-3 is blocked by MG132 or Alogliptin (Fig. S23), suggesting CD26 is degraded via proteasomal pathway. The inhibitory effect of degraders on NSCLC cells The effects of Lenalidomide, Alogliptin and degraders on cell viabilities of H460, H1299, A549 and PC9 cells were studied (Fig. S24). It is shown that degraders have stronger inhibitory abilities toward H460 and H1299 cells than those of Lenalidomide and Alogliptin, while the cell viabilities of A549 and PC9 cells are still above 50% after treatment with degraders. This result could be attributed to the different expression levels of CD26 among these cells (Fig. S25), i.e. the sensitivities of H460 and H1299 cells to degraders are positively related to the expressions of CD26. Then the half maximal inhibitory concentration (IC 50 ) values of degraders toward cells were investigated. As shown in Fig. 2 a, P4- 3 has lower IC 50 value toward H460 cells at 48 hours and suitable IC 50 value at 72 hours, suggesting its powerful therapeutic effect. Importantly, P4-3 has no effect on the cell viabilities on the BEAS-2B cells (Fig. S26a), meaning P4-3 has almost no extra toxicity toward normal lung cells. And the proliferations of BEAS-2B cells are not affected by the knock-down of CD26 (Fig. S26b). To study the effect of P4-3 on migrations of cancer cells, the transwell assays were performed. As shown in Fig. 2 b, the migrations of H460 cells are mightily suppressed by P4-3 or P4-4 , which suggests the degradation of CD26 can cause the inhibition of migration of NSCLC cells. This conclusion is further confirmed by small interfering RNA (siRNA) interference results, i.e. the migrations of H460 cells are inhibited by knock-down of CD26 (Fig. 2 c, d). Lastly, it is observed that the proliferations of H460 cells are also repressed by the knock-down of CD26 (Fig. 2 e). It is no surprising that the same phenomenon is observed in H1299 cells (Fig. S27). These entire results exhibit that P4-3 is a promising agent with no extra toxicity for treatment of NSCLC and CD26 can be used as a target for NSCLC. The mechanism of therapeutic effect in vitro The above results show that proliferations and migrations of NSCLC cells were inhibited by CD26 degrader: P4-3 , the mechanism of such effects was then studied. It is reported that most chemotherapeutics can elevate the intracellular levels of reactive oxygen species (ROS) 33 , and high level of ROS could promote DNA damage and inhibit the cell proliferation 34 , 35 . So the ROS generation induced by P4-3 was first studied. As shown in Fig. 3 a, the addition of P4-3 causes the production of ROS in H460 cells and it is found that knock-down of CD26 can also elevate the intracellular ROS level (Fig. 3 a). At the same time, the contents of intracellular malondialdehyde (MDA, one important oxidative stress indicator) are increased by P4-3 and the knock-down of CD26 (Fig. 3 b). As mentioned above, the high ROS level could promote the DNA damage, as shown in Fig. 3 c, degradation and knock-down of CD26 lead to the serious DNA damage in H460 cells. Bennett et al suggested that mitochondrial dysfunction was related to DNA damage 36 , so as shown in Fig. 3 d, addition of P4-3 and knock-down of CD26 can lead to the reduce of red fluorescence of H460 cells, which means the decline of mitochondrial membrane potential, suggesting the occurrence of early apoptosis. Meanwhile, we observe that some cavitation are appeared in mitochondria and the length of mitochondria become shorted under the treatment of P4-3 or after the knock-down of CD26 (Fig. 3 e), suggesting the functions of mitochondria are destroyed. Then, the Annexin V/Propidium Iodide (PI) kits were performed to verify arises of apoptosis. As shown in Figs. 3 f and 3 g, cell ratios of Annexin V + /PI + are increased with the P4-3 treating time, which means the apoptosis are induced by degradation of CD26. And the knock-down of CD26 can also result in apoptosis (Figs. 3 f and 3 g). In order to further confirm the mechanism, some important biomarkers in apoptosis process were analysed. As shown in Fig. 3 h, cytochrome C (Cyt c), which are usually distributed in the mitochondrial inner membrane are high expressed after the treatment of P4-3 or knock-down of CD26, suggesting the damage of mitochondria. It is shown that Bcl2 (one kind of apoptosis inhibitory factor) are down expressed in the P4-3 treated or CD26 knock-down group (Fig. 3 h). On the contrary, Bax (one key protein in the mitochondrial apoptosis pathway) are high expressed in the P4-3 treated or CD26 knock-down group (Fig. 3 h), which indicates the degradation/knock-down of CD26 can lead to the mitochondria-mediated apoptosis. And this is affirmed by the high expression of cleaved caspase 3 (cle. cas3, Fig. 3 h). The apoptosis induced by P4-3 or knock-down of CD26 are also observed in H1299 cells (Fig. S28). As mentioned above, P4-3 exhibited almost no extra toxicity toward normal lung cells (BEAS-2B cells), the reason for this phenomenon was explored. As shown in Fig. S29, addition of P4-3 or knock-down of CD26 causes no increase of ROS and MDA levels in cells, as well as no DNA damages (Fig. S30). All above results suggest that the degradation of CD26 leads to the mitochondria-mediated apoptosis of NSCLC cells, which is clearly elaborated by the usage of P4-3 . Gene sequencing and proteomics result of different groups of cells To further investigate the potential biological mechanism of the treatment effects toward NSCLC cells, whole genome RNA expression sequencing (RNA-seq) in H460 cells was conducted. Afterward, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to obtain biological process in different samples. As shown in Fig. 4 a, 426 up-regulated genes and 705 down-regulated genes are found in the P4-3 treated group compared to the control group (DMSO group). Based on this result, several biological processes are significantly affected by P4-3 , such as apoptosis and apoptosis-multiple species (Fig. 4 b). While the reason of the superiority of P4-3 to Alogliptin in cell suppression is also missing, so proteomics in H460 cells treated without or with P4-3 at early stage was conducted. It is reported that up-regulated proliferating cell nuclear antigen clamp associated factor (PCLAF) promotes cancer cell growth and the attenuation of expression by siRNA suppresses cell proliferation 37 . The intervention of P4-3 results in the down-regulation of PCLAF (P/D, P means P4-3 group, D means DMSO group), suggesting the growth of H460 cells are inhibited. In addition, neuronal pentraxin II (NPTX2) is considered as one kind of tumor-suppressors 38 , and the up-regulation of it induced by P4-3 may be another pathway of cell suppression (Fig. 1 e). Although ras-related protein (RAB34) related to cell migration and invasion 39 , and adhesion G-protein coupled receptor D1 (ADGRD1) which is a potential prognostic biomarker in NSCLC 40 are down-regulated by the use of Alogliptin, two proteins: BolA family member 2 (BOLA2) and NADPH oxidase organizer 1 (NOXO1) which can promote tumor proliferation 41 , 42 are up-regulated in the Alogliptin-treating group (A/D, A means Alogliptin group, D means DMSO group), suggesting the potential risks of Alogliptin (Fig. 4 c). Importantly, the down-regulations of chromosome 1 open reading frame 122 (C1orf122, its silencing can reduce growth of the cancer cell line 43 ) and galectin-7 (LGALS7, its expression can increases the probability of lung metastases 44 ) are observed in the P4-3 -treating group instead of Alogliptin group (P/A). Meanwhile, P4-3 can also reduce the DNA resistance to external stress (endonuclease/exonuclease/phosphatase family domain containing 1, EEPD1 can provide DNA resistance to replication stress 45 ). Interestingly, succinate dehydrogenase complex assembly factor 2 (SDHAF2, a tumor suppressor 46 ) is activated by P4-3 instead of Alogliptin (Fig. 4 d). These results suggest use of P4-3 leads to the activation of some key cell death pathways, which can explain the superiority of P4-3 to Alogliptin in cell suppression. Furthermore, the changes of proteins induced by P4-3 are closely related to the process of cell growth and death (Fig. 4 e) and the pathways such as DNA damage and the occurrence of oxidative stress are the main events after treatment of P4-3 (Fig. 4 f). All above results suggest that the degradation of CD26 can lead to the mitochondria-mediated apoptosis of NSCLC cells, which is clearly elaborated by the usage of P4-3 . Antitumor effects in vivo Considering the excellent therapeutic effect of P4-3 against NSCLC cells mentioned above, it is necessary to explore its anti-tumor performance in vivo . The biosafety of P4-3 was firstly studied. As depicted in Fig. S31, the hematoxylin and eosin (H&E) staining results exhibit that no tissue damages are emerged in major organs of mice treated with P4-3 . Meanwhile, the weight bodies, alanine transaminase (ALT), creatinine (CREA) and α-amylase concentrations of mice are unaffected by P4-3 (Fig. S31b). It should be pointed out that an increased risk of pancreatitis is associated with the usage of CD26 inhibitors, so we conducted a thorough examination of the pancreatic tissue in our mice models treated with P4-3 . And no obvious pancreatic damage (Fig. S31) or inflammation (Fig. S31b) are observed, indicating P4-3 has a favorable safety profile. In addition, the types of T cells in mice spleen and concentrations of monocytes in mice serums are also unchanged upon the treatment of P4-3 (Fig. S32). The numbers of lymphocytes increase a little but are within reference range, which is the normal response against P4-3 in vivo (Fig. S32b). These results indicate that P4-3 is a safe potential drug. Next, we constructed H460 cell line-derived xenograft (CDX) subcutaneous tumor model to investigate the anti-tumor effect of P4-3 . As shown in Fig. 5 a, the mice bearing subcutaneous tumors were intravenous (I.V.) injected with P4-3 every 3 days one time (0.6 mg/kg) for 4 times. The volumes of tumors treated with P4-3 were significantly smaller than those of control group (Fig. 5 b), and the body weights of mice were almost unchanged (Fig. 5 b). It can be clearly seen that the tumors from mice treated with P4-3 are obviously suppressed (Fig. 5 c). As mentioned above, P4-3 could induce apoptosis of NSCLC cells, so the tumor repression mechanism was explored. After treated with P4-3 , the MDA levels in tumors is increased (Fig. S33), suggesting the tumors may be inhibited through the apoptosis manners. As illustrated in Fig. 5 d, we observe that there exist some complete mitochondria in tumors from control groups (Fig. 5 d), while nuclear chromatin is present in the central position in tumors from P4-3 treated mice (Fig. 5 d), further implying the tumors are in the state of apoptosis. This guess was further proved by the H&E and TUNEL staining results (Figs, 5d and 5e). In order to clarify the role of CD26 in tumor apoptosis, we employed lentivirus loaded small hairpin RNAs (shRNA) to knock down CD26 in NSCLC cells (H460 and H1299 cells). As shown in Fig. S34, the expressions of CD26 in cells are knocked down effectively through shRNA3 (Abbreviation: shRNA in the further experiments). Then the E. V. and shRNA-transfected H460 cell were subcutaneous injected to construct CDX model (Fig. 5 f), and it can be seen that the CD26 were also low expressed in tumors (Fig. S35). Interestingly, the tumors constructed by the shRNA-transfected H460 cells grow slowly (Figs. 5 g and 5 h) accompanied by the high levels of MDA concentrations in shRNA group (Fig. S36). The apoptosis of tumors are proved by several evidences (Figs. 5 i, 5 j), i.e. down-regulation of BCl2 and up-regulations of Cyt c, Bax and Cle. cas3 are also observed in the tumor from P4-3 treated mice (Figs. 5 k- 5 n), which confirms our supposition. Furthermore, we explored the therapeutic effect of P4-3 against lung metastases. The lung metastases constructed through normal and shRNA-transfected H460 cells were established successively (Fig. S37). It can be seen that tumors in lung are repressed successfully by P4-3 through apoptosis manner (Figs. S37b and S37c). And the tumors in lung derived from shRNA-transfected H460 cells grow little and apoptosis in tumors are also observed (Figs. S37e and S37f). The body weights are almost unchanged (Fig. S38). All above results indicate that P4-3 is a powerful agent for NSCLC therapy in vivo . The inhibitory performance of P4-3 against organoids To investigate the clinical application potential of P4-3 , we further extended our study to a more translational model by utilizing patient-derived organoids. Firstly, it is necessary to study the expressions of CD26 in patients’ NSCLC tumor tissues. As shown in Figs. 6 a and 6 b, the expression levels of CD26 in tumor are higher than those of paracancer (P.C.) tissues, suggesting the clinical significance of CD26 in NSCLC. Then the degradation effects of P4-3 on CD26 in tumor organoids were explored. As described in Fig. 6 c, CD26 are degradated obviously by P4-3 , and the higher dose of degrader can be attributed to the fact that organoids are composed of numerous tumor cells. Surprisingly, it is found that organoids are significantly suppressed by P4-3 and staurosporine (Figs. 6 d and S39). Meanwhile, the tumor organoids are inhibited by alogliptin slightly (Fig. S40), demonstrating the effective curative effect of P4-3 . Without doubt, the MDA concentrations in tumor organoids are increased under the treatment of P4-3 (Fig. 6 e). TEM and western blot results further confirm that tumor organoids undergo the apoptotic processes induced by P4-3 (Figs. 6 f and 6 g). The biosafety of P4-3 for P.C. organoids is of great importance. It is shown that the inhibition rates of P4-3 are significantly lower than those of staurosporine (Fig. S41), indicating that P4-3 could be a safe agent clinically. Together, all the results above confirm that P4-3 is a promising treatment strategy for NSCLC. In this study, several potential CD26 degraders were developed and the molecular structures of were optimized. Remarkably, CD26 can be degraded by P4-3 obviously at low dose (~ 500 nM) without the degradation of CD26 isoenzymes, which was independent of autophagy pathway. The proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299) and tumors were significantly inhibited by P4-3 which demonstrated suitable biosafety. Interestingly, the powerful proliferation inhibition capabilities of P4-3 for organoids were observed. Lastly and importantly, a possible mechanism of P4-3 for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC (Fig. 7 ). Materials and methods Materials Lenalidomide (Catalogue number: L0332-1g, CAS number: 191732-72-6), ethyl oxalyl monochloride (Catalogue number: C1066-25G, CAS number: 4755-77-5), succinic anhydride (Catalogue number: S0107-25G, CAS number: 108-30-5) and N-hydroxysuccinimide (NHS, Catalogue number: H0623-5G, CAS number: 6066-82-6) were purchased from Tokyo Chemical Industry (TCI) Co. Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl, Catalogue number: A10807-1g, CAS number: 25952-53-8) was purchased from Alfa Aesar organics products. Hexanedioic anhydride (Catalogue number: A0110740010, CAS number: 2035-75-8), suberic acid (Catalogue number: A0104830250, CAS number: 505-48-6), crystal violet (Catalogue number: D051109-5g, CAS number: 548-62-9) and dimethyl sulfoxide-d6 (DMSO-d6, catalogue number: E090002-0.6ml×10, CAS number: 2206-27-1) were purchased from Energy Chemical. Chloroform-d (CDCl 3 , catalogue number: 240987, CAS number: 865-49-6), super-dry pyridine (Catalogue number: 299669, CAS number: 110-86-1) and super-dry N, N-Dimethylformamide (DMF, catalogue number: 983353, CAS number: 68-12-2) were purchased from J&K Scientific. Staurosporine (Catalogue number: HY-15141, CAS number: 62996-74-1), alogliptin (Catalogue number: HY-A0023A, CAS number: 850649-61-5), MG132 (Catalogue number: HY-13259, CAS number: 133407-82-6) and chloroquine (Catalogue number: HY-17589A, CAS number: 54-05-7) were purchased from MedChemExpress. Dimethyl sulfoxide (DMSO) for molecular biology (Catalogue number: D8418, CAS number: 67-68-5) was purchased from Millipore Sigma. Dipeptidyl peptidase IV (DPP4) inhibitor screening assay kit (Catalogue number: ab133081) was purchased from Abcam. Reactive oxygen species (ROS) assay kit (Catalogue number: S0033M), DNA damage assay kit by γ-H 2 AX immunofluorescence (Catalogue number: C2035S), mitochondrial membrane potential assay kit with JC-1 (Catalogue number: C2006), lipid peroxidation MDA assay kit (Catalogue number: S0131M), annexin V-FITC apoptosis detection Kit (Catalogue number: C1062L), western blot transfer buffer (Catalogue number: P0021B), cell lysis buffer for western blot and IP (Catalogue number: P0013) and BeyoGold™ transwell permeable supports (Catalogue number: FTW010-24Ins) were purchased from Beyotime. CellTiter-Glo® 3D cell viability assay kit (Catalogue number: G9683) was purchased from Promega Corporation. Monoclonal anti-β-actin antibody (Catalogue number: 66009-1-Ig), monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Catalogue number: 60004-1-Ig), monoclonal anti-CD26 antibody (Catalogue number: 68383-1-Ig), polyclonal anti-dipeptidyl peptidase 8 (DPP8) antibody (Catalogue number: 12752-1-AP), polyclonal anti-bax antibody (Catalogue number: 50599-2-lg), polyclonal anti-Bcl2 human antibody (Catalogue number: 12789-1-AP), polyclonal anti-cytochrome c human antibody (Catalogue number: 10993-1-AP), cell counting kit-8 (CCK-8, Catalogue number: PF00004) and membrane protein extraction kit (Catalogue number: PK10015) were purchased from ProteinTech. Recombinant anti-fibroblast activation protein (FAP) antibody (Catalogue number: 66562S) was purchased from Cell Signaling Technology. Polyclonal anti-DPP7 antibody (Catalogue number: YT5125) was purchased from ImmunoWay Biotechnology. Recombinant anti-DPP9 antibody (Catalogue number: Ab302903) and recombinant anti-cleaved caspase-3 antibody (Catalogue number: Ab32042) were purchased from Abcam. Anti-ATP1B1 antibody (Catalogue number: CY8105) was purchased from Abways. Recombinant anti-Bax antibody for immunofluorescence (Catalogue number: GB154122-100), recombinant anti-Bcl2 antibody for immunofluorescence (Catalogue number: GB154380-100) and anti-DPP4/CD26 rabbit pAb for immunofluorescence (Catalogue number: GB114937-100) were purchased from ServiceBio. Co.Ltd. Small interfering RNA (siRNA: sense 5’-3’: GCACAGCACACCAACAUAUTT, antisense 5’-3’: AUAUGUUGGUGUGCUGUGCTT) for CD26 was purchased from GenePharma. Lentivirus loaded small hairpin RNAs kits (shRNA, Table S1 exhibited the sequences of shRNAs) were purchased from Genechem Co. Ltd. Lipofectamine™ 3000 transfection reagent (Catalogue number: L3000001) and fetal bovine serum (FBS, catalogue number: 10091155) were purchased from Thermo Fisher Scientific. Allophycocyanin (APC) anti-mouse CD3 antibody (Catalogue number: 100236), phycoerythrin (PE) anti-mouse CD8 antibody (Catalogue number: 104708) and fluorescein isothiocyanate (FITC) anti-mouse CD4 antibody (Catalogue number: 100406) were purchased from Biolegend. All other chemical or biological reagents were local. Synthetic routes of all compounds All Nuclear Magnetic Resonance (NMR) spectra were performed with a Bruker Avance 300 spectrometer. P4-1 . Lenalidomide (2.59 g, 10 mmol) was dissolved in 10 mL of pyridine, and ethyl oxalyl monochloride (2.4 g, 20 mmol) were added slowly into the solution. The reaction mixture was stirred at room temperature overnight and pyridine was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of methanol to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain P4-1-A (2.2 g, yield ~ 60%). The 1 H and 13 C NMR spectra are shown in Figure S1 and S2. 1 H NMR (300 MHz, DMSO-d6, δ): 10.99 (s, 1H), 10.88 (s, 1H), 7.51–7.67 (m, 3H), 5.14 (s, 1H), 4.26–4.39 (m, 4H), 2.83–2.95 (m, 1H), 2.29–2.60 (m, 3H), 1.96–2.02 (q, 1H), 1.28–1.32 (t, 2H). 13 C NMR (75 MHz, DMSO-d6, δ): 173.45, 171.59, 168.20, 160.96, 156.27, 136.53, 133.62, 132.55, 129.42, 128.08, 121.58, 63.15, 52.30, 47.20, 31.88, 23.21, 14.53. P4-1-A (0.5 g, 1.4 mmol) and alogliptin (0.67 g, 2.0 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was heated to reflux for 16 hours. Then the mixture was cooled and added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. At last, the solid was dried with phosphorus pentoxide for 24 hours to obtain P4-1 (white solid, 0.2 g, yield ~ 25%). The 1 H and 13 C NMR spectra are shown in Figure S3 and S4. 1 H NMR (300 MHz, DMSO-d6, δ): 11.09 (s, 1H), 10.78 (s, 1H), 8.95 (s, 1H), 7.80 (s, 1H), 7.62 (s, 4H), 7.42 (s, 2H), 7.24 (s, 1H), 5.36 (s, 1H), 5.15 (s, 3H), 4.40 (s, 2H), 3.79 (s, 1H), 3.33 (s, 3H), 2.48–2.60, 1.56–1.98 (m, 12H). 13 C NMR (75 MHz, DMSO-d6, δ): 173.48, 171.57, 168.25, 162.80, 159.96, 159.04, 152.64, 141.72, 136.47, 134.15, 133.82, 133.55, 132.84, 128.62, 127.73, 117.88, 110.64, 90.11, 54.86, 51.66, 47.15, 46.76, 46.64, 29.28, 28.06, 23.76, 23.21. HR-ESI-MS, calcd for C 34 H 33 N 7 O 7 [M] + : m/z 651.2441, found [M + Na] + : m/z 674.2337. P4-2. Lenalidomide (2.59 g, 10 mmol) was dissolved in 10 mL of DMF, and succinic anhydride (1.5 g, 15 mmol) was added slowly into the solution. The reaction mixture was stirred at 60°C overnight and pyridine was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain P4-2-A (2.0 g, yield ~ 50%). The 1 H and 13 C NMR spectra are shown in Figure S5 and S6. 1 H NMR (300 MHz, DMSO-d6, δ): 12.10 (s, 2H), 10.97 (s, 1H), 9.82 (s, 1H), 7.80–7.81 (d, 1H, J = 3 Hz), 7.78–7.79 (d, 2H), 5.09–5.15 (q, 1H), 4.33–4.41 (t, 2H), 2.84–2.97 (m, 1H), 2.48–2.62 (m, 4H), 2.25–2.39 (m, 1H), 1.99–2.04 (m, 1H). 13 C NMR (75 MHz, DMSO-d6, δ): 174.40, 173.48, 171.69, 170.99, 168.51, 134.44, 134.42, 133.36, 129.34, 125.79, 119.66, 52.20, 47.14, 31.90, 31.36, 29.67, 23.36. P4-2-A (0.5 g, 1.4 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with methylene chloride (CH 2 Cl 2 ) /ethyl acetate (v/v, 100/1) to CH 2 Cl 2 /methanol (v/v, 50/1) and then re-crystallized with water, affording P4-2 as a white solid (0.08 g, yeild ~ 14%). The 1 H and 13 C NMR spectra are shown in Figure S7 and S8. 1 H NMR (300 MHz, DMSO-d6, δ): 9.66 (s, 1H), 9.27 (s, 1H), 7.82 (s, 1H), 7.63–7.65 (d, 1H, J = 6 Hz), 7.51–7.58 (m, 2H), 7.38 (s, 1H), 7.35 (s, 1H), 7.22 (s, 1H), 6.65 (s, 1H), 5.20–5.22 (d, 2H), 5.15 (s, 1H), 4.26–4.34 (t, 2H), 3.98 (s, 1H), 3.22–3.24 (d, 3H), 2,55-2.72 (d, 8H), 2.27 (s, 1H), 2.02 (s, 5H), 1.70 (s, 2H), 1.53 (s, 2H). 13 C NMR (75 MHz, CDCl 3 , δ): 172.49, 171.53, 170.87, 169.35, 163.48, 159.92, 152.67, 140.86, 133.66, 132.65, 129.28, 128.40, 125.73, 120.46, 117.89, 110.83, 90.31, 55.34, 52.17, 46.80, 39.59, 37.61, 37.33, 34.62, 33.87, 32.97, 32.18, 30.28, 29.95, 29.62, 28.22, 27.33, 24.70, 22.95, 19.97, 14.40. HR-ESI-MS, calcd for C 36 H 37 N 7 O 7 [M] + : m/z 679.2754, found [M + Na] + : m/z 702.2643. P4-3. Hexanedioic anhydride (1.5 g, 15 mmol) and lenalidomide (2.59 g, 10 mmol) were dissolved in 25 mL of DMF, the reaction mixture was stirred at 60°C overnight and DMF was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain P4-3-A (1.5 g, yield ~ 35%). The 1 H and 13 C NMR spectra are shown in Figure S9 and S10. 1 H NMR (300 MHz, DMSO-d6, δ): 12.03 (s, 1H), 11.02 (s, 1H), 9.78–9.82 (d, 1H, J = 12 Hz), 7.79 (s, 1H), 7.48 (s, 2H), 5.11–5.15 (d, 1H, J = 12 Hz), 4.34 (s, 2H), 3.56 (s, 1H), 2.06–2.86 (m, 8H), 1.47–1.57 (d, 3H). 13 C NMR (75 MHz, DMSO-d6, δ): 175.20, 173.47, 171.88, 171.68, 168.52, 134.48, 133.36, 129.29, 126.01, 119.71, 52.21, 47.22, 36.22, 34.42, 31.90, 29.69, 25.38, 24.93, 23.31. P4-3-A (0.5 g, 1.3 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with CH 2 Cl 2 /ethyl acetate (v/v, 100/1) to CH 2 Cl 2 /methanol (v/v, 65/1) and then re-crystallized with water, affording P4-3 as a white-gray solid (0.04 g, yeild ~ 8%). The 1 H and 13 C NMR spectra are shown in Figure S11 and S12. 1 H NMR (300 MHz, DMSO-d6, δ): 9.72–9.79 (d, 1H, J = 21 Hz), 9.11 (s, 1H), 7.70–7.75 (t, 1H), 7.61–7.64 (d, 1H, J = 9 Hz), 7.50–7.54 (t, 2H), 7.35 (s, 2H), 7.18–7.20 (d, 1H, J = 6 Hz), 6.58–6.60 (d, 1H, J = 6 Hz), 5.20–5.28 (d, 3H), 5.02–5.04 (t, 1H), 4.33 (s, 2H), 3.95 (s, 1H), 3.20 (s, 3H), 2.02–2.66 (m, 12H), 1.43–1.79 (t, 9H). 13 C NMR (75 MHz, CDCl 3 , δ): 173.20, 172.56, 172.45, 170.89, 169.40, 163.50, 159.96, 152.66, 140.74, 134.40, 134.29, 133.63, 133.49, 133.42, 132.66, 129.21, 128.39, 127.80, 117.72, 110.78, 90.19, 55.37, 52.15, 46.82, 45.55, 36.35, 35.97, 31.68, 29.93, 29.28, 28.22, 25.19, 25.05, 23.26. HR-ESI-MS, calcd for C 38 H 41 N 7 O 7 [M] + : m/z 707.3067, found [M + Na] + : m/z 730.2962. P4-4. Suberic acid (8.7 g, 50 mmol) and acetic anhydride were stirred at 140°C overnight. And then the acetic anhydride was removed, the residue was beat by 100 mL of acetonitrile to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain suberic anhydride (4.4 g, yield ~ 50%). Suberic anhydride (1.7 g, 11 mmol) and lenalidomide (2.59 g, 10 mmol) were dissolved in 25 mL of DMF. The reaction mixture was stirred at 60°C overnight and DMF was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain P4-4-A (1.0 g, yield ~ 25%). The 1 H and 13 C NMR spectra are shown in Figure S13 and S14. 1 H NMR (300 MHz, DMSO-d6, δ): 11.98 (s, 1H), 11.02 (s, 1H), 9.76 (s, 1H), 7.78–7.93 (t, 1H), 7.43–7.50 (q, 2H), 5.10–5.16 (q, 1H), 4.27–4.41 (q, 2H), 2.86–2.90 (d, 2H), 2.71 (s, 1H), 2.00-2.71 (m, 7H), 1.29–1.98 (m, 6H). 13 C NMR (75 MHz, DMSO-d6, δ): 173.47, 172.03, 171.70, 168.51, 134.49, 133.35, 129.29, 125.85, 119.73, 52.36, 52.19, 47.20, 36.47, 31.88, 29.14, 25.69, 23.34. P4-4-A (0.5 g, 1.2 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with CH 2 Cl 2 /ethyl acetate (v/v, 100/1) to CH 2 Cl 2 /methanol (v/v, 60/1) and then re-crystallized with water, affording P4-4 as a gray solid (0.05 g, yeild ~ 10%). The 1 H and 13 C NMR spectra are shown in Figure S15 and S16. 1 H NMR (300 MHz, DMSO-d6, δ): 9.70–9.79 (d, 1H, J = 27 Hz), 9.11 (s, 1H), 7.68–7.74 (t, 1H), 7.60–7.63 (d, 1H, J = 9Hz), 7.49–7.54 (t, 2H), 7.31–7.36 (t, 2H), 7.16–7.19 (d, 1H, J = 9Hz), 6.49 (s, 1H), 5.27 (s, 2H), 5.19 (s, 1H), 4.97–5.01 (d, 1H, J = 12Hz), 4.32 (s, 2H), 3.94 (s, 1H), 3.19 (s, 3H), 2.01–2.69 (m, 11H), 1.18–1.79 (m, 13H). 13 C NMR (75 MHz, CDCl 3 , δ): 173.46, 172.75, 172.44, 170.78, 169.43, 163.46, 159.94, 152.61, 140.67, 134.54, 134.45, 133.58, 133.41, 132.66, 129.18, 128.38, 127.79, 120.57, 117.66, 110.87, 90.19, 55.53, 53.76, 52.06, 46.76, 45.48, 36.78, 36.38, 31.67, 29.00, 28.20, 25.65, 23.31. HR-ESI-MS, calcd for C 40 H 45 N 7 O 7 [M] + : m/z 735.3380, found [M + Na] + : m/z 758.3273. General procedures for cell viability tests Unless otherwise stated, all the cell viability tests were performed according to the following procedure. Cells (5.0 × 10 3 cells/well) were cultured in 96-well plates for 24 hours. Different compounds with suitable final concentrations were added in to each well (0.5% DMSO was used as co-solvent, and no compound was added in cell proliferation experiment), and cells were cultured for different time. The culture medium was then discarded and cell viabilities were measured according to the CCK-8 protocols. The absorbance value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent). EC 50 values The half-maximal effect concentration (EC 50 ) values of P4-1 to P4-4 were determined according to the protocols of dipeptidyl peptidase IV (DPP4, CD26) inhibitor screening assay kit. The fluorescence value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent). Extracting membrane protein and cytoplasmic protein Cells were lysed with lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice for 10 min, and then centrifuged at 13,000 rpm for 15 min. For membrane protein and cytoplasmic protein extraction, the cells were collected at 4°C and washed with pre-cooled phosphate buffered saline (PBS, 0.1 M). The membrane protein and cytoplasmic protein were extracted using a membrane protein extraction kit according the protocols. Protease or Chloroquine inhibition test Cells were incubated with MG132 (5 µM) or Alogliptin (5 µM) for 9 hours only, or followed by addition of the P4-3 (1 µM) for an additional 9 hours. Subsequently, proteins were extracted, and the degradation efficacy was evaluated using western blot assays. The procedures of effects of Chloroquine on the CD26 degradation efficacy were similar. Key parameters of western blot The protein samples were prepared according to the demand, and the protein concentrations were determined through the bicinchoninic acid assay (BCA). The current for film transfer was 250 mA and the transfer time was determined based on protein molecular weight. The blot imaging was performed through Amersham ImageQuant™ 800 western blot imaging systems (Cytiva). Transwell assays Unless otherwise stated, all transwell assays were performed according to the following procedure. Cells (1.0 × 10 5 cells/well) were cultured without FBS in the upper chamber of 6-transwell well, culture medium with FBS was placed in the lower chamber. And different compounds with suitable final concentrations were added in to upper chamber (0.5% DMSO was used as co-solvent, and no compound was added siRNA-transfected cells). Then cells were cultured for different time. The culture medium was discarded and the cells in the lower chamber were stained with crystal violet, counted and photographed. siRNA transfection Cells (NCI-H460, NCI-H1299 and BEAS-2B cells, 1 × 10 5 cells/well) were cultured in 6-well plates for 24 hours. And CD26 siRNA and Lipofectamine™ 3000 were mixed to prepared transfection. The mixed solutions were then added into each well and the final concentration of siRNA was controlled to 100 nM, at the same time, CD26-negative control was also transfected. Cells were cultured for further 24 hours and prepared to western blot experiments to verify the transfection efficiency. The successfully-transfected cells were used for further experiments. shRNA transfection Cells (H460 and H1299 cells, 1 × 10 4 cells/well) were cultured in 96-well plates for 24 hours. The multiplicities of infection (MOI) values of both cells were about 100. The transfection processes were strictly complied with the protocols provided by Genechem. Cells were performed to fluorescent image and western blot experiments to verify the transfection efficiency. The successfully-transfected cells were used for further experiments. ROS fluorescent imaging Cells (H460, H1299 and BEAS-2B cells, 1 × 10 5 cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with P4-3 for 24 hours (siRNA-transfected cells were treated without P4-3 ) and cultured with DCFH-DA (Final concentrations: 10 µM) according to the ROS assay kit protocols for 1 hour. Lastly, the cells were prepared to fluorescent image. DNA damage fluorescent imaging Cells (H460, H1299 and BEAS-2B cells, 1 × 10 5 cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with P4-3 for 24 hours (siRNA-transfected cells were treated without P4-3 ) and cultured with γ-H 2 AX (Final concentrations: 50 µM) according to the DNA damage assay kit protocols. Lastly, the cells were prepared to fluorescent image. Mitochondrial membrane potential confocal fluorescent imaging Cells (H460 and H1299 cells, 5 × 10 4 cells/mL) were cultured in confocal dishes) for 24 hours. Then cells were treated with P4-3 for 24 hours and cultured with JC-1 probe according to the mitochondrial membrane potential assay kit protocols. Lastly, the cells were placed to confocal fluorescent image through Leica TCS SP5 confocal laser scanning microscope. MDA assay Cells (H460, H1299 and BEAS-2B cells, 1 × 10 5 cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with P4-3 for 24 hours (siRNA-transfected cells were treated without P4-3 ) and lysed. The MDA concentrations in lysates were determined according to the lipid peroxidation MDA assay kit protocols. It should be noted that the MDA concentrations in organoids and animal samples were detected through the similar procedures. The absorbance value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent). Flow fluorescence analysis Cells (H460 and H1299 cells, 1 × 10 5 cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with P4-3 for 24 hours (siRNA-transfected cells were treated without P4-3 ) and placed to stain with annexin V-FITC according to the kit protocols. Lastly, the stained cells were analyzed through NovoCyte flow cytometer (Agilent). Transmission electron microscopy (TEM) The cells were treated without or with P4-3 (9 µM, 0.5% DMSO was used as co-solvent) for 24 hours. Then cells were centrifuged and the precipitations were re-suspended in the fixative and then fixed at 4℃ for 4 hours. The fixed cells were centrifuged and washed with 0.1 M PBS for 3 times and were wrapped in the 1% agarose. The samples were then fixed with 1% OsO 4 (in PBS) for 2 hours at room temperature without light. After that, the samples were washed with 0.1 M PBS for 3 times. Samples were then dehydrated at room temperature and embedding at 37°C overnight. The embedding models were polymerized at 60°C for 48 hours. After that, the samples were cut into flakes (60–80 nm) and were fished out onto the 150 meshes cuprum grids. Samples were successively stained with 2% uranium acetate for 8 min without light and 2.6% lead citrate without CO 2 for 8 min for experiments. The images were obtained through a HT7800/HT7700 TEM (Hitachi) with accelerating voltage of 80.0 kV. The procedures for tumor tissues and organoids TEM images were similar. Proteomics or RNA sequencing (RNA-seq) sample preparation H460 cells were seeded in a 6-well plate. Based on different interventions, the following groups were established: P4-3 group (1 µM with 0.5% DMSO as co-solvent), DMSO group (0.5% DMSO), and Alogliptin group (1 µM with 0.5% DMSO as co-solvent). The cells were incubated with different drug for 9 hours, and were washed 2–3 times with pre-cooled PBS at 4°C. The cells were then digested with trypsin and collected and rapid frozen in liquid nitrogen for proteomics or RNA-seq analysis. For RNA-seq analysis, the procedures were similar apart from the concentration and treating time of P4-3 . RNA-seq Total RNA was extracted from the cells using TRIzol® Reagent according the manufacturer’s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. according to the manufacturer’s instructions (Illumina, San Diego, CA). The human RNA-seq transcriptome library was prepared following Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1 µg of total RNA. Messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Then double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. Libraries were selected for cDNA target fragments of 300 bp on 2% low range ultra agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After being quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq 6000 sequencer (2 × 150 bp read length). To identify differential expression genes (DEGs) between different groups, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. Essentially, differential expression analysis was performed using the DESeq2 or DEGseq method. DEGs with |log2FC| ≧ 1 and FDR ≤ 0.05 (DESeq2) or FDR ≤ 0.001 (DEGseq) were considered to be significantly different expressed genes. In addition, functional-enrichment analysis: Kyoto Encyclopedia of Genes (KEGG) was performed to identify which DEGs were significantly enriched in metabolic pathways at Bonferroni-corrected P-value ≤ 0.05 compared with the whole-transcriptome background. KEGG pathway analysis was carried out by KEGG orthology based annotation system (KOBAS). Proteomics The samples in liquid nitrogen were transferred to a 5-mL centrifuge tube. After that, four volumes of lysis buffer (with 1% of protease inhibitor: cocktail) was added to the cell powder, followed by sonication three minutes on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 min. Finally, the supernatant was collected and the protein concentration was determined with BCA kit. The protein solutions were reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature without light. The protein samples were then diluted by adding 200 mM triethanolamine borate (TEAB) to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for thefirst digestion overnight and 1:100 trypsin-to-protein mass ratios for a second 4 h-digestion. Finally, the peptides were desalted by Strata X SPE column. The tryptic peptides were dissolved in solvent A, directly loaded onto a home-made reversed-phase analytical column (25-cm length, 100 µm i.d.). The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile/in water) and solvent B (0.1% formic acid in acetonitrile). Peptides were separated with following gradient: 0–9 min, 6%-24%B;9–11 min, 24%-35%B༛11–13 min, 35%-80%B༛13–15 min, 80%B, and all at a constant flow rate of 500 nl/minon a NanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the TIMS-TOF Pro mass spectrometry. The electrospray voltage applied was 1.75 kV. Precursors and fragments were analyzed at the TOF detector. The timsTOF Pro was operated in data independent parallel accumulation serial fragmentation (dia-PASEF) mode. The full MS scan was set as300-1500 (MS/MS scan range) and 20PASEF (MS/MS mode)-MS/MS scans were acquired per cycle. The MS/MS scan range was set as 400–850 and isolation window was set as7 m/z. After that, the DIA data were processed using DIA-NN search engine (v.1.8). Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20231220.fasta concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 1 missing cleavages. Excision on N-term Met and carbamidomethyl on Cys were specified as fixed modification. FDR was adjusted to < 5%. The structural domain of a protein is a specific protein region in a protein that is conserved in sequence and can generally perform a function independently, and is a structural component of molecular function, generally consisting of 25 to 500 amino acids. These areas are relatively spatially compact, structurally stable, and capable of being independently folded into functional structures. A protein may have multiple domains, and a domain may alsoexist in multiple proteins. In the project data, protein structural domain annotation was performed on the identified proteins based on the Pfam database and the corresponding PfamScan tool. The KEGG integrates currently known protein-protein interaction network information, such as pathways and related complexes (Pathway database), genes and gene products (Gene database), biological complexes and related reactions (Compound and Reaction databases), and other information. The KEGG pathways mainly include metabolism, genetic information processing, environmental information processing, cellular processes, human diseases and drug development. We annotate protein pathways based on the KEGG pathway database, and identify proteins through BLAST comparison (blastp, evalue ≤ 1e − 4 ), for each sequence, the annotation is based on the top-scoring comparison result. Lastly, we used PSORTb software to perform subcellular structure prediction analysis of proteins identified in eukaryotes. Biosafety evaluation of P4-3 in vivo The uses of animals were permitted by the institutional animal ethics committee of Xi’an Jiaotong University (Approval number: XJTUAE2023-1611). Briefly, C57BL/6 mice (healthy, 5–6 weeks) were provided from Laboratory Animal Center (LAC) of Xi'an Jiaotong University and were randomly divided into 2 groups (N = 5): control group and P4-3 group. The mice in P4-3 group were intravenous injected with P4-3 (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times) and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without P4-3 . Subsequently, mice were sacrificed and the main organs: heart, liver, spleen, lung, kidney and pancreas were sliced for hematoxylin and eosin (H&E) staining experiments to evaluate the biosafety of P4-3 . Whole blood was collected for complete blood count (CBC). Serum was collected to measure relevant biochemical indicators. The spleen was excised and grinded. The grinding solution was filtered through a 40-µm cell strainer (Biosharp) to obtain a single-cell suspension for flow cytometry experiments (BioTek Synergy LX multimode micro-plate reader, Agilent). The anti-tumor evaluation effects on subcutaneous tumor The animal experiments were permitted by the institutional animal ethics committee of Xi’an Jiaotong University (Approval number: XJTUAE2023-1611 and XJTUAE2024-1849). For P4-3 intervention experiment, BALB/c nude mice (healthy, 5–6 weeks) were randomly divided into 2 groups (N = 5): control group and P4-3 group. The mice in P4-3 group were firstly subcutaneous injected with H460 cells (3 × 10 6 cells, 100 µL) and mice were feed for 10 days. After tumor formation, mice in P4-3 group were intravenous injected with P4-3 (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times), and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without P4-3 . The weights and tumor volumes were recorded every 3 days. After treatment, mice were sacrificed and the tumors were weighed and sliced or mashed for further experiments. For shRNA-transfected injecting experiment, BALB/c nude mice (healthy, 5–6 weeks) were randomly divided into 2 groups (N = 5): E. V. group and shRNA group. The mice in shRNA group were firstly subcutaneous injected with shRNA-transfected H460 cells (3 × 10 6 cells, 100 µL) and mice in E. V. group were firstly subcutaneous injected with E. V.-transfected H460 cells (3 × 10 6 cells, 100 µL). Mice were feed for 10 days and the weights were recorded every 3 days. After 17 days, mice were sacrificed and the tumors were weighed and sliced for further experiments. The anti-tumor evaluation effects on metastases For P4-3 intervention experiment, BALB/c nude mice (healthy, 5–6 weeks) were randomly divided into 2 groups (N = 5): control group and P4-3 group. The mice in P4-3 group were firstly thoracic injected with H460 cells (3 × 10 6 cells, 100 µL) and mice were feed for 10 days. After tumor formation, mice in P4-3 group were intravenous injected with P4-3 (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times), and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without P4-3 . The weights were recorded every 3 days. After treatment, mice were sacrificed and the tumors were weighed and sliced or mashed for further experiments. For shRNA-transfected injecting experiment, BALB/c nude mice (healthy, 5–6 weeks) were randomly divided into 2 groups (N = 5): E. V. group and shRNA group. The mice in shRNA group were firstly thoracic injected with shRNA-transfected H460 cells (3 × 10 6 cells, 100 µL) and mice in E. V. group were firstly intravenous injected with E. V.-transfected H460 cells (3 × 10 6 cells, 100 µL). Mice were feed for 10 days and the weights were recorded every 3 days. After 17 days, mice were sacrificed and the tumor volumes were recorded and tumors were sliced for further experiments. The anti-cancer evaluation effects of P4-3 on organoids The use of human tumor and paracancer tissues of non-small cell lung cancer were strictly complied with the rules of institutional animal ethics committee of Xi’an Jiaotong University (Approval number: XJTU1AF2021LSK-484) and the informed consent has been signed by patients. All organoids were obtained from Shaanxi Weiyuan Biomedical Research Institute Co., Ltd. 3 cases were randomly selected for anti-cancer evaluation. Firstly, tumor organoids were treated with P4-3 with different concentrations for 9 hours and then the degradation of CD26 was tested. After that, tumor and paracancer organoids were treated with different compounds for different time, the inhibition rates of cells were performed through CellTiter-Glo® 3D cell viability assay kit. And the lysates were prepared to perform other experiments. H&E Major organs or tumors were successively fixed, embedded, and sectioned. The samples were preprocessed with pretreatment liquid for 1 min and then stained with Hematoxylin for 5 min. After that, samples were washed and stained with Eosin for 15 s. Samples were then dehydrated and sealed up for imaging (Eclipse E100 optical microscope, Nikon). Immunofluorescence or Tunel or Immunohistochemical staining The tumors were collected and paraffin sectioned. The samples were then repaired with corresponding primary and secondary antibodies. After that, the samples were sealed with 3% bovine serum albumin (BSA) or 10% donkey serum. The samples were incubated with corresponding primary antibodies at 4°C overnight. Subsequently, samples were washed with PBS for 3 times and incubated with corresponding secondary antibodies for 50 min without light. Lastly, the samples were washed with PBS for 3 times and stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min without light. The samples were washed with PBS for 3 times firstly and auto-fluorescence quencher for imaging (Eclipse C1 fluorescence microscope, Nikon). The procedures for immunohistochemical staining were similar and the procedures for Tunel staining were also similar without sealing and incubation of antibodies. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.0 and Origin 9 software. All data are determined from at least three independent experiments and presented as mean ± standard deviation (SD). Statistical significance between two groups was calculated using Two-tailed test. Differences between multiple groups were assessed by ANOVA followed by Dunnett post-hoc test (ANOVA Dunnett’t test). P-values of less than 0.05 were considered as statistically significance. Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability Source data are provided with this paper. Competing interests The authors have declared that no competing interest exists. Additional information Supplementary information The online version containssupplementary material available at Reprints and permissions information is available at Author contributions G. J. Zhang, B. H. Liu and Q. Y. Gong conceptualized and designed the project. B. H. Liu, D. Q. Qiao, X. Z. Zhu and J. Q. Huang performed the experiments and analyzed the results. R. Gao and L. J. Zhang provided suggestions on cell line and animal models construction. Y. J. Huang provided suggestions on compound synthesis. G. J. Zhang, J. Z. Wang, Y. J. Huang and Q. Y. Gong supervised the project. G. J. Zhang, B. H. Liu and Q. Y. Gong wrote the manuscript. All authors discussed and commented on the manuscript. Acknowledgements Authors thank the support from the center for translational medicine, the First Affiliated Hospital of Xi’an Jiaotong University and Shaanxi Weiyuan Biomedical Research Institute Co., Ltd. Q. Gong thank the funds supporting from the Key Research and Development Project of Shaanxi Province (2023-YBSF-292), the opening foundation (M2022-3) from Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, Ministry of Education, Qingdao University of Science and Technology. J. Wang thanks the fund supporting from the National Natural Science Foundation of China (82102976). Y. Huang thanks the fund supporting from the National Natural Science Foundation of China (52203337) and the Youth Top Talent Program (11301223010722) from Xi’an Jiaotong University. G. 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Heliyon 10:e29285 Geleta AB, Dong LF, Rohlena J, Neuzil J (2016) The assembly factor SDHAF2 is dispensable for flavination of the catalytic subunit of mitochondrial complex II in breast cancer cells. J Biol Chem 291:21414–21420 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5702716","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399336458,"identity":"475e6e78-f5b6-48d8-993d-a4c7462f1391","order_by":0,"name":"Qiuyu Gong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYNACAyBmbwBTDAwHiNbCc4DB4ADxWkBAIgGqmpAWeffewy/eFBxO3HDzjUHxxzYGOb4bCYyfC/BoMTxzLs1yjgFQy+20BIODbQzGkjcSmKVn4NMyI8fMmAesJfkASEvihhsJbMw8RGm5ebABpKWeoBZ5iRzjx2AtN5jBtiQYENJiwHPGjHGOQbrxzDNAv5w5J2E488zDZmm8trT3GH9488datu/4GTODijIbeb7jyQc/47XlAAObBFCBYwMDAxswRiWAYowNeDQAbWlgYP4A1GIPZDM/wKt0FIyCUTAKRiwAAO+jU6sn3R8OAAAAAElFTkSuQmCC","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":true,"prefix":"","firstName":"Qiuyu","middleName":"","lastName":"Gong","suffix":""},{"id":399336459,"identity":"a28ea182-2cd4-4e98-b4d8-be3860eb8baa","order_by":1,"name":"Zhang Guangjian","email":"","orcid":"","institution":"Department of Thoracic Surgery, the First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Guangjian","suffix":""},{"id":399336460,"identity":"16902eee-e0e6-445e-8598-fae4eb8c329d","order_by":2,"name":"Bohao Liu","email":"","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":false,"prefix":"","firstName":"Bohao","middleName":"","lastName":"Liu","suffix":""},{"id":399336461,"identity":"53f0b0ee-0abf-410a-a170-696da9ee473e","order_by":3,"name":"Deqian Qiao","email":"","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":false,"prefix":"","firstName":"Deqian","middleName":"","lastName":"Qiao","suffix":""},{"id":399336462,"identity":"bfa552e9-5f1d-4bdf-a98d-c8c4ea3cae74","order_by":4,"name":"Xingzhuo Zhu","email":"","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":false,"prefix":"","firstName":"Xingzhuo","middleName":"","lastName":"Zhu","suffix":""},{"id":399336463,"identity":"dbe0c71b-52bc-4cf8-b4f0-5febc56b0769","order_by":5,"name":"Jiaqi Huang","email":"","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Huang","suffix":""},{"id":399336464,"identity":"791ba0e5-f6bb-4fd1-b0a4-4d7d3f6aedea","order_by":6,"name":"Rui Gao","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Gao","suffix":""},{"id":399336465,"identity":"892ec1f2-0ac0-4943-89df-99948b0e89af","order_by":7,"name":"Linjuan Zhang","email":"","orcid":"","institution":"xi`an jiaotong university","correspondingAuthor":false,"prefix":"","firstName":"Linjuan","middleName":"","lastName":"Zhang","suffix":""},{"id":399336466,"identity":"98c029a3-b5bf-492b-bcc0-ec95d764571a","order_by":8,"name":"Jizhao Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jizhao","middleName":"","lastName":"Wang","suffix":""},{"id":399336467,"identity":"6205a993-b9ae-4471-baa2-d2c3df077888","order_by":9,"name":"Yinjuan Huang","email":"","orcid":"","institution":"Nanyang Technological University","correspondingAuthor":false,"prefix":"","firstName":"Yinjuan","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-12-24 02:05:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5702716/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5702716/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73356210,"identity":"4d44d31d-5dd2-4293-b960-fb2c8af73d0c","added_by":"auto","created_at":"2025-01-09 08:07:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18508187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe design of CD26 candidate degraders and their degradation performances.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) The molecular formula of VHL ligands-based and Lenalidomide-based degraders. (\u003cstrong\u003eb\u003c/strong\u003e) The binding modes and bind energies of VHL ligands-based degraders and degraders in the manuscript toward CD26 (PDB: 3G0B). (\u003cstrong\u003ec\u003c/strong\u003e) The western blot of CD26 from H460 cells. Cells were treated with different concentrations of candidate degraders for 9 hours at 37 °C. 0.5 % DMSO was used as co-solvent. (\u003cstrong\u003ed\u003c/strong\u003e) The western blot of DPP7, DPP8, DPP9 and FAP from H460 cells. Cells were treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003e for 9 hours at 37 °C. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (\u003cstrong\u003ee\u003c/strong\u003e) The protein volcano maps from different groups: P/D means\u003cstrong\u003e P4-3 \u003c/strong\u003egroup vs DMSO group. H460 cells were treated with\u003cstrong\u003eP4-3 \u003c/strong\u003e(1 μM, 9 hours, 0.5 % DMSO was used as co-solvent) or Alogliptin (1 μM, 9 hours, 0.5 % DMSO was used as co-solvent). (\u003cstrong\u003ef\u003c/strong\u003e) The relative expression levels of DPP3, DPP7, DPP8, DPP9, DPP10 and FAP from H460 cells treated with \u003cstrong\u003eP4-3\u003c/strong\u003e (1.0 μΜ for 9 hours) determined from proteomics.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/2658a4d5e807db34cf71f840.png"},{"id":73356206,"identity":"99bbca1c-7671-44c1-8a92-36eaf5c0bcb4","added_by":"auto","created_at":"2025-01-09 08:07:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16493568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe therapeutic effect of degraders toward H460 cells\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Cell viabilities of H460 cells treated with different concentrations of degraders for different time. N=3. 5 % DMSO was used as co-solvent. (\u003cstrong\u003eb\u003c/strong\u003e) Left: the representative photos of cresyl violet-staining H460 cells treated with \u003cstrong\u003eP4-3\u003c/strong\u003e (final concentration: 9 μM, time: 24 hours, DMSO was used as co-solvent) for 24 hours. Scale bar 25 μm. Right: the statistics of cell migration numbers of different systems. n.s.: no significance. *: p \u0026lt; 0.05. **: p \u0026lt; 0.01 compared to cells group. ANOVA Dunnett’t test. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (\u003cstrong\u003ec\u003c/strong\u003e) The western blot of CD26 of H460 cells, negative control (NC) and siRNA-transfected H460 cells. (\u003cstrong\u003ed\u003c/strong\u003e) Left: the representative photos of cresyl violet-staining H460 cells, NC and siRNA-transfected H460 cells. Scale bar 50 μm. Right: the statistics of cell migration numbers of different systems. n.s.: no significance. ANOVA Dunnett’t test. (\u003cstrong\u003ee\u003c/strong\u003e) The proliferative curves of H460 cells, NC and siRNA-transfected H460 cells. N=3, **: p \u0026lt; 0.01 compared to cells and NC groups. ANOVA Dunnett’t test. Note: the siRNA experiments here can be used as positive controls.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/ddbb1337820c9c8168472dfa.png"},{"id":73356212,"identity":"77779d4a-bae8-4e0b-971c-a8bd8c3f0416","added_by":"auto","created_at":"2025-01-09 08:07:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3618516,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of therapeutic effect of P4-3 against H460 cells. (a) The representative fluorescent images of H460 cells under different conditions treated with DCFH-DA (10 μM). Scale bar 25 μm. P4-3 final concentration: 9 μM, time: 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (b) The MDA concentrations of H460 cells under different conditions. n.s.: no significance. ANOVA Dunnett’t test for left panel, two-tailed test for right panel. (c) The representative fluorescent images of H460 cells under different conditions treated with g-H\u003csub\u003e2\u003c/sub\u003eAX (50 μM). Scale bar, 75 μm. P4-3 final concentration: 9 μM, time: 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (d) The representative confocal fluorescent images of H460 cells under different conditions treated with JC-1 probe. Scale bar 10 μm. P4-3 final concentration: 9 μM, time: 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (e) Left: the representative transmission electron microscopy (TEM) images mitochondria in H460 cells under different conditions. P4-3 final concentration: 9 μM, time: 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. Scale bar 1 μm. Right: the statistics of mitochondrial lengths of different systems. ANOVA Dunnett’t test. (f) Flow cytometry results of H460 cells treated with P4-3 (30 nM) for different time/H460 cells treated with NC and siRNA. (g) The statistics of H460 cell ratios of PI\u003csup\u003e+\u003c/sup\u003e/Annexin\u003csup\u003e+\u003c/sup\u003e under different conditions. ANOVA Dunnett’t test. (h) The western blot of cytochrome C, Bcl2, Bax and cleaved caspase 3 in H460 cells of different groups. P4-3 final concentration: 9 μM, time: 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. Note: the siRNA experiments here can be used as positive controls.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/f3abd5f9e763ac48d638ffe2.png"},{"id":73356218,"identity":"ce082374-c283-4756-af0c-38360a862dc6","added_by":"auto","created_at":"2025-01-09 08:07:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12821655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene sequencing and proteomics result of different groups of cells.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Differential gene volcano map represented the number of up-regulated or down-regulated gene expressions between control (DMSO) and \u003cstrong\u003eP4-3\u003c/strong\u003egroups (9 μM, 24 hours, 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers). (\u003cstrong\u003eb\u003c/strong\u003e) Enrichment analysis of KEGG between different samples. (\u003cstrong\u003ec,d\u003c/strong\u003e) The protein volcano maps from different groups: A/D means Alogliptin group vs DMSO group, P/A means\u003cstrong\u003e P4-3 \u003c/strong\u003egroup vs Alogliptin group. H460 cells were treated with\u003cstrong\u003e P4-3 \u003c/strong\u003e(1 μM, 9 hours, 0.5 % DMSO was used as co-solvent) or Alogliptin (1 μM, 9 hours, 0.5 % DMSO was used as co-solvent).\u003cstrong\u003e \u003c/strong\u003eDMSO group represents cells were treated with 0.5 % DMSO. (\u003cstrong\u003ee\u003c/strong\u003e) The down-regulated protein numbers in KEGG pathways. (\u003cstrong\u003ef\u003c/strong\u003e) The protein numbers in subcellular level (P/A down-regulated proteins).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/055640859506cd4cad86a8a4.png"},{"id":73356204,"identity":"ded9a8ec-2175-4fce-a5ff-0802c4474814","added_by":"auto","created_at":"2025-01-09 08:07:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7342770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntitumor effects of P4-3 in vivo.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) The schematic diagram of CDX subcutaneous tumor establishment and treatment by \u003cstrong\u003eP4-3\u003c/strong\u003e. (\u003cstrong\u003eb\u003c/strong\u003e) The changes of tumor volumes and body weights of different groups with time. N=5, **\u0026lt;0.01. ANOVA Dunnett’t test. (\u003cstrong\u003ec\u003c/strong\u003e) The photos and weights of tumors of different groups after 17 days. Two-tailed test. (\u003cstrong\u003ed\u003c/strong\u003e) The representative TEM and H\u0026amp;E staining images of tumors of different groups. Scale bar of TEM 1.0 μm. Scale bar of H\u0026amp;E staining images 50 μm. (\u003cstrong\u003ee\u003c/strong\u003e) The representative TUNEL staining images of tumors of different groups. Scale bar 20 μm. (\u003cstrong\u003ef\u003c/strong\u003e) The schematic diagram of CDX subcutaneous tumor model constructed by E. V. and shRNA-transfected H460 cells. (\u003cstrong\u003eg\u003c/strong\u003e) The changes of tumor volumes and body weights of different groups with time. N=5, **\u0026lt;0.01. ANOVA Dunnett’t test. (\u003cstrong\u003eh\u003c/strong\u003e) The photos and weights of tumors of different groups after 17 days. Two-tailed test. (\u003cstrong\u003ei\u003c/strong\u003e) The representative TEM and H\u0026amp;E staining images of tumors of different groups. Scale bar of TEM 1.0 μm. Scale bar of H\u0026amp;E staining images 50 μm. (\u003cstrong\u003ej\u003c/strong\u003e) The representative TUNEL staining images of tumors of different groups. Scale bar 20 μm. (\u003cstrong\u003ek\u003c/strong\u003e) The representative Cty C, Bax, Bcl2 and cleaved caspase 3 (Cle. Cas3) immunofluorescence images of tumors of different groups. Scale bar 50 μm. (\u003cstrong\u003el\u003c/strong\u003e) The statistics of fluorescence intensities of Cty C, Bax, Bcl2 and Cle. Cas3 immunofluorescence images of different groups. Two-tailed test. Control group means the mice bearing subcutaneous tumors I.V. injected by buffers containing 1.0 % DMSO. P4-3 group means the mice bearing subcutaneous tumors I.V. injected by P4-3 containing 1.0 % DMSO as co-solvent. (\u003cstrong\u003em\u003c/strong\u003e) The representative Cty C, Bax, Bcl2 and cleaved caspase 3 (Cle. cas3) immunofluorescence images of tumors of different groups. Scale bar 50 μm. (\u003cstrong\u003en\u003c/strong\u003e) The statistics of fluorescence intensities of Cty C, Bax, Bcl2 and Cle. cas3 immunofluorescence images of different groups. Two-tailed test. E. V. group means the mice bearing subcutaneous tumors constructed by E. V.-transfected H460 cells. shRNA group means the mice bearing subcutaneous tumors constructed by shRNA-transfected H460 cells.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/9e5beb9dd5c219d991cf64e0.png"},{"id":73357121,"identity":"ef43f523-40c6-4288-95d7-c6952eef6bba","added_by":"auto","created_at":"2025-01-09 08:15:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5840813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe inhibitory performance of P4-3 against organoids.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThe western blot of CD26 in tumor and paracancer (P.C.) tissues of different patients. (\u003cstrong\u003eb\u003c/strong\u003e) The representative immunohistochemical (IHC) staining results of tumor and P.C. tissues of different patients. Scale bar 20 μm. (\u003cstrong\u003ec\u003c/strong\u003e) The western blot of CD26 in tumor organoids derived from patients treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003e for 9 hours. 0.5 % DMSO was used as co-solvent. 0 represents 0.5 % DMSO in buffers. (\u003cstrong\u003ed\u003c/strong\u003e) Up: the representative tumor organoids photos derived from patients treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003efor 24 hours. Scale bar 25 μm. Down: the statistics of inhibition rates of \u003cstrong\u003eP4-3\u003c/strong\u003eand staurosporine. ANOVA Dunnett’t test. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (\u003cstrong\u003ee\u003c/strong\u003e) The MDA concentrations in tumor organoids derived from patients treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003e for 24 hours. ANOVA Dunnett’t test. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (\u003cstrong\u003ef\u003c/strong\u003e) The representative TEM images of tumor organoids derived from patients treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003e for 24 hours. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers. (\u003cstrong\u003eg\u003c/strong\u003e) The western blot of cyt c, Bcl2, Bax and cle. cas3 in tumor organoids derived from patients treated with different concentrations of \u003cstrong\u003eP4-3\u003c/strong\u003e for 24 hours. 0.5 % DMSO was used as co-solvent. DMSO represents 0.5 % DMSO in buffers.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/30dd2a1387048acc3fa4ad8d.png"},{"id":73356216,"identity":"a0a052d4-16ba-49a8-bce5-513a72c1dbe7","added_by":"auto","created_at":"2025-01-09 08:07:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7434699,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic illustration of developed CD26 degraders for targeted therapy of NSCLC.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/2729d911ea4ffc4283998b9b.png"},{"id":75590140,"identity":"3a7e8d67-b4d4-408e-9ccd-7e83c439941b","added_by":"auto","created_at":"2025-02-06 07:03:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":74239480,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/e298da7f-3889-4399-ac52-698974509562.pdf"},{"id":73356220,"identity":"4927f413-5196-4efc-9278-022db8d12fc5","added_by":"auto","created_at":"2025-01-09 08:07:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44855301,"visible":true,"origin":"","legend":"Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5702716/v1/505d2a487b96eec3b63b0857.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is a very aggressive and highly prevalent disease worldwide, with the leading cause of death among male malignant tumors, and its mortality rate among female malignant tumors ranks second only to breast cancer\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is reported that lung cancer was one of the most frequently diagnosed cancers (about 2.5\u0026nbsp;million new cases worldwide) in 2022\u003csup\u003e3\u003c/sup\u003e, which brings great challenges to global health issues. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which accounts for approximately 85% of all lung cancer cases\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The treatment of NSCLC is therefore extremely urgent. The most traditional NSCLC therapy method is chemotherapy and the representative drugs include cisplatin, carboplatin, taxanes and gemcitabine\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Besides, radiotherapy is a standard means for NSCLC treatment, which could improve overall survival rate of patients\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the drug resistance of chemotherapy and side effects of radiotherapy may cause pains to patients, restricting their further applications. Molecular targeted therapy (MTT) is another ideal way for NSCLC treatment, and several representative drugs, such as epidermal growth factor receptor (EGFR) inhibitors (gefitinib, erlotinib, dacomitinib, osimertinib)\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and anaplastic lymphoma kinase (ALK) inhibitors (crizotinib, alectinib, and brigatinib)\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, have been used successfully. In recent years, pembrolizumab\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and atezolizumab\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e targeting PD-L1 have been developed for immunotherapy for NSCLC, which brings hope to NSCLC treatment. Unfortunately, drug resistance still remains an obstacle to overcome in MTT and immunotherapy, which makes the development of NSCLC treatment strategies with low drug resistance imperative.\u003c/p\u003e \u003cp\u003eProteolysis-targeting chimeras (PROTACs) with the advantages of lower drug-resistance, better selectivity and catalytic dosage compared to classic inhibitors, has emerged as a promising approach for MTT of cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. For NSCLC, several PROTAC degraders have been developed. For example, some degraders targeting kinases (EGFR tyrosine kinase, ALK and Kirsten rat sarcoma viral oncogene homolog (KRAS)) were utilized for NSCLC cells inhibition, which exhibits a great potential for NSCLC treatment\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Unfortunately, the developments kinase-based NSCLC degraders remain huge challenges due to the features of high structural conservation of kinase ATP binding sites. Therefore, developing other cell surface proteases (low conserved binding sites) PROTAC degraders attracts our great interest.\u003c/p\u003e \u003cp\u003eCD26 is a 110 kDa cell surface glycoprotein with an extracellular domain with dipeptidyl peptidase 4 (DPP4) activities, which owns diverse biological functions\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. CD26 has been considered as potential biomarkers and targets for several cancers, including NSCLC\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, Wesley et al. proposed that CD26 acted as the tumor suppressor in NSCLC cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, which is contrary to the above-mentioned point. The reason for this inconsistency may be that powerful molecular probe tools have not yet been reported. So developing a degrader based on CD26 PROTAC may help to eliminate our confusion.\u003c/p\u003e \u003cp\u003eHerein, a series of PROTAC degraders (\u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003e4\u003c/b\u003e) targeting cell-surface CD26 have been developed. To achieve the efficient degradation of cell surface proteins, which is a huge challenge, the molecular structures of degraders were rational designed and optimized. Remarkably, CD26 can be degraded by \u003cb\u003eP4-3\u003c/b\u003e obviously at low dose (~\u0026thinsp;500 nM) without degrading CD26 isoenzymes, which was independent of autophagy pathway. Surprisingly, the proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299) and tumors were significantly inhibited by \u003cb\u003eP4-3\u003c/b\u003e, and no toxicity of \u003cb\u003eP4-3\u003c/b\u003e for BEAS-2B cells (human lung normal epithelial cells) were obtained. More interestingly, the powerful proliferation inhibition capabilities of \u003cb\u003eP4-3\u003c/b\u003e for organoids were observed. In addition, a mechanism of \u003cb\u003eP4-3\u003c/b\u003e for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC.\u003c/p\u003e"},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening of CD26 degraders\u003c/h2\u003e \u003cp\u003eThe CD26 PROTAC-based degraders are designed according to the previous strategies. The candidate CD26 degraders (named \u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003eP4-4\u003c/b\u003e, von Hippel-Lindau-P1: \u003cb\u003eVHL-P1\u003c/b\u003e to \u003cb\u003eVHL-P4\u003c/b\u003e) contain three parts: protein-of-interest (POI) ligand (Alogliptin were used here due to its superior binding capability\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e), a linker and E3 ligase ligand (Lenalidomide or VHL ligand here, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The linker structure is optimized to obtain the molucule with the best degradation performance. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the bind energies of all VHL ligands-based degraders are higher than the Lenalidomide-based degraders, suggesting the degraders \u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003eP4-4\u003c/b\u003e are more suitable here. The detailed syntheses routes are described in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The structures of \u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003eP4-4\u003c/b\u003e and related products are well characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (Figs. S2-S17). To evaluate the binding capabilities of candidate degraders, the half-maximal effect concentration (EC\u003csub\u003e50\u003c/sub\u003e) were performed. As shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e8, the EC\u003csub\u003e50\u003c/sub\u003e values of \u003cb\u003eP4-3\u003c/b\u003e and \u003cb\u003eP4-4\u003c/b\u003e are 86.95 nM and 10.32 nM, respectively. These results suggest that \u003cb\u003eP4-3\u003c/b\u003e may be a favorite degrader. Next, the degradation abilities of all candidates\u0026rsquo; degraders are explored. The distribution of CD26 was first studied, as shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e9, CD26 is mainly exists in cell membranes (or cell surfaces), and this trend is unaffected by the use of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). So unless otherwise mentioned, the western blot results of CD26 later refer to the expressions of cell membrane CD26. Then, NCl-H460, NCl-H1299 (two representative NSCLC cells) and BEAS-2B cells (human lung normal epithelial cells) were selected as models. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, with concentration increases of degraders, CD26 from H460 cells are degraded and it is found that \u003cb\u003eP4-3\u003c/b\u003e has the best degradation capility (effective concentration is ~\u0026thinsp;500 nM). At the same time, it is found that CD26 can be degraded by \u003cb\u003eP4-3\u003c/b\u003e with 9 hours (Fig. S20). Furthermore, CD26 from H1299 and BEAS-2B cells are also degraded by \u003cb\u003eP4-3\u003c/b\u003e as well (Fig. S21). Undoubtedly, the selectivity of \u003cb\u003eP4-3\u003c/b\u003e is another important issue, so the degradations of several isoenzymes by \u003cb\u003eP4-3\u003c/b\u003e were studied. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and S21, DPP7, DPP8, DPP9 and fibroblast activation protein (FAP) from H460, H1299 and BEAS-2B cells are almost unaffected by \u003cb\u003eP4-3\u003c/b\u003e, which means \u003cb\u003eP4-3\u003c/b\u003e has extremely excellent selectivity toward CD26. Moreover, the specificity of \u003cb\u003eP4-3\u003c/b\u003e was confirmed by proteomics. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, the changed proteins (Fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5) from H460 cells occupy a little (\u0026lt;\u0026thinsp;1%). Meanwhile, the relative expressions of DPP3, DPP7, DPP8, DPP9, DPP10 and FAP from cells treated with \u003cb\u003eP4-3\u003c/b\u003e are almost unchanged compared with DMSO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Interestingly, CD26 was not detected in our mass spectrometry-based proteomic analysis. This observation may be attributed to the inherent limitations of mass spectrometry, such as the instrument\u0026rsquo;s detection threshold and dynamic range constraints, which can impact the detection of proteins with lower abundance, indicating that the selectivity of \u003cb\u003eP4-3\u003c/b\u003e is not affected by the high expressions of DPP3, DPP7, DPP8, DPP9, DPP10 and FAP. And these results further confirms that CD26 degrader with superior capacity is successfully constructed and the good performances of \u003cb\u003eP4-3\u003c/b\u003e can be attributed the rational design and optimization of molecular structures. In addition, the possible degradation pathways of \u003cb\u003eP4-3\u003c/b\u003e are investigated. As shown in Fig. S22, the addition of chloroquine (one kind of autophagy inhibitor) has no effect on the degradation results, suggesting CD26 degradation is independent of autophagy pathway. Furthermore, it can be seen that the degradation capacity of \u003cb\u003eP4-3\u003c/b\u003e is blocked by MG132 or Alogliptin (Fig. S23), suggesting CD26 is degraded via proteasomal pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe inhibitory effect of degraders on NSCLC cells\u003c/h3\u003e\n\u003cp\u003eThe effects of Lenalidomide, Alogliptin and degraders on cell viabilities of H460, H1299, A549 and PC9 cells were studied (Fig. S24). It is shown that degraders have stronger inhibitory abilities toward H460 and H1299 cells than those of Lenalidomide and Alogliptin, while the cell viabilities of A549 and PC9 cells are still above 50% after treatment with degraders. This result could be attributed to the different expression levels of CD26 among these cells (Fig. S25), i.e. the sensitivities of H460 and H1299 cells to degraders are positively related to the expressions of CD26. Then the half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of degraders toward cells were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, P4-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e has lower IC\u003csub\u003e50\u003c/sub\u003e value toward H460 cells at 48 hours and suitable IC\u003csub\u003e50\u003c/sub\u003e value at 72 hours, suggesting its powerful therapeutic effect. Importantly, \u003cb\u003eP4-3\u003c/b\u003e has no effect on the cell viabilities on the BEAS-2B cells (Fig. S26a), meaning \u003cb\u003eP4-3\u003c/b\u003e has almost no extra toxicity toward normal lung cells. And the proliferations of BEAS-2B cells are not affected by the knock-down of CD26 (Fig. S26b). To study the effect of \u003cb\u003eP4-3\u003c/b\u003e on migrations of cancer cells, the transwell assays were performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the migrations of H460 cells are mightily suppressed by \u003cb\u003eP4-3\u003c/b\u003e or \u003cb\u003eP4-4\u003c/b\u003e, which suggests the degradation of CD26 can cause the inhibition of migration of NSCLC cells. This conclusion is further confirmed by small interfering RNA (siRNA) interference results, i.e. the migrations of H460 cells are inhibited by knock-down of CD26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Lastly, it is observed that the proliferations of H460 cells are also repressed by the knock-down of CD26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). It is no surprising that the same phenomenon is observed in H1299 cells (Fig. S27). These entire results exhibit that \u003cb\u003eP4-3\u003c/b\u003e is a promising agent with no extra toxicity for treatment of NSCLC and CD26 can be used as a target for NSCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe mechanism of therapeutic effect\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe above results show that proliferations and migrations of NSCLC cells were inhibited by CD26 degrader: \u003cb\u003eP4-3\u003c/b\u003e, the mechanism of such effects was then studied. It is reported that most chemotherapeutics can elevate the intracellular levels of reactive oxygen species (ROS)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and high level of ROS could promote DNA damage and inhibit the cell proliferation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. So the ROS generation induced by \u003cb\u003eP4-3\u003c/b\u003e was first studied. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the addition of \u003cb\u003eP4-3\u003c/b\u003e causes the production of ROS in H460 cells and it is found that knock-down of CD26 can also elevate the intracellular ROS level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At the same time, the contents of intracellular malondialdehyde (MDA, one important oxidative stress indicator) are increased by \u003cb\u003eP4-3\u003c/b\u003e and the knock-down of CD26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As mentioned above, the high ROS level could promote the DNA damage, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, degradation and knock-down of CD26 lead to the serious DNA damage in H460 cells. Bennett et al suggested that mitochondrial dysfunction was related to DNA damage\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, so as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, addition of \u003cb\u003eP4-3\u003c/b\u003e and knock-down of CD26 can lead to the reduce of red fluorescence of H460 cells, which means the decline of mitochondrial membrane potential, suggesting the occurrence of early apoptosis. Meanwhile, we observe that some cavitation are appeared in mitochondria and the length of mitochondria become shorted under the treatment of \u003cb\u003eP4-3\u003c/b\u003e or after the knock-down of CD26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), suggesting the functions of mitochondria are destroyed. Then, the Annexin V/Propidium Iodide (PI) kits were performed to verify arises of apoptosis. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, cell ratios of Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e are increased with the \u003cb\u003eP4-3\u003c/b\u003e treating time, which means the apoptosis are induced by degradation of CD26. And the knock-down of CD26 can also result in apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). In order to further confirm the mechanism, some important biomarkers in apoptosis process were analysed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, cytochrome C (Cyt c), which are usually distributed in the mitochondrial inner membrane are high expressed after the treatment of \u003cb\u003eP4-3\u003c/b\u003e or knock-down of CD26, suggesting the damage of mitochondria. It is shown that Bcl2 (one kind of apoptosis inhibitory factor) are down expressed in the \u003cb\u003eP4-3\u003c/b\u003e treated or CD26 knock-down group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). On the contrary, Bax (one key protein in the mitochondrial apoptosis pathway) are high expressed in the \u003cb\u003eP4-3\u003c/b\u003e treated or CD26 knock-down group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), which indicates the degradation/knock-down of CD26 can lead to the mitochondria-mediated apoptosis. And this is affirmed by the high expression of cleaved caspase 3 (cle. cas3, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The apoptosis induced by \u003cb\u003eP4-3\u003c/b\u003e or knock-down of CD26 are also observed in H1299 cells (Fig. S28). As mentioned above, \u003cb\u003eP4-3\u003c/b\u003e exhibited almost no extra toxicity toward normal lung cells (BEAS-2B cells), the reason for this phenomenon was explored. As shown in Fig. S29, addition of \u003cb\u003eP4-3\u003c/b\u003e or knock-down of CD26 causes no increase of ROS and MDA levels in cells, as well as no DNA damages (Fig. S30). All above results suggest that the degradation of CD26 leads to the mitochondria-mediated apoptosis of NSCLC cells, which is clearly elaborated by the usage of \u003cb\u003eP4-3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGene sequencing and proteomics result of different groups of cells\u003c/h3\u003e\n\u003cp\u003eTo further investigate the potential biological mechanism of the treatment effects toward NSCLC cells, whole genome RNA expression sequencing (RNA-seq) in H460 cells was conducted. Afterward, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to obtain biological process in different samples. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, 426 up-regulated genes and 705 down-regulated genes are found in the \u003cb\u003eP4-3\u003c/b\u003e treated group compared to the control group (DMSO group). Based on this result, several biological processes are significantly affected by \u003cb\u003eP4-3\u003c/b\u003e, such as apoptosis and apoptosis-multiple species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). While the reason of the superiority of \u003cb\u003eP4-3\u003c/b\u003e to Alogliptin in cell suppression is also missing, so proteomics in H460 cells treated without or with \u003cb\u003eP4-3\u003c/b\u003e at early stage was conducted.\u003c/p\u003e \u003cp\u003eIt is reported that up-regulated proliferating cell nuclear antigen clamp associated factor (PCLAF) promotes cancer cell growth and the attenuation of expression by siRNA suppresses cell proliferation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The intervention of \u003cb\u003eP4-3\u003c/b\u003e results in the down-regulation of PCLAF (P/D, P means \u003cb\u003eP4-3\u003c/b\u003e group, D means DMSO group), suggesting the growth of H460 cells are inhibited. In addition, neuronal pentraxin II (NPTX2) is considered as one kind of tumor-suppressors\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and the up-regulation of it induced by \u003cb\u003eP4-3\u003c/b\u003e may be another pathway of cell suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Although ras-related protein (RAB34) related to cell migration and invasion\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and adhesion G-protein coupled receptor D1 (ADGRD1) which is a potential prognostic biomarker in NSCLC\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e are down-regulated by the use of Alogliptin, two proteins: BolA family member 2 (BOLA2) and NADPH oxidase organizer 1 (NOXO1) which can promote tumor proliferation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e are up-regulated in the Alogliptin-treating group (A/D, A means Alogliptin group, D means DMSO group), suggesting the potential risks of Alogliptin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Importantly, the down-regulations of chromosome 1 open reading frame 122 (C1orf122, its silencing can reduce growth of the cancer cell line\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e) and galectin-7 (LGALS7, its expression can increases the probability of lung metastases\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e) are observed in the \u003cb\u003eP4-3\u003c/b\u003e-treating group instead of Alogliptin group (P/A). Meanwhile, \u003cb\u003eP4-3\u003c/b\u003e can also reduce the DNA resistance to external stress (endonuclease/exonuclease/phosphatase family domain containing 1, EEPD1 can provide DNA resistance to replication stress\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e). Interestingly, succinate dehydrogenase complex assembly factor 2 (SDHAF2, a tumor suppressor\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e) is activated by \u003cb\u003eP4-3\u003c/b\u003e instead of Alogliptin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These results suggest use of \u003cb\u003eP4-3\u003c/b\u003e leads to the activation of some key cell death pathways, which can explain the superiority of \u003cb\u003eP4-3\u003c/b\u003e to Alogliptin in cell suppression. Furthermore, the changes of proteins induced by \u003cb\u003eP4-3\u003c/b\u003e are closely related to the process of cell growth and death (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) and the pathways such as DNA damage and the occurrence of oxidative stress are the main events after treatment of \u003cb\u003eP4-3\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). All above results suggest that the degradation of CD26 can lead to the mitochondria-mediated apoptosis of NSCLC cells, which is clearly elaborated by the usage of \u003cb\u003eP4-3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntitumor effects\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConsidering the excellent therapeutic effect of \u003cb\u003eP4-3\u003c/b\u003e against NSCLC cells mentioned above, it is necessary to explore its anti-tumor performance \u003cem\u003ein vivo\u003c/em\u003e. The biosafety of \u003cb\u003eP4-3\u003c/b\u003e was firstly studied. As depicted in Fig. S31, the hematoxylin and eosin (H\u0026amp;E) staining results exhibit that no tissue damages are emerged in major organs of mice treated with \u003cb\u003eP4-3\u003c/b\u003e. Meanwhile, the weight bodies, alanine transaminase (ALT), creatinine (CREA) and α-amylase concentrations of mice are unaffected by \u003cb\u003eP4-3\u003c/b\u003e (Fig. S31b). It should be pointed out that an increased risk of pancreatitis is associated with the usage of CD26 inhibitors, so we conducted a thorough examination of the pancreatic tissue in our mice models treated with \u003cb\u003eP4-3\u003c/b\u003e. And no obvious pancreatic damage (Fig. S31) or inflammation (Fig. S31b) are observed, indicating \u003cb\u003eP4-3\u003c/b\u003e has a favorable safety profile. In addition, the types of T cells in mice spleen and concentrations of monocytes in mice serums are also unchanged upon the treatment of \u003cb\u003eP4-3\u003c/b\u003e (Fig. S32). The numbers of lymphocytes increase a little but are within reference range, which is the normal response against \u003cb\u003eP4-3\u003c/b\u003e \u003cem\u003ein vivo\u003c/em\u003e (Fig. S32b). These results indicate that \u003cb\u003eP4-3\u003c/b\u003e is a safe potential drug. Next, we constructed H460 cell line-derived xenograft (CDX) subcutaneous tumor model to investigate the anti-tumor effect of \u003cb\u003eP4-3\u003c/b\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the mice bearing subcutaneous tumors were intravenous (I.V.) injected with \u003cb\u003eP4-3\u003c/b\u003e every 3 days one time (0.6 mg/kg) for 4 times. The volumes of tumors treated with \u003cb\u003eP4-3\u003c/b\u003e were significantly smaller than those of control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), and the body weights of mice were almost unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). It can be clearly seen that the tumors from mice treated with \u003cb\u003eP4-3\u003c/b\u003e are obviously suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). As mentioned above, \u003cb\u003eP4-3\u003c/b\u003e could induce apoptosis of NSCLC cells, so the tumor repression mechanism was explored. After treated with \u003cb\u003eP4-3\u003c/b\u003e, the MDA levels in tumors is increased (Fig. S33), suggesting the tumors may be inhibited through the apoptosis manners. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, we observe that there exist some complete mitochondria in tumors from control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), while nuclear chromatin is present in the central position in tumors from \u003cb\u003eP4-3\u003c/b\u003e treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), further implying the tumors are in the state of apoptosis. This guess was further proved by the H\u0026amp;E and TUNEL staining results (Figs, 5d and 5e). In order to clarify the role of CD26 in tumor apoptosis, we employed lentivirus loaded small hairpin RNAs (shRNA) to knock down CD26 in NSCLC cells (H460 and H1299 cells). As shown in Fig. S34, the expressions of CD26 in cells are knocked down effectively through shRNA3 (Abbreviation: shRNA in the further experiments). Then the E. V. and shRNA-transfected H460 cell were subcutaneous injected to construct CDX model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), and it can be seen that the CD26 were also low expressed in tumors (Fig. S35). Interestingly, the tumors constructed by the shRNA-transfected H460 cells grow slowly (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh) accompanied by the high levels of MDA concentrations in shRNA group (Fig. S36). The apoptosis of tumors are proved by several evidences (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej), i.e. down-regulation of BCl2 and up-regulations of Cyt c, Bax and Cle. cas3 are also observed in the tumor from \u003cb\u003eP4-3\u003c/b\u003e treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en), which confirms our supposition. Furthermore, we explored the therapeutic effect of \u003cb\u003eP4-3\u003c/b\u003e against lung metastases. The lung metastases constructed through normal and shRNA-transfected H460 cells were established successively (Fig. S37). It can be seen that tumors in lung are repressed successfully by \u003cb\u003eP4-3\u003c/b\u003e through apoptosis manner (Figs. S37b and S37c). And the tumors in lung derived from shRNA-transfected H460 cells grow little and apoptosis in tumors are also observed (Figs. S37e and S37f). The body weights are almost unchanged (Fig. S38). All above results indicate that \u003cb\u003eP4-3\u003c/b\u003e is a powerful agent for NSCLC therapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe inhibitory performance of P4-3 against organoids\u003c/h3\u003e\n\u003cp\u003eTo investigate the clinical application potential of \u003cb\u003eP4-3\u003c/b\u003e, we further extended our study to a more translational model by utilizing patient-derived organoids. Firstly, it is necessary to study the expressions of CD26 in patients\u0026rsquo; NSCLC tumor tissues. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, the expression levels of CD26 in tumor are higher than those of paracancer (P.C.) tissues, suggesting the clinical significance of CD26 in NSCLC. Then the degradation effects of \u003cb\u003eP4-3\u003c/b\u003e on CD26 in tumor organoids were explored. As described in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, CD26 are degradated obviously by \u003cb\u003eP4-3\u003c/b\u003e, and the higher dose of degrader can be attributed to the fact that organoids are composed of numerous tumor cells. Surprisingly, it is found that organoids are significantly suppressed by \u003cb\u003eP4-3\u003c/b\u003e and staurosporine (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and S39). Meanwhile, the tumor organoids are inhibited by alogliptin slightly (Fig. S40), demonstrating the effective curative effect of \u003cb\u003eP4-3\u003c/b\u003e. Without doubt, the MDA concentrations in tumor organoids are increased under the treatment of \u003cb\u003eP4-3\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). TEM and western blot results further confirm that tumor organoids undergo the apoptotic processes induced by \u003cb\u003eP4-3\u003c/b\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The biosafety of \u003cb\u003eP4-3\u003c/b\u003e for P.C. organoids is of great importance. It is shown that the inhibition rates of \u003cb\u003eP4-3\u003c/b\u003e are significantly lower than those of staurosporine (Fig. S41), indicating that \u003cb\u003eP4-3\u003c/b\u003e could be a safe agent clinically. Together, all the results above confirm that \u003cb\u003eP4-3\u003c/b\u003e is a promising treatment strategy for NSCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, several potential CD26 degraders were developed and the molecular structures of were optimized. Remarkably, CD26 can be degraded by \u003cb\u003eP4-3\u003c/b\u003e obviously at low dose (~\u0026thinsp;500 nM) without the degradation of CD26 isoenzymes, which was independent of autophagy pathway. The proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299) and tumors were significantly inhibited by \u003cb\u003eP4-3\u003c/b\u003e which demonstrated suitable biosafety. Interestingly, the powerful proliferation inhibition capabilities of \u003cb\u003eP4-3\u003c/b\u003e for organoids were observed. Lastly and importantly, a possible mechanism of \u003cb\u003eP4-3\u003c/b\u003e for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eLenalidomide (Catalogue number: L0332-1g, CAS number: 191732-72-6), ethyl oxalyl monochloride (Catalogue number: C1066-25G, CAS number: 4755-77-5), succinic anhydride (Catalogue number: S0107-25G, CAS number: 108-30-5) and N-hydroxysuccinimide (NHS, Catalogue number: H0623-5G, CAS number: 6066-82-6) were purchased from Tokyo Chemical Industry (TCI) Co. Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl, Catalogue number: A10807-1g, CAS number: 25952-53-8) was purchased from Alfa Aesar organics products. Hexanedioic anhydride (Catalogue number: A0110740010, CAS number: 2035-75-8), suberic acid (Catalogue number: A0104830250, CAS number: 505-48-6), crystal violet (Catalogue number: D051109-5g, CAS number: 548-62-9) and dimethyl sulfoxide-d6 (DMSO-d6, catalogue number: E090002-0.6ml\u0026times;10, CAS number: 2206-27-1) were purchased from Energy Chemical. Chloroform-d (CDCl\u003csub\u003e3\u003c/sub\u003e, catalogue number: 240987, CAS number: 865-49-6), super-dry pyridine (Catalogue number: 299669, CAS number: 110-86-1) and super-dry N, N-Dimethylformamide (DMF, catalogue number: 983353, CAS number: 68-12-2) were purchased from J\u0026amp;K Scientific. Staurosporine (Catalogue number: HY-15141, CAS number: 62996-74-1), alogliptin (Catalogue number: HY-A0023A, CAS number: 850649-61-5), MG132 (Catalogue number: HY-13259, CAS number: 133407-82-6) and chloroquine (Catalogue number: HY-17589A, CAS number: 54-05-7) were purchased from MedChemExpress. Dimethyl sulfoxide (DMSO) for molecular biology (Catalogue number: D8418, CAS number: 67-68-5) was purchased from Millipore Sigma. Dipeptidyl peptidase IV (DPP4) inhibitor screening assay kit (Catalogue number: ab133081) was purchased from Abcam. Reactive oxygen species (ROS) assay kit (Catalogue number: S0033M), DNA damage assay kit by γ-H\u003csub\u003e2\u003c/sub\u003eAX immunofluorescence (Catalogue number: C2035S), mitochondrial membrane potential assay kit with JC-1 (Catalogue number: C2006), lipid peroxidation MDA assay kit (Catalogue number: S0131M), annexin V-FITC apoptosis detection Kit (Catalogue number: C1062L), western blot transfer buffer (Catalogue number: P0021B), cell lysis buffer for western blot and IP (Catalogue number: P0013) and BeyoGold\u0026trade; transwell permeable supports (Catalogue number: FTW010-24Ins) were purchased from Beyotime. CellTiter-Glo\u0026reg; 3D cell viability assay kit (Catalogue number: G9683) was purchased from Promega Corporation. Monoclonal anti-β-actin antibody (Catalogue number: 66009-1-Ig), monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Catalogue number: 60004-1-Ig), monoclonal anti-CD26 antibody (Catalogue number: 68383-1-Ig), polyclonal anti-dipeptidyl peptidase 8 (DPP8) antibody (Catalogue number: 12752-1-AP), polyclonal anti-bax antibody (Catalogue number: 50599-2-lg), polyclonal anti-Bcl2 human antibody (Catalogue number: 12789-1-AP), polyclonal anti-cytochrome c human antibody (Catalogue number: 10993-1-AP), cell counting kit-8 (CCK-8, Catalogue number: PF00004) and membrane protein extraction kit (Catalogue number: PK10015) were purchased from ProteinTech. Recombinant anti-fibroblast activation protein (FAP) antibody (Catalogue number: 66562S) was purchased from Cell Signaling Technology. Polyclonal anti-DPP7 antibody (Catalogue number: YT5125) was purchased from ImmunoWay Biotechnology. Recombinant anti-DPP9 antibody (Catalogue number: Ab302903) and recombinant anti-cleaved caspase-3 antibody (Catalogue number: Ab32042) were purchased from Abcam. Anti-ATP1B1 antibody (Catalogue number: CY8105) was purchased from Abways. Recombinant anti-Bax antibody for immunofluorescence (Catalogue number: GB154122-100), recombinant anti-Bcl2 antibody for immunofluorescence (Catalogue number: GB154380-100) and anti-DPP4/CD26 rabbit pAb for immunofluorescence (Catalogue number: GB114937-100) were purchased from ServiceBio. Co.Ltd. Small interfering RNA (siRNA: sense 5\u0026rsquo;-3\u0026rsquo;: GCACAGCACACCAACAUAUTT, antisense 5\u0026rsquo;-3\u0026rsquo;: AUAUGUUGGUGUGCUGUGCTT) for CD26 was purchased from GenePharma. Lentivirus loaded small hairpin RNAs kits (shRNA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e exhibited the sequences of shRNAs) were purchased from Genechem Co. Ltd. Lipofectamine\u0026trade; 3000 transfection reagent (Catalogue number: L3000001) and fetal bovine serum (FBS, catalogue number: 10091155) were purchased from Thermo Fisher Scientific. Allophycocyanin (APC) anti-mouse CD3 antibody (Catalogue number: 100236), phycoerythrin (PE) anti-mouse CD8 antibody (Catalogue number: 104708) and fluorescein isothiocyanate (FITC) anti-mouse CD4 antibody (Catalogue number: 100406) were purchased from Biolegend. All other chemical or biological reagents were local.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthetic routes of all compounds\u003c/h3\u003e\n\u003cp\u003eAll Nuclear Magnetic Resonance (NMR) spectra were performed with a Bruker Avance 300 spectrometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-1\u003c/b\u003e. Lenalidomide (2.59 g, 10 mmol) was dissolved in 10 mL of pyridine, and ethyl oxalyl monochloride (2.4 g, 20 mmol) were added slowly into the solution. The reaction mixture was stirred at room temperature overnight and pyridine was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of methanol to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain \u003cb\u003eP4-1-A\u003c/b\u003e (2.2 g, yield\u0026thinsp;~\u0026thinsp;60%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 10.99 (s, 1H), 10.88 (s, 1H), 7.51\u0026ndash;7.67 (m, 3H), 5.14 (s, 1H), 4.26\u0026ndash;4.39 (m, 4H), 2.83\u0026ndash;2.95 (m, 1H), 2.29\u0026ndash;2.60 (m, 3H), 1.96\u0026ndash;2.02 (q, 1H), 1.28\u0026ndash;1.32 (t, 2H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, DMSO-d6, δ): 173.45, 171.59, 168.20, 160.96, 156.27, 136.53, 133.62, 132.55, 129.42, 128.08, 121.58, 63.15, 52.30, 47.20, 31.88, 23.21, 14.53.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-1-A\u003c/b\u003e (0.5 g, 1.4 mmol) and alogliptin (0.67 g, 2.0 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was heated to reflux for 16 hours. Then the mixture was cooled and added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. At last, the solid was dried with phosphorus pentoxide for 24 hours to obtain \u003cb\u003eP4-1\u003c/b\u003e (white solid, 0.2 g, yield\u0026thinsp;~\u0026thinsp;25%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S3 and S4. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 11.09 (s, 1H), 10.78 (s, 1H), 8.95 (s, 1H), 7.80 (s, 1H), 7.62 (s, 4H), 7.42 (s, 2H), 7.24 (s, 1H), 5.36 (s, 1H), 5.15 (s, 3H), 4.40 (s, 2H), 3.79 (s, 1H), 3.33 (s, 3H), 2.48\u0026ndash;2.60, 1.56\u0026ndash;1.98 (m, 12H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, DMSO-d6, δ): 173.48, 171.57, 168.25, 162.80, 159.96, 159.04, 152.64, 141.72, 136.47, 134.15, 133.82, 133.55, 132.84, 128.62, 127.73, 117.88, 110.64, 90.11, 54.86, 51.66, 47.15, 46.76, 46.64, 29.28, 28.06, 23.76, 23.21. HR-ESI-MS, calcd for C\u003csub\u003e34\u003c/sub\u003eH\u003csub\u003e33\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M]\u003csup\u003e+\u003c/sup\u003e: m/z 651.2441, found [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: m/z 674.2337.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-2.\u003c/b\u003e Lenalidomide (2.59 g, 10 mmol) was dissolved in 10 mL of DMF, and succinic anhydride (1.5 g, 15 mmol) was added slowly into the solution. The reaction mixture was stirred at 60\u0026deg;C overnight and pyridine was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain \u003cb\u003eP4-2-A\u003c/b\u003e (2.0 g, yield\u0026thinsp;~\u0026thinsp;50%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S5 and S6. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 12.10 (s, 2H), 10.97 (s, 1H), 9.82 (s, 1H), 7.80\u0026ndash;7.81 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 Hz), 7.78\u0026ndash;7.79 (d, 2H), 5.09\u0026ndash;5.15 (q, 1H), 4.33\u0026ndash;4.41 (t, 2H), 2.84\u0026ndash;2.97 (m, 1H), 2.48\u0026ndash;2.62 (m, 4H), 2.25\u0026ndash;2.39 (m, 1H), 1.99\u0026ndash;2.04 (m, 1H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, DMSO-d6, δ): 174.40, 173.48, 171.69, 170.99, 168.51, 134.44, 134.42, 133.36, 129.34, 125.79, 119.66, 52.20, 47.14, 31.90, 31.36, 29.67, 23.36.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-2-A\u003c/b\u003e (0.5 g, 1.4 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with methylene chloride (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e) /ethyl acetate (v/v, 100/1) to CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/methanol (v/v, 50/1) and then re-crystallized with water, affording \u003cb\u003eP4-2\u003c/b\u003e as a white solid (0.08 g, yeild\u0026thinsp;~\u0026thinsp;14%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S7 and S8. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 9.66 (s, 1H), 9.27 (s, 1H), 7.82 (s, 1H), 7.63\u0026ndash;7.65 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 Hz), 7.51\u0026ndash;7.58 (m, 2H), 7.38 (s, 1H), 7.35 (s, 1H), 7.22 (s, 1H), 6.65 (s, 1H), 5.20\u0026ndash;5.22 (d, 2H), 5.15 (s, 1H), 4.26\u0026ndash;4.34 (t, 2H), 3.98 (s, 1H), 3.22\u0026ndash;3.24 (d, 3H), 2,55-2.72 (d, 8H), 2.27 (s, 1H), 2.02 (s, 5H), 1.70 (s, 2H), 1.53 (s, 2H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e, δ): 172.49, 171.53, 170.87, 169.35, 163.48, 159.92, 152.67, 140.86, 133.66, 132.65, 129.28, 128.40, 125.73, 120.46, 117.89, 110.83, 90.31, 55.34, 52.17, 46.80, 39.59, 37.61, 37.33, 34.62, 33.87, 32.97, 32.18, 30.28, 29.95, 29.62, 28.22, 27.33, 24.70, 22.95, 19.97, 14.40. HR-ESI-MS, calcd for C\u003csub\u003e36\u003c/sub\u003eH\u003csub\u003e37\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M]\u003csup\u003e+\u003c/sup\u003e: m/z 679.2754, found [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: m/z 702.2643.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-3.\u003c/b\u003e Hexanedioic anhydride (1.5 g, 15 mmol) and lenalidomide (2.59 g, 10 mmol) were dissolved in 25 mL of DMF, the reaction mixture was stirred at 60\u0026deg;C overnight and DMF was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain \u003cb\u003eP4-3-A\u003c/b\u003e (1.5 g, yield\u0026thinsp;~\u0026thinsp;35%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S9 and S10. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 12.03 (s, 1H), 11.02 (s, 1H), 9.78\u0026ndash;9.82 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12 Hz), 7.79 (s, 1H), 7.48 (s, 2H), 5.11\u0026ndash;5.15 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12 Hz), 4.34 (s, 2H), 3.56 (s, 1H), 2.06\u0026ndash;2.86 (m, 8H), 1.47\u0026ndash;1.57 (d, 3H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, DMSO-d6, δ): 175.20, 173.47, 171.88, 171.68, 168.52, 134.48, 133.36, 129.29, 126.01, 119.71, 52.21, 47.22, 36.22, 34.42, 31.90, 29.69, 25.38, 24.93, 23.31.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-3-A\u003c/b\u003e (0.5 g, 1.3 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/ethyl acetate (v/v, 100/1) to CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/methanol (v/v, 65/1) and then re-crystallized with water, affording \u003cb\u003eP4-3\u003c/b\u003e as a white-gray solid (0.04 g, yeild\u0026thinsp;~\u0026thinsp;8%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S11 and S12. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 9.72\u0026ndash;9.79 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21 Hz), 9.11 (s, 1H), 7.70\u0026ndash;7.75 (t, 1H), 7.61\u0026ndash;7.64 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9 Hz), 7.50\u0026ndash;7.54 (t, 2H), 7.35 (s, 2H), 7.18\u0026ndash;7.20 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 Hz), 6.58\u0026ndash;6.60 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 Hz), 5.20\u0026ndash;5.28 (d, 3H), 5.02\u0026ndash;5.04 (t, 1H), 4.33 (s, 2H), 3.95 (s, 1H), 3.20 (s, 3H), 2.02\u0026ndash;2.66 (m, 12H), 1.43\u0026ndash;1.79 (t, 9H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e, δ): 173.20, 172.56, 172.45, 170.89, 169.40, 163.50, 159.96, 152.66, 140.74, 134.40, 134.29, 133.63, 133.49, 133.42, 132.66, 129.21, 128.39, 127.80, 117.72, 110.78, 90.19, 55.37, 52.15, 46.82, 45.55, 36.35, 35.97, 31.68, 29.93, 29.28, 28.22, 25.19, 25.05, 23.26. HR-ESI-MS, calcd for C\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e41\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M]\u003csup\u003e+\u003c/sup\u003e: m/z 707.3067, found [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: m/z 730.2962.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-4.\u003c/b\u003e Suberic acid (8.7 g, 50 mmol) and acetic anhydride were stirred at 140\u0026deg;C overnight. And then the acetic anhydride was removed, the residue was beat by 100 mL of acetonitrile to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain suberic anhydride (4.4 g, yield\u0026thinsp;~\u0026thinsp;50%). Suberic anhydride (1.7 g, 11 mmol) and lenalidomide (2.59 g, 10 mmol) were dissolved in 25 mL of DMF. The reaction mixture was stirred at 60\u0026deg;C overnight and DMF was removed by evaporation under reduced pressure. Then the mixture was beat by 100 mL of ice water to remove impurities. The products were filtered and dried with phosphorus pentoxide for 24 hours to obtain \u003cb\u003eP4-4-A\u003c/b\u003e (1.0 g, yield\u0026thinsp;~\u0026thinsp;25%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S13 and S14. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 11.98 (s, 1H), 11.02 (s, 1H), 9.76 (s, 1H), 7.78\u0026ndash;7.93 (t, 1H), 7.43\u0026ndash;7.50 (q, 2H), 5.10\u0026ndash;5.16 (q, 1H), 4.27\u0026ndash;4.41 (q, 2H), 2.86\u0026ndash;2.90 (d, 2H), 2.71 (s, 1H), 2.00-2.71 (m, 7H), 1.29\u0026ndash;1.98 (m, 6H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, DMSO-d6, δ): 173.47, 172.03, 171.70, 168.51, 134.49, 133.35, 129.29, 125.85, 119.73, 52.36, 52.19, 47.20, 36.47, 31.88, 29.14, 25.69, 23.34.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP4-4-A\u003c/b\u003e (0.5 g, 1.2 mmol), alogliptin (0.67 g, 2.0 mmol), EDC-HCl (0.6 g, 3 mmol) and NHS (0.33 g, 3 mmol) were dissolved in 7.5 mL of DMF. The reaction mixture was stirred at room temperature for 16 hours. Then the mixture was added into 200 mL of ice water. The precipitated solid was collected and washed with deionized water for 3 times. The residue as a crude product was firstly purified by flash silica gel chromatography eluted with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/ethyl acetate (v/v, 100/1) to CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/methanol (v/v, 60/1) and then re-crystallized with water, affording \u003cb\u003eP4-4\u003c/b\u003e as a gray solid (0.05 g, yeild\u0026thinsp;~\u0026thinsp;10%). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra are shown in Figure S15 and S16. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, DMSO-d6, δ): 9.70\u0026ndash;9.79 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27 Hz), 9.11 (s, 1H), 7.68\u0026ndash;7.74 (t, 1H), 7.60\u0026ndash;7.63 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9Hz), 7.49\u0026ndash;7.54 (t, 2H), 7.31\u0026ndash;7.36 (t, 2H), 7.16\u0026ndash;7.19 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9Hz), 6.49 (s, 1H), 5.27 (s, 2H), 5.19 (s, 1H), 4.97\u0026ndash;5.01 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12Hz), 4.32 (s, 2H), 3.94 (s, 1H), 3.19 (s, 3H), 2.01\u0026ndash;2.69 (m, 11H), 1.18\u0026ndash;1.79 (m, 13H). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (75 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e, δ): 173.46, 172.75, 172.44, 170.78, 169.43, 163.46, 159.94, 152.61, 140.67, 134.54, 134.45, 133.58, 133.41, 132.66, 129.18, 128.38, 127.79, 120.57, 117.66, 110.87, 90.19, 55.53, 53.76, 52.06, 46.76, 45.48, 36.78, 36.38, 31.67, 29.00, 28.20, 25.65, 23.31. HR-ESI-MS, calcd for C\u003csub\u003e40\u003c/sub\u003eH\u003csub\u003e45\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M]\u003csup\u003e+\u003c/sup\u003e: m/z 735.3380, found [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: m/z 758.3273.\u003c/p\u003e\n\u003ch3\u003eGeneral procedures for cell viability tests\u003c/h3\u003e\n\u003cp\u003eUnless otherwise stated, all the cell viability tests were performed according to the following procedure. Cells (5.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) were cultured in 96-well plates for 24 hours. Different compounds with suitable final concentrations were added in to each well (0.5% DMSO was used as co-solvent, and no compound was added in cell proliferation experiment), and cells were cultured for different time. The culture medium was then discarded and cell viabilities were measured according to the CCK-8 protocols. The absorbance value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEC\u003csub\u003e50\u003c/sub\u003e values\u003c/h2\u003e \u003cp\u003eThe half-maximal effect concentration (EC\u003csub\u003e50\u003c/sub\u003e) values of \u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003eP4-4\u003c/b\u003e were determined according to the protocols of dipeptidyl peptidase IV (DPP4, CD26) inhibitor screening assay kit. The fluorescence value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExtracting membrane protein and cytoplasmic protein\u003c/h2\u003e \u003cp\u003eCells were lysed with lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice for 10 min, and then centrifuged at 13,000 rpm for 15 min. For membrane protein and cytoplasmic protein extraction, the cells were collected at 4\u0026deg;C and washed with pre-cooled phosphate buffered saline (PBS, 0.1 M). The membrane protein and cytoplasmic protein were extracted using a membrane protein extraction kit according the protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eProtease or Chloroquine inhibition test\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCells were incubated with MG132 (5 \u0026micro;M) or Alogliptin (5 \u0026micro;M) for 9 hours only, or followed by addition of the \u003cb\u003eP4-3\u003c/b\u003e (1 \u0026micro;M) for an additional 9 hours. Subsequently, proteins were extracted, and the degradation efficacy was evaluated using western blot assays. The procedures of effects of Chloroquine on the CD26 degradation efficacy were similar.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKey parameters of western blot\u003c/h2\u003e \u003cp\u003e The protein samples were prepared according to the demand, and the protein concentrations were determined through the bicinchoninic acid assay (BCA). The current for film transfer was 250 mA and the transfer time was determined based on protein molecular weight. The blot imaging was performed through Amersham ImageQuant\u0026trade; 800 western blot imaging systems (Cytiva).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assays\u003c/h2\u003e \u003cp\u003eUnless otherwise stated, all transwell assays were performed according to the following procedure. Cells (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured without FBS in the upper chamber of 6-transwell well, culture medium with FBS was placed in the lower chamber. And different compounds with suitable final concentrations were added in to upper chamber (0.5% DMSO was used as co-solvent, and no compound was added siRNA-transfected cells). Then cells were cultured for different time. The culture medium was discarded and the cells in the lower chamber were stained with crystal violet, counted and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfection\u003c/h2\u003e \u003cp\u003eCells (NCI-H460, NCI-H1299 and BEAS-2B cells, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 6-well plates for 24 hours. And CD26 siRNA and Lipofectamine\u0026trade; 3000 were mixed to prepared transfection. The mixed solutions were then added into each well and the final concentration of siRNA was controlled to 100 nM, at the same time, CD26-negative control was also transfected. Cells were cultured for further 24 hours and prepared to western blot experiments to verify the transfection efficiency. The successfully-transfected cells were used for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eshRNA transfection\u003c/h2\u003e \u003cp\u003eCells (H460 and H1299 cells, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were cultured in 96-well plates for 24 hours. The multiplicities of infection (MOI) values of both cells were about 100. The transfection processes were strictly complied with the protocols provided by Genechem. Cells were performed to fluorescent image and western blot experiments to verify the transfection efficiency. The successfully-transfected cells were used for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eROS fluorescent imaging\u003c/h2\u003e \u003cp\u003eCells (H460, H1299 and BEAS-2B cells, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with \u003cb\u003eP4-3\u003c/b\u003e for 24 hours (siRNA-transfected cells were treated without \u003cb\u003eP4-3\u003c/b\u003e) and cultured with DCFH-DA (Final concentrations: 10 \u0026micro;M) according to the ROS assay kit protocols for 1 hour. Lastly, the cells were prepared to fluorescent image.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDNA damage fluorescent imaging\u003c/h2\u003e \u003cp\u003eCells (H460, H1299 and BEAS-2B cells, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with \u003cb\u003eP4-3\u003c/b\u003e for 24 hours (siRNA-transfected cells were treated without \u003cb\u003eP4-3\u003c/b\u003e) and cultured with γ-H\u003csub\u003e2\u003c/sub\u003eAX (Final concentrations: 50 \u0026micro;M) according to the DNA damage assay kit protocols. Lastly, the cells were prepared to fluorescent image.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential confocal fluorescent imaging\u003c/h2\u003e \u003cp\u003eCells (H460 and H1299 cells, 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL) were cultured in confocal dishes) for 24 hours. Then cells were treated with \u003cb\u003eP4-3\u003c/b\u003e for 24 hours and cultured with JC-1 probe according to the mitochondrial membrane potential assay kit protocols. Lastly, the cells were placed to confocal fluorescent image through Leica TCS SP5 confocal laser scanning microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMDA assay\u003c/h2\u003e \u003cp\u003eCells (H460, H1299 and BEAS-2B cells, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with \u003cb\u003eP4-3\u003c/b\u003e for 24 hours (siRNA-transfected cells were treated without \u003cb\u003eP4-3\u003c/b\u003e) and lysed. The MDA concentrations in lysates were determined according to the lipid peroxidation MDA assay kit protocols. It should be noted that the MDA concentrations in organoids and animal samples were detected through the similar procedures. The absorbance value of each well was recorded through the BioTek Synergy LX multimode micro-plate reader (Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFlow fluorescence analysis\u003c/h2\u003e \u003cp\u003eCells (H460 and H1299 cells, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 6-well plates for 24 hours. Then cells were treated with \u003cb\u003eP4-3\u003c/b\u003e for 24 hours (siRNA-transfected cells were treated without \u003cb\u003eP4-3\u003c/b\u003e) and placed to stain with annexin V-FITC according to the kit protocols. Lastly, the stained cells were analyzed through NovoCyte flow cytometer (Agilent).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eThe cells were treated without or with \u003cb\u003eP4-3\u003c/b\u003e (9 \u0026micro;M, 0.5% DMSO was used as co-solvent) for 24 hours. Then cells were centrifuged and the precipitations were re-suspended in the fixative and then fixed at 4℃ for 4 hours. The fixed cells were centrifuged and washed with 0.1 M PBS for 3 times and were wrapped in the 1% agarose. The samples were then fixed with 1% OsO\u003csub\u003e4\u003c/sub\u003e (in PBS) for 2 hours at room temperature without light. After that, the samples were washed with 0.1 M PBS for 3 times. Samples were then dehydrated at room temperature and embedding at 37\u0026deg;C overnight. The embedding models were polymerized at 60\u0026deg;C for 48 hours. After that, the samples were cut into flakes (60\u0026ndash;80 nm) and were fished out onto the 150 meshes cuprum grids. Samples were successively stained with 2% uranium acetate for 8 min without light and 2.6% lead citrate without CO\u003csub\u003e2\u003c/sub\u003e for 8 min for experiments. The images were obtained through a HT7800/HT7700 TEM (Hitachi) with accelerating voltage of 80.0 kV. The procedures for tumor tissues and organoids TEM images were similar.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eProteomics or RNA sequencing (RNA-seq) sample preparation\u003c/h2\u003e \u003cp\u003eH460 cells were seeded in a 6-well plate. Based on different interventions, the following groups were established: \u003cb\u003eP4-3\u003c/b\u003e group (1 \u0026micro;M with 0.5% DMSO as co-solvent), DMSO group (0.5% DMSO), and Alogliptin group (1 \u0026micro;M with 0.5% DMSO as co-solvent). The cells were incubated with different drug for 9 hours, and were washed 2\u0026ndash;3 times with pre-cooled PBS at 4\u0026deg;C. The cells were then digested with trypsin and collected and rapid frozen in liquid nitrogen for proteomics or RNA-seq analysis. For RNA-seq analysis, the procedures were similar apart from the concentration and treating time of \u003cb\u003eP4-3\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eRNA-seq\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the cells using TRIzol\u0026reg; Reagent according the manufacturer\u0026rsquo;s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. according to the manufacturer\u0026rsquo;s instructions (Illumina, San Diego, CA). The human RNA-seq transcriptome library was prepared following Illumina\u0026reg; Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1 \u0026micro;g of total RNA. Messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Then double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation and \u0026lsquo;A\u0026rsquo; base addition according to Illumina\u0026rsquo;s library construction protocol. Libraries were selected for cDNA target fragments of 300 bp on 2% low range ultra agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After being quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq 6000 sequencer (2 \u0026times; 150 bp read length). To identify differential expression genes (DEGs) between different groups, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. Essentially, differential expression analysis was performed using the DESeq2 or DEGseq method. DEGs with |log2FC| ≧ 1 and FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05 (DESeq2) or FDR\u0026thinsp;\u0026le;\u0026thinsp;0.001 (DEGseq) were considered to be significantly different expressed genes. In addition, functional-enrichment analysis: Kyoto Encyclopedia of Genes (KEGG) was performed to identify which DEGs were significantly enriched in metabolic pathways at Bonferroni-corrected P-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 compared with the whole-transcriptome background. KEGG pathway analysis was carried out by KEGG orthology based annotation system (KOBAS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eProteomics\u003c/h2\u003e \u003cp\u003eThe samples in liquid nitrogen were transferred to a 5-mL centrifuge tube. After that, four volumes of lysis buffer (with 1% of protease inhibitor: cocktail) was added to the cell powder, followed by sonication three minutes on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4\u0026deg;C for 10 min. Finally, the supernatant was collected and the protein concentration was determined with BCA kit. The protein solutions were reduced with 5 mM dithiothreitol for 30 min at 56\u0026deg;C and alkylated with 11 mM iodoacetamide for 15 min at room temperature without light. The protein samples were then diluted by adding 200 mM triethanolamine borate (TEAB) to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for thefirst digestion overnight and 1:100 trypsin-to-protein mass ratios for a second 4 h-digestion. Finally, the peptides were desalted by Strata X SPE column. The tryptic peptides were dissolved in solvent A, directly loaded onto a home-made reversed-phase analytical column (25-cm length, 100 \u0026micro;m i.d.). The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile/in water) and solvent B (0.1% formic acid in acetonitrile). Peptides were separated with following gradient: 0\u0026ndash;9 min, 6%-24%B;9\u0026ndash;11 min, 24%-35%B༛11\u0026ndash;13 min, 35%-80%B༛13\u0026ndash;15 min, 80%B, and all at a constant flow rate of 500 nl/minon a NanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the TIMS-TOF Pro mass spectrometry. The electrospray voltage applied was 1.75 kV. Precursors and fragments were analyzed at the TOF detector. The timsTOF Pro was operated in data independent parallel accumulation serial fragmentation (dia-PASEF) mode. The full MS scan was set as300-1500 (MS/MS scan range) and 20PASEF (MS/MS mode)-MS/MS scans were acquired per cycle. The MS/MS scan range was set as 400\u0026ndash;850 and isolation window was set as7 m/z. After that, the DIA data were processed using DIA-NN search engine (v.1.8). Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20231220.fasta concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 1 missing cleavages. Excision on N-term Met and carbamidomethyl on Cys were specified as fixed modification. FDR was adjusted to \u0026lt;\u0026thinsp;5%. The structural domain of a protein is a specific protein region in a protein that is conserved in sequence and can generally perform a function independently, and is a structural component of molecular function, generally consisting of 25 to 500 amino acids. These areas are relatively spatially compact, structurally stable, and capable of being independently folded into functional structures. A protein may have multiple domains, and a domain may alsoexist in multiple proteins. In the project data, protein structural domain annotation was performed on the identified proteins based on the Pfam database and the corresponding PfamScan tool. The KEGG integrates currently known protein-protein interaction network information, such as pathways and related complexes (Pathway database), genes and gene products (Gene database), biological complexes and related reactions (Compound and Reaction databases), and other information. The KEGG pathways mainly include metabolism, genetic information processing, environmental information processing, cellular processes, human diseases and drug development. We annotate protein pathways based on the KEGG pathway database, and identify proteins through BLAST comparison (blastp, evalue\u0026thinsp;\u0026le;\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e), for each sequence, the annotation is based on the top-scoring comparison result. Lastly, we used PSORTb software to perform subcellular structure prediction analysis of proteins identified in eukaryotes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiosafety evaluation of P4-3\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003e The uses of animals were permitted by the institutional animal ethics committee of Xi\u0026rsquo;an Jiaotong University (Approval number: XJTUAE2023-1611). Briefly, C57BL/6 mice (healthy, 5\u0026ndash;6 weeks) were provided from Laboratory Animal Center (LAC) of Xi'an Jiaotong University and were randomly divided into 2 groups (N\u0026thinsp;=\u0026thinsp;5): control group and \u003cb\u003eP4-3\u003c/b\u003e group. The mice in \u003cb\u003eP4-3\u003c/b\u003e group were intravenous injected with \u003cb\u003eP4-3\u003c/b\u003e (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times) and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without \u003cb\u003eP4-3\u003c/b\u003e. Subsequently, mice were sacrificed and the main organs: heart, liver, spleen, lung, kidney and pancreas were sliced for hematoxylin and eosin (H\u0026amp;E) staining experiments to evaluate the biosafety of \u003cb\u003eP4-3\u003c/b\u003e. Whole blood was collected for complete blood count (CBC). Serum was collected to measure relevant biochemical indicators. The spleen was excised and grinded. The grinding solution was filtered through a 40-\u0026micro;m cell strainer (Biosharp) to obtain a single-cell suspension for flow cytometry experiments (BioTek Synergy LX multimode micro-plate reader, Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eThe anti-tumor evaluation effects on subcutaneous tumor\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e The animal experiments were permitted by the institutional animal ethics committee of Xi\u0026rsquo;an Jiaotong University (Approval number: XJTUAE2023-1611 and XJTUAE2024-1849). For \u003cb\u003eP4-3\u003c/b\u003e intervention experiment, BALB/c nude mice (healthy, 5\u0026ndash;6 weeks) were randomly divided into 2 groups (N\u0026thinsp;=\u0026thinsp;5): control group and \u003cb\u003eP4-3\u003c/b\u003e group. The mice in \u003cb\u003eP4-3\u003c/b\u003e group were firstly subcutaneous injected with H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L) and mice were feed for 10 days. After tumor formation, mice in \u003cb\u003eP4-3\u003c/b\u003e group were intravenous injected with \u003cb\u003eP4-3\u003c/b\u003e (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times), and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without \u003cb\u003eP4-3\u003c/b\u003e. The weights and tumor volumes were recorded every 3 days. After treatment, mice were sacrificed and the tumors were weighed and sliced or mashed for further experiments. For shRNA-transfected injecting experiment, BALB/c nude mice (healthy, 5\u0026ndash;6 weeks) were randomly divided into 2 groups (N\u0026thinsp;=\u0026thinsp;5): E. V. group and shRNA group. The mice in shRNA group were firstly subcutaneous injected with shRNA-transfected H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L) and mice in E. V. group were firstly subcutaneous injected with E. V.-transfected H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L). Mice were feed for 10 days and the weights were recorded every 3 days. After 17 days, mice were sacrificed and the tumors were weighed and sliced for further experiments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eThe anti-tumor evaluation effects on metastases\u003c/h2\u003e \u003cp\u003eFor \u003cb\u003eP4-3\u003c/b\u003e intervention experiment, BALB/c nude mice (healthy, 5\u0026ndash;6 weeks) were randomly divided into 2 groups (N\u0026thinsp;=\u0026thinsp;5): control group and \u003cb\u003eP4-3\u003c/b\u003e group. The mice in \u003cb\u003eP4-3\u003c/b\u003e group were firstly thoracic injected with H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L) and mice were feed for 10 days. After tumor formation, mice in \u003cb\u003eP4-3\u003c/b\u003e group were intravenous injected with \u003cb\u003eP4-3\u003c/b\u003e (0.6 mg/kg, 1.0% DMSO was used as co-solvent, every 3 days one time, 4 times), and mice in control group were intravenous injected with phosphate buffered saline containing 1.0% DMSO without \u003cb\u003eP4-3\u003c/b\u003e. The weights were recorded every 3 days. After treatment, mice were sacrificed and the tumors were weighed and sliced or mashed for further experiments. For shRNA-transfected injecting experiment, BALB/c nude mice (healthy, 5\u0026ndash;6 weeks) were randomly divided into 2 groups (N\u0026thinsp;=\u0026thinsp;5): E. V. group and shRNA group. The mice in shRNA group were firstly thoracic injected with shRNA-transfected H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L) and mice in E. V. group were firstly intravenous injected with E. V.-transfected H460 cells (3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, 100 \u0026micro;L). Mice were feed for 10 days and the weights were recorded every 3 days. After 17 days, mice were sacrificed and the tumor volumes were recorded and tumors were sliced for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eThe anti-cancer evaluation effects of P4-3 on organoids\u003c/h2\u003e \u003cp\u003eThe use of human tumor and paracancer tissues of non-small cell lung cancer were strictly complied with the rules of institutional animal ethics committee of Xi\u0026rsquo;an Jiaotong University (Approval number: XJTU1AF2021LSK-484) and the informed consent has been signed by patients. All organoids were obtained from Shaanxi Weiyuan Biomedical Research Institute Co., Ltd. 3 cases were randomly selected for anti-cancer evaluation. Firstly, tumor organoids were treated with \u003cb\u003eP4-3\u003c/b\u003e with different concentrations for 9 hours and then the degradation of CD26 was tested. After that, tumor and paracancer organoids were treated with different compounds for different time, the inhibition rates of cells were performed through CellTiter-Glo\u0026reg; 3D cell viability assay kit. And the lysates were prepared to perform other experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eH\u0026E\u003c/h3\u003e\n\u003cp\u003eMajor organs or tumors were successively fixed, embedded, and sectioned. The samples were preprocessed with pretreatment liquid for 1 min and then stained with Hematoxylin for 5 min. After that, samples were washed and stained with Eosin for 15 s. Samples were then dehydrated and sealed up for imaging (Eclipse E100 optical microscope, Nikon).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence or Tunel or Immunohistochemical staining\u003c/h2\u003e \u003cp\u003eThe tumors were collected and paraffin sectioned. The samples were then repaired with corresponding primary and secondary antibodies. After that, the samples were sealed with 3% bovine serum albumin (BSA) or 10% donkey serum. The samples were incubated with corresponding primary antibodies at 4\u0026deg;C overnight. Subsequently, samples were washed with PBS for 3 times and incubated with corresponding secondary antibodies for 50 min without light. Lastly, the samples were washed with PBS for 3 times and stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min without light. The samples were washed with PBS for 3 times firstly and auto-fluorescence quencher for imaging (Eclipse C1 fluorescence microscope, Nikon). The procedures for immunohistochemical staining were similar and the procedures for Tunel staining were also similar without sealing and incubation of antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 9.0 and Origin 9 software. All data are determined from at least three independent experiments and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance between two groups was calculated using Two-tailed test. Differences between multiple groups were assessed by ANOVA followed by Dunnett post-hoc test (ANOVA Dunnett\u0026rsquo;t test). P-values of less than 0.05 were considered as statistically significance.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n\u003ch2\u003eReporting summary\u003c/h2\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eSource data are provided with this paper.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version containssupplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u003c/strong\u003e is available at\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eG. J. Zhang, B. H. Liu and Q. Y. Gong conceptualized and designed the project. B. H. Liu, D. Q. Qiao, X. Z. Zhu and J. Q. Huang performed the experiments and analyzed the results. R. Gao and L. J. Zhang provided suggestions on cell line and animal models construction. Y. J. Huang provided suggestions on compound synthesis. G. J. Zhang, J. Z. Wang, Y. J. Huang and Q. Y. Gong supervised the project. G. J. Zhang, B. H. Liu and Q. Y. Gong wrote the manuscript. All authors discussed and commented on the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eAuthors thank the support from the center for translational medicine, the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University and Shaanxi Weiyuan Biomedical Research Institute Co., Ltd. Q. Gong thank the funds supporting from the Key Research and Development Project of Shaanxi Province (2023-YBSF-292), the opening foundation (M2022-3) from Key Laboratory of Optic-Electric Sensing and Analytical Chemistry for Life Science, Ministry of Education, Qingdao University of Science and Technology. J. Wang thanks the fund supporting from the National Natural Science Foundation of China (82102976). Y. Huang thanks the fund supporting from the National Natural Science Foundation of China (52203337) and the Youth Top Talent Program (11301223010722) from Xi\u0026rsquo;an Jiaotong University. G. Zhang thanks the fund supporting from the Key Research and Development Project of Shaanxi Province (2024SF-ZDCYL-02-09), Capacity Improvement Plan of Shaanxi Health Committee (2024PT-09)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeiter A, Veluswamy RR, Wisnivesky JP (2023) The global burden of lung cancer: current status and future trends. 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Heliyon 10:e29285\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeleta AB, Dong LF, Rohlena J, Neuzil J (2016) The assembly factor SDHAF2 is dispensable for flavination of the catalytic subunit of mitochondrial complex II in breast cancer cells. J Biol Chem 291:21414\u0026ndash;21420\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CD26, non-small cell lung cancer, proteolysis-targeting chimeras, molecular targeted therapy, organoids","lastPublishedDoi":"10.21203/rs.3.rs-5702716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5702716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMolecular targeted therapy (MTT) for non-small cell lung cancer (NSCLC) has been a central issue for a long time. However, drug resistance and extra toxicity have limited its further clinical applications. Herein, taking advantages of the proteolysis-targeting chimeras (PROTACs), a series of PROTAC degraders (\u003cb\u003eP4-1\u003c/b\u003e to \u003cb\u003e4\u003c/b\u003e) targeting cell-surface CD26 (a potential target for NSCLC) have been developed for MTT of NSCLC. To achieve the efficient degradation of cell surface proteins, which is a huge challenge, the molecular structures of degraders were rational designed and optimized. Remarkably, CD26 can be degraded by \u003cb\u003eP4-3\u003c/b\u003e evidently at low dose (~\u0026thinsp;500 nM) without degrading CD26 isoenzymes, which was independent of autophagy pathway. Surprisingly, the proliferation of representative NSCLC cells (NCl-H460 and NCl-H1299 cells) and tumors were significantly inhibited by \u003cb\u003eP4-3\u003c/b\u003e, and no toxicity of \u003cb\u003eP4-3\u003c/b\u003e for BEAS-2B cells (human lung normal epithelial cells) were obtained. More interestingly, the powerful proliferation inhibition capabilities of \u003cb\u003eP4-3\u003c/b\u003e for organoids were observed. Moreover, a mechanism of \u003cb\u003eP4-3\u003c/b\u003e for treatment of NSCLC was proposed, which is the degradation of CD26 that induce the mitochondria-mediated apoptosis of NSCLC cells, tumors and organoids. Our exciting findings put forward instructive thoughts for the development of promising treatment strategies for NSCLC.\u003c/p\u003e","manuscriptTitle":"Harnessing cell-surface CD26 proteolysis-targeting chimeras for molecular targeted therapy against non-small cell lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 08:07:07","doi":"10.21203/rs.3.rs-5702716/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf45aff2-788d-4aef-a836-ca687a5c0161","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42548680,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"},{"id":42548681,"name":"Biological sciences/Chemical biology/Small molecules"}],"tags":[],"updatedAt":"2025-06-11T05:31:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-09 08:07:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5702716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5702716","identity":"rs-5702716","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}