Synergistic effects of XPO1 inhibitors combined with CD19 CAR-T cells in TP53-mutated DLBCL

Synergistic effects of XPO1 inhibitors combined with CD19 CAR-T cells in TP53-mutated DLBCL | 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 Synergistic effects of XPO1 inhibitors combined with CD19 CAR-T cells in TP53-mutated DLBCL Zhimin Bai, Xiaoyu Huang, Xinfeng Wang, Yong Zhou, Zenghua Lin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5946503/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 Purpose Diffuse large B-cell lymphoma (DLBCL) is mostly curable by chemotherapy, but p53 mutations limit the therapeutic effect of DLBCL. Although chimeric antigen receptor (CAR) T cells have made revolutionary progress in the treatment of DLBCL, p53 mutations still lead to drug resistance and/or relapse of DLBCL, affecting the prognosis of lymphoma. Therefore, the project aim to explore additional therapeutic strategies to improve the prognosis of DLBCL with p53 mutations. Materials and Methods We investigated the correlation between XPO1 and mut-P53 employing qRT-PCR, WB, CCK8 and flow cytometry. Then, we conduct XPO1 inhibitor (KPT-330) to explore the apoptotic effect on DLBCL. Through the TCGA database, there is a clear correlation between XPO1-related genes and the PI3K-AKT pathway. Finally, the effect of KPT-330 on the killing ability of CAR-T cells was evaluated by CCK8, flow cytometry and ELISA. Results In this study, we showed that XPO1 inhibitor (KPT-330) synergized with CAR-T to reduce the viability of DLBCL cells and enhance the killing effect of CAR-T cells. As expected, KPT-330 combined with CAR-T therapy slowed tumor growth and reduced tumor burden in DLBCL with p53 mutations. Mechanistically, XPO1 inhibitor KPT-330 can cooperate with CAR-T in the treatment of DLBCL by activating the PI3K pathway. Then, in vitro cytotoxicity assays revealed that the KPT-330 combined with CAR-T group significantly enhanced the secretion of effector cytokines IFN-γ, TNF-α, and IL-2, and activated the immune system. Conclusions The XPO1 inhibitor KPT-330 exerts anti-cancer effects through dual mechanisms (stabilizing p53 and inhibiting the PI3K-AKT pathway), providing a molecular basis for DLBCL treatment. We may provide a potential promising combination therapy for the treatment of DLBCL with p53 mutations. Biological sciences/Immunology Health sciences/Oncology XPO1 DLBCL p53 CAR-T PI3K-AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous type of aggressive non-Hodgkin lymphoma (NHL). About 60% of patients reported a complete and sustained response with traditional chemotherapy with rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). However, up to 40% of patients develop refractory or recurrent (R/R) after treatment 1 , the most common cause of which is gene mutation. Approximately 20–30% of DLBCL have TP53 mutations 2 – 5 , which leads to a worse prognosis of DLBCL 6 , 7 . TP53 is a tumor suppressor gene that plays an important role in cell cycle and proliferation. One of its main functions is to induce apoptosis in cells induced by DNA damage. TP53 gene mutations can destroy genetic stability and lead to uncontrolled proliferation of cancer cells 8 . The role of TP53 mutations as a negative prognostic factor has been well established in many malignancies, including DLBCL. Therefore, the search for other effective treatment strategies is an unmet medical need for DLBCL patients with TP53 mutations. XPO1 (exportin 1), also termed chromosome region maintenance 1 (CRM1), is a nuclear export receptor involved in the transportation of proteins such as histones, polymerases, transcription factors and/or RNA from the nucleus into the cytosol and responsible for the nuclear-cytoplasmic transport and cellular homeostasis of up to 220 cargoes, including the tumor suppressors p53 and I-κB 9 . XPO1 over-expression has been observed in many malignancies, and elevated XPO1 levels have been associated with poor clinical prognosis 10 – 13 . Thus, down-regulation of XPO1 constitutes an interesting therapeutic strategy. Notably, inhibiting XPO1 by selective inhibitors of nuclear export (SINEs) has been demonstrated in hematological malignancies including multiple myeloma (MM), ALL, NHL, acute myeloid leukemia (AML). The XPO1 inhibitor selinexor was approved for the treatment of adults with r/r MM by the U.S. Food and Drug Administration (FDA) in September 2019, and by the European Medicines Agency (EMA) in December 2019. Moreover, selinexor is currently under clinical evaluation for treatment of DLBCL, r/r AML as well as myelodysplastic syndrome (MDS) 14 , 15 . Researches have found that p53 and XPO1 are inextricably linked. In some human tumor cells, wild-type p53 has been reported to be abnormally isolated in the cytoplasm. Nuclear export of p53 is mediated by both MDM2 and XPO1. MDM2 activates nuclear export signaling (NES) in p53 through its E3 ubiquitin ligase activity, causing conformational changes in p53 that expose the NES domain of p53. After ubiquitination, XPO1 recognizes p53’s NES and exports the protein from the nucleus to the cytoplasm, thereby limiting p53-mediated transcriptional activity and inhibiting the ability of p53 to trigger apoptosis 16 – 18 . A promising treatment for relapsed/refractory (r/r) DLBCL patients is chimeric antigen receptor (CAR) T-cell therapy, which modifies the natural course of chemorefractory DLBCL. In the laboratory, T cells are extracted from the patient's blood, equipped with so-called CARs to help to recognize and destroy DLBCL cells, and then delivered back into the patient's blood. However, 25–50% of patients with r/r DLBCL still do not achieve remission after CD19 CAR-T therapy and survive for less than one year 19 . Many factors influencing the poor prognosis of DLBCL have been published in the literature, among which TP53 alterations and double expression are the most factors associated with poor prognosis of the r/r DLBCL patients and have a lower likelihood of achieving complete remission after treatment with CD19 CAR-T 20 . Previous studies have shown that XPO1 blocking promotes p53 nuclear retention and can have a powerful anti-lymphoma effect on HGBCL-DH cells with or without TP53 mutations 21 , 22 .The present study investigated the effectiveness of combining CD19-CAR-T cells with the XPO1 inhibitor KPT-330 administration. The results of this study suggest that this combinatorial strategy is superior to CAR-T monotherapy against DLBCL. Materials and Methods 1. Cell lines and primary samples The DLBCL cell lines OCI-Ly19 was generously gifted by Dr. GQ. Song (Affiliated Hospital of Nantong University, Jiangsu, China). OCI-Ly19 cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% heat-inactivated fetal bovine serum (Gibco), 100U/ml penicillin and 100ug/ml streptomycin (NCM Biotech) at 37℃ in a 5% CO2 incubator. The medium was changed every 2–3 days to maintain the cell density between 2×10^5 and 2×10^6 cells/ml. 2. CAR-T cells generation and manufacturing Peripheral blood lymphocytes were obtained and collected from patients by density gradient separation. T lymphocytes were further sorted with anti-CD3 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and activated with 5 ug/mL monoclonal anti-CD3/CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for 48 h. Then T cells were transfected with a lentivirus encoding CD19/22-4-1BB-CD3z transgene, and the T cells were treated in AIM-V medium supplemented with 10% autologous serum, 100 IU/ml IL-2, 5 ng/ml IL-7, and 5 ng/ml IL-15 (Gibco, New York, New York, USA) for 12 to 20 days until the number of cells reached the preset value. All of these CAR-T products were provided by the Taihe Chunyu biotechnology co. (Hebei, China). The study protocol was conducted in accordance with the amended Declaration of Helsinki and was approved by the Ethics Committee of Affiliated Hospital of Nantong University (2025-L092). All patients gave informed written consent for participation in the study. 3. Flow cytometry The treated OCI-LY19 cells were washed twice with PBS, and stained with the AnnexinV-PE/7AAD double staining kit (BD Biosciences) for 15–20 min. After incubation in the dark for 15 minutes, 1X Binding Buffer was added, and the cells were resuspended and detected by flow cytometry (BD FACSCalibur). The proportion of early and late apoptotic cells was analyzed using FlowJoVX software. 4. siRNA and plasmid transfection siRNA and the plasmid vector (pCMV) were purchased from Genepharma (Shanghai, China) and carried the target gene. The transfection steps were as follows: OCI-LY19 cells were seeded in a 6-well plate at a density of 5×10^5 cells/mL, siRNA transfection was performed according to the steps of the Ribo kit, and the overexpression plasmid was transfected using Lipofectamine3000 reagent (Invitrogen, USA). After 48 hours, the cells were extracted for subsequent experimental analysis. The siRNA sequences were: XPO1-homo-3285: forward primer, GGCUGUCAAUUCUCAUUGUTT. Reverse primer, ACAAUGAGAAUUGACAGCCTT. XPO1-homi-1854: forward primer, GGCUGCUGAACUCUAUAGATT. Reverse primer, UCUAUAGAGUUCAGCAGCCTT. 5. Determination of IC50 and cytotoxicity assay KPT-330 (Selinexor) (Selleck, USA) OCI-LY19 cells were seeded in 96-well plates at 1×10^5 cells/well and cultured for 48 hours using medium containing different concentrations (concentration range:0.00nM to 400nM) of KPT-330 (Selleck, USA). After culture, 10ul CCK-8 reagent (Dojindo) was added to each well and incubated for another 2 hours. The optical density (OD450) was measured at a wavelength of 450nm using a microplate reader (Bio-Rad). The half inhibitory concentration (IC50) was calculated based on the measured data to evaluate the cytotoxicity of KPT-330. The target cells, OCI-Ly19, were co-cultured with CAR-T cells at different E/T ratio (0.5:1, 1:1, 2:1). CCK8 was added to each well at different time points (0h.12h.24h.48h.72h), and the optical density (OD) (BioTek) was measured after 2 hours. The OD450 value was measured by a microplate reader to calculate the survival rate of tumor cells and the killing rate of CAR-T cells. The killing rate was calculated by the following equation: [1-(OD of the effector target cell well)-(OD of the effector cell well)/OD of the target cell well]. 6. Cell invasion assay The cell invasion assay was performed using a Transwell chamber (Corning). The pre-treated OCI-LY19 cell suspension (2×10^5 cells/100µL) was added to the upper chamber, and RPMI-1640 medium containing 20% fetal bovine serum was added to the lower chamber, and incubated at 37°C for 24 hours. After the incubation, the non-invaded cells in the upper chamber were removed, and the lower chamber was photographed under an optical microscope. At the same time, the cells in the lower chamber were collected and counted using a cell counting plate, and the experiment was repeated 3 times. 7. RT-qPCR detection of mRNA transcription levels OCI-LY19 cells were treated with KPT-330 (82.4 nmol) for OCI-LY19 cells, alone or in combination for 24h. Total RNA was extracted using TRIzol reagent (Invitrogen) as directed by the manufacturer. cDNA was then synthesized by reverse transcription using PrimeScript RT Reagent Kit (Takara, Dalian, China). qPCR SYBR Green PCR Master Mix (Vazyme) was used for fast and real-time quantification in TOUCH DEEP WELL with CFX96 PCR system (Bio-rad,USA). The relative expression of each gene was calculated using the 2^-ΔΔCt method. GAPDH was the internal reference gene, and mut-P53 and XPO1 were the target genes. The primer sequences were as follows: XPO1: forward primer, TCTCATTGTTTCCCAGCATTCCTTG. reverse primer: TAAGCCCGTATCTGCGACATTCC. 8. Western Blot 8. Western Blot Cells were collected 72 hours after transfection, and proteins in the cells were isolated using RIPA lysis buffer (Beyotime), and protein concentrations were determined using the BCA assay (Vazyme). Next, proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane (Beyotime). Then, the membranes were blocked with a rapid blocking solution for 30 minutes at room temperature. Primary antibodies were incubated according to standard procedures, and incubated overnight at 4°C, and appropriate HRP-conjugated secondary antibodies were incubated. After washing, the membranes were developed using a luminescence imaging system (Tanon5200). 9. CFDA-SE cell proliferation CFDA-SE (Invitrogen) dye was used to detect CAR-T cell proliferation. CAR-T cells were stained with CFDA-SE, and CAR-T cells (1×10 6 cells/tube) were stained with CSFE (Beyotime, China) at 37°C. After washing, the same number of target cells were co-cultured with CAR-T cells, and CAR-T cells were collected after 5 days, and fluorescence was detected by flow cytometry. The changes in fluorescence intensity at different time points were detected by flow cytometry to analyze cell proliferation. 10. Cytokine analysis CAR-T cells and OCI-LY19 cells were cultured at a 1:1 effector-target ratio for 24 hours, and the culture medium was collected by centrifugation. The levels of inflammatory factors (IL-2, IL-4, IL-6, IL-10, TNF-α, TNF-β) were quantitatively detected using an ELISA kit (eBioscience). Sample incubation, enzyme-labeled secondary antibody reaction, and color development were performed according to the kit instructions, and the absorbance was finally measured at a wavelength of 450 nm using an ELISA kit. 11. Statistical analysis Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software Inc.). P‑values were calculated using the parametric two‑way t‑test between two groups, and the one‑way analysis of variance (ANOVA) with Bonferroni's multiple comparison test for three or four groups. P < 0.05 was considered statistically significant. When not otherwise indicated, results were represented as mean ± standard deviation (SD). IC50s were presented as mean ± standard error of the mean (SEM). Graphs and tables were designed using GraphPad Prism 6. Results 1. XPO1 expression levels in pan-cancer: Unpaired comparisons of the levels of XPO1 expression levels between tumor tissues from TCGA database and normal tissues from TCGA database (*p < 0.05, **p < 0.01, ***p < 0.001, ns = no significance). Reported studies have reported increased expression of XPO1 in a variety of cancers. We use the TCGA pan-cancer database to evaluate the expression of XPO1 mRNA in various tumors. The analysis results suggest that XPO1 is highly expressed in 21 types of tumors, including adrenocortical carcinoma (ACC), bladder cancer (BLCA), breast cancer (BRCA), cervical cancer (CESC), cholangiocarcinoma (CHOL), and colon adenocarcinoma (COAD), diffuse large B-cell lymphoma (DLBC), esophageal cancer (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), renal chromophobe cell carcinoma (KICH), renal papillary cell cancer (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), rectal adenocarcinoma (READ), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), and thymoma (THYM) (Fig. 1 A). Notably, XPO1 was highly expressed in diffuse large B-cell lymphoma in TCGA unpaired samples ( Fig. 1 A ) . At the same time, we used the GEO database to analyze the correlation between XPO1 expression and DLBCL prognosis, and found that high XPO1 expression was significantly associated with poor prognosis and shortened overall survival in DLBCL patients ( Fig. 1 B ) . These indicate that XPO1 may play an important role in the development of this tumor and deserves further study. 2. Correlation between XPO1 and mut-TP53 expression in DLBCL patients The mutation rate of TP53 in DLBCL is 10%-20% 23 . Abnormal regulation of the TP53 pathway can make the DLBCL genome unstable, leading to resistance to the chemotherapy regimens and disease progression. Therefore, we further studied the correlation between XPO1 and mut-TP53. We collected peripheral blood form 61 patients with DLBCL in the Department of Hematology, Affiliated Hospital of Nantong University in the past 3 years for experimental analysis. We analyzed the relative expression levels of XPO1 and mut-TP53 in clinical patients by Polymerase Chain Reaction (PCR), and analyzed the correlation between XPO1 and mut-TP53. The results showed that the expression of XPO1 was closely related to the expression of mut-TP53, and the expression of mut-TP53 was positively correlated with the expression of XPO1 ( Fig. 1 C ) . This shows that in DLBCL, a high mutation rate of TP53 will affect the high expression of XPO1, leading to poor prognosis. 3. Correlation between XPO1 and mut-TP53 expression in OCI-LY19 cells and the role of inhibitor KPT-330 We used XPO1 overexpression plasmid HE and si-RNA in OCI-LY cells to study the correlation between XPO1 expression level and mut-TP53 in diffuse large B-cell lymphoma cells. Fluorescence quantitative PCR detection showed that when XPO1 was highly expressed, it was positively correlated with mut-TP53 ( Fig. 2 A ) , and when XPO1 was lowly expressed, it was negatively correlated with mut-TP53 ( Fig. 2 B ) . We set up a concentration gradient of KPT-330 to treat OCI-LY19 for 48 hours, calculated the half-inhibitory concentration by CCK8 detection, and used KPT-330 at a concentration of 82.4nm to kill tumor cells ( Fig. 2 C ) . The results showed that the use of XPO1 inhibitor KPT-330 in OCI-LY19 cells inhibited XPO1 expression, and mut-TP53 decreased accordingly, and the correlation decreased ( Fig. 2 D ) . 4. KPT‑330 treatment reduces XPO1 levels and inhibits OCI-LY19 cell growth in vitro XPO1 is highly expressed after plasmid transfection, XPO1 is low-expressed after siRNA transfection, and XPO1 is highly expressed and treated with KPT-330. After culturing the cells for 24h, 48h and 72h, CCK-8 experiments showed that over-expression of XPO1 significantly promoted the proliferation of DLBCL cells, low expression of XPO1 inhibited the proliferation of tumor cells, and KPT-330 inhibited the over-expression of Proliferation of OCI-LY19 cells by XPO1 ( Fig. 3 A ) . Transwell is used to detect the migration ability of OCI-LY19. Compared with the control group, when XPO1 is over-expressed, the migration ability of OCI-LY19 is strong, and when XPO1 is low expressed, the migration ability is weak. However, the use of KPT-330 significantly inhibited OCI-LY19 migration ability ( Fig. 2 A ) . At the same time, flow cytometry analysis showed that compared with the control group, the apoptosis rate of OCI-LY19 cells with XPO1 over-expression increased, and the apoptosis rate of cells with low XPO1 expression decreased. The use of KPT-330 indicated the apoptosis rate of tumor cells increased ( Fig. 3 C ) . The above results suggest that high expression of XPO1 will significantly promote the proliferation of OCI-LY19 cells and reduce cell apoptosis. However, the XPO1 inhibitor KPT-330 can inhibit the proliferation of tumor cells that highly express XPO1 and induce tumor cell apoptosis. KPT-330 inhibited the proliferation of OCI-LY19 cells over-expressing XPO1. 5. Correlation and enrichment analysis of XPO1 To further clarify the role of XPO1 in DLBCL, we analyzed the genes that were positively correlated with XPO1 expression in the TCGA database. We selected genes associated with XPO1 for enrichment analysis and displayed the top 30 genes (ranked by correlation) in a volcano plot ( Fig. 4 A ) . At the same time, the gene ontology (GO) enrichment and KEGG pathways analysis were performed on the top 30 genes associated with XPO1. GO BP analysis revealed that the associated genes were markedly enriched in regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-templated and protein phosphorylation. The top three significantly enriched CC terms included nucleoplasm, cytosol and nucleus. For GO MF analysis, the top significantly enriched term was the metal ion binding ( Fig. 4 B ) . Furthermore, and the gene display map corresponding to the KEGG pathway showed that XPO1, as an important transporter, is involved in regulating the transport of intracellular proteins and is essential for biological processes such as cell cycle and cellular gene expression. KEGG pathways analysis suggested that XPO1 causes nuclear retention and activation of tumor suppressor proteins and other growth regulatory proteins, while downregulating the levels of multiple tumor suppressor proteins in the cytoplasm through the PI3K-AKT pathway ( Fig. 4 C-D ) . 6. XPO1 expression levels and the effect of KPT-330 on P53 P53 is a carrier protein of XPO1, so we used western blot to analyze the changes of P53 protein levels to clarify the status of P53 under the treatment of KPT-330. The results showed that when XPO1 was highly expressed, the protein expression level of P53 decreased, and conversely, when it was lowly expressed, the expression level of P53 increased ( Fig. 5 A ) . When the XPO1 inhibitor KPT-330 was used, XPO1 was gradually inhibited as the inhibitor action time increased, and the expression level of P53 protein increased accordingly ( Fig. 5 B ) . The above suggests that XPO1 inhibitor KPT-330 promotes the accumulation of P53 in cells and reduces its degradation rate, activating the function of P53 to exert anti-cancer effects. 7. Western blot analysis of the effect of XPO1 expression on the PI3K-AKT pathway The PI3K-AKT pathway is a key pathway regulating cell proliferation and apoptosis, and is closely related to the occurrence, proliferation, transformation, apoptosis, and drug resistance of tumors. We performed KEGG enrichment analysis on the up-regulated genes that were positively correlated with XPO1, and found that XPO1-related genes were significantly correlated with the PI3K-AKT pathway. Therefore, we further investigated whether XPO1 is involved in regulating the PI3K-AKT pathway. We used western blot to detect that when si-RNA was transfected to reduce the expression of XPO1 and the XPO1 inhibitor KPT-330 was used, the protein levels of p-PI3K and P-AKT decreased, indicating that when XPO1 was inhibited, the activation of the PI3K-AKT pathway was inhibited ( Fig. 5 C ) . The inhibition of the PI3K-AKT pathway can also reduce the degradation of P53. 8. Killing rate and cytokine release levels of CAR T cells treated with KPT-330 We evaluated the killing ability of CAR-T cell treated with KPT-330. We treated with tumor cells with the half maximal inhibitory concentration (IC50) of KPT-330, via CCK8 killing experiment to detect the killing of KPT-330 combined with CAR-T cells on OCI-LY19. The efficacy target ratio and co-culture time were selected, and finally we chose 24 hours of co-culture and 1:1 efficacy target ratio for subsequent experiments ( Fig. 6 A ) . And the results also showed that the killing rate of tumor cells was higher after pretreatment with KPT-330 and combined with CAR-T cells. We detected by flow cytometry that the use of KPT-330 treatment combined with CAR-T increased the apoptosis rate of tumors ( Fig. 6 B ) . To investigate the effector function of KPT-330 combined with CAR-T, a group of cytokines were detected in the in vitro cytotoxicity assay. Compared with the control group, KPT-330 combined with CD19CAR-T displayed higher cytokine secretion levels when co-cultured with OCI-LY19. We could observe increased secretion of effector cytokines and chemokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) ( Fig. 6 C ) . Discussion P53 is a well-known transcription factor that participates in a variety of cellular processes including apoptosis, cell cycle, and DNA damage response, and plays an anti-cancer role in the cell nucleus. TP53 mutation is a poor prognostic factor for diffuse large B-cell lymphoma. Clinical studies have shown that salvage therapy and ASCT have limited efficacy in patients with TP53 mutations and have failed to improve the prognosis of patients with TP53 mutations. In patients with lymphoid malignancies, although CD19 CAR-T cell therapy is more effective than other second-line therapies or new drugs, relapse and resistance after CAR-T cell therapy still pose a challenge. Patients with TP53 mutations are less likely to achieve complete remission after CD19 CAR-T therapy, which still limits and hinders the efficacy and long-term survival of relapsed and refractory DLBCL 19 , 22 . To address this problem, several studies are being explored. In order to improve the persistence and efficacy of CAR-T cells, there are currently multiple researches to enhance the production of CAR-T cells. For example, PI3K δ inhibitors were found to enhance in vivo function of CAR-T cells made from T lymphocytes of CLL patients 24 . In addition, armored CAR-T cells have also been developed. Additional genetic modifications can be made to CAR-T cells to express additional costimulatory ligands or cytokines to enhance the response of CAR-T cells 25 . In addition, in order to reduce tumor escape during CAR-T cell therapy, a variety of studies have been discovered that can enhance the targeting of CAR-T cells to kill tumor cells. For example, the combination of CAR-T cells with PD-1/PD-L1 inhibitors can enhance the efficacy of CAR-T cells and improve the treatment effect of patients 26 , 27 . CAR-T cells combined with reactive oxygen species (ROS) accelerators can overcome the therapeutic resistance mediated by the tumor micro-environment 28 . Recent clinical studies have found that the combination of BDK inhibitors, such as ibrutinib and CAR-T cell therapy, has better clinical responses in CLL patients 29 . In human xenotransplantation models, the combination of ibrutinib and CAR-T cells can promote the proliferation and anti-tumor efficacy of CAR-T cells and reduce the incidence of cytokine release syndrome (CRS) 30 , 31 . XPO1 is the only export protein that mediates the transport of multiple proteins, including tumor suppressor, growth regulatory, and anti-apoptotic proteins as well as several mRNAs and ribosomal proteins essential for ribosome biogenesis. The nuclear export of p53 is mediated by XPO1, which recognizes p53’s NES and exports the protein from the nucleus to the cytoplasm, where it is unable to perform transcriptional activity to regulate cell fate 32 . Due to XPO1 inhibitor general anti-tumor effect, we tried to combine XPO1 inhibitors (KPT-330) to improve the efficacy of CAR-T cell therapy. In the present study, we observed that the XPO1 inhibitor (KPT-330), synergized with CAR-T to reduce the viability of DLBCL cells and enhance the killing effect of CAR-T cells. In the present study, we found that XPO1 is highly expressed in diffuse large B-cell lymphoma through TCGA database analysis. At the same time, we used the GEO database to analyze the correlation between XPO1 expression and the prognosis of DLBCL, and found that high XPO1 expression was significantly related with poor prognosis in DLBCL patients. Since XOP1 is the nuclear export protein of P53, it can transport P53 from the nucleus to the cytoplasm, thereby losing its transcriptional activity and being unable to regulate cell functions. Therefore, we envision combining XOP1 inhibitors for diffuse large B-cell lymphoma with P53 mutations. KPT330 inhibits the expression of XPO1 and reduces the nuclear export of p53, thereby participating in regulating various cellular processes such as apoptosis, cell cycle and DNA damage response, exerting anti-cancer effects in the nucleus, and enhancing the killing effect of CAR-T on DLBCL. We applied the XPO1 inhibitor KPT-330 to DLBCL cell lines, and detected the decrease in XPO1 expression through fluorescence quantitative PCR, and the expression of mut-P53 also decreased, indicating that high expression of XPO1 is positively correlated with mut-P53. Application of the XPO1 inhibitor can reduce the expression of mut-P53. Simultaneous application of the XPO1 inhibitor KPT-330 can inhibit the proliferation of DLBCL tumor cells that highly express XPO1 and induce their apoptosis. We also conducted in-depth research on the mechanism by which XPO1 inhibitors inhibit DLBCL cell proliferation and induce and promote tumor cell apoptosis. Through the TCGA database, we screened out differential genes that were positively correlated with XPO1, and performed GO and KEGG enrichment analysis on the differential genes. The results show that XPO1, as an important transporter, participates in mediating intracellular protein transport and is crucial to biological processes such as cell cycle and cellular gene expression. At the same time, there is a clear correlation between XPO1-related genes and the PI3K-AKT pathway. Inhibiting the expression of XPO1 can inhibit the activation of the PI3K-AKT pathway and simultaneously down-regulate the levels of multiple tumor suppressor proteins in the cytoplasm. In addition, for potential combination methods, we chose KPT-330 with a half-inhibitory concentration of 82.4 nmol to treat tumor cells, so that KPT-330 and CAR-T cells can achieve the best synergy, which should be able to effectively inhibit target tumor cells without affecting CAR-T cells. Our research data showed that pretreatment with KPT-330 enhanced the anti-tumor cytotoxicity of CAR-T cells and promoted tumor apoptosis. At the same time, the combined application of KPT-330 also increased the cytotoxicity of CAR-T cells and enhanced the cytokine release of CAR-T cells. Therefore, presensitizing tumor cells with KPT-330 may presensitize the tumor cells (without killing them completely) in preparation for treatment with CAR-T cells. All in all, this finding has clinical promise and supports the combined application of KPT-330 for TP53-mutated DLBCL, which may inhibit tumor proliferation by inhibiting the activation of the PI3K-AKT pathway, thereby enhancing the killing function of CAR-T cells. In summary, this study shows that the XPO1 inhibitor KPT-330 can overcome the resistance of TP53-mutated DLBCL to CAR-T therapy. The enhancement of the anti-tumor function of CAR-T cells is attributed to the fact that KPT-330 decreases the expression of XPO1 and inhibits the activation of the PI3K-AKT pathway, thereby enhancing the killing function of CAR-T cells and also enhancing the biological functions of CAR-T cells, significantly. It significantly improves the therapeutic effect of TP53-mutated DLBCL and provides new ideas and directions for future clinical research and practice of CAR-T. Declarations Ethical Statement The experiment in this study was approved by the Ethical and Welfare Committee of the Affiliated Hospital of Nantong University (2024-L034) Author Contributions Conceived and designed the analysis: Bai Z, Lin Z Collected the data: Huang X, Wang X Contributed data or analysis tools: Huang X Performed the analysis: Huang X, Zhou Y, Wrote the paper: Bai Z Funding Acquisition: Bai Z,Liu H Conflicts of Interest Conflict of interest relevant to this article was not reported. Acknowledgments This work was funded by Jiangsu Funding Program for Excellent Postdoctoral Talent, 2022ZB894; Social and Livelihood Science and Technology Project of Nantong, MSZ2023057. Data availability The datasets generated during and/or analysed during the current study are available in The Cancer Genome Atlas(TCGA) database, and the survival datasets can be obtain at the GEO repository with the accession number GSE10846 and the link https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10846. References Crump, M. et al. Outcomes in refractory diffuse large B-cell lymphoma: results from the international SCHOLAR-1 study. Blood 130 , 1800–1808. 10.1182/blood-2017-03-769620 (2017). Miao, Y., Medeiros, L. J., Li, Y., Li, J. & Young, K. H. Genetic alterations and their clinical implications in DLBCL. Nat. Rev. Clin. Oncol. 16 , 634–652. 10.1038/s41571-019-0225-1 (2019). Huang, P. et al. [Prognostic evaluation of P53 and BCL2 proteins in MYC/BCL2 double expression DLBCL]. Zhonghua Xue Ye Xue Za Zhi . 40 , 589–593. 10.3760/cma.j.issn.0253-2727.2019.07.010 (2019). Pascual, M. et al. PD-1/PD-L1 immune checkpoint and p53 loss facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas. Blood 133 , 2401–2412. 10.1182/blood.2018889931 (2019). Schiefer, A. I. et al. Impact of Single or Combined Genomic Alterations of TP53, MYC, and BCL2 on Survival of Patients With Diffuse Large B-Cell Lymphomas: A Retrospective Cohort Study. Med. (Baltim). 94 , e2388. 10.1097/MD.0000000000002388 (2015). Gebauer, N. et al. TP53 mutations are frequent events in double-hit B-cell lymphomas with MYC and BCL2 but not MYC and BCL6 translocations. Leuk. Lymphoma . 56 , 179–185. 10.3109/10428194.2014.907896 (2015). Deng, M. et al. Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents. Mol. Cancer Res. 19 , 249–260. 10.1158/1541-7786.MCR-20-0466 (2021). Bernard, E. et al. Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes. Nat. Med. 26 , 1549–1556. 10.1038/s41591-020-1008-z (2020). Azizian, N. G. & Li, Y. XPO1-dependent nuclear export as a target for cancer therapy. J. Hematol. Oncol. 13 , 61. 10.1186/s13045-020-00903-4 (2020). Bahlis, N. J. et al. Selinexor plus low-dose bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma. Blood 132 , 2546–2554. 10.1182/blood-2018-06-858852 (2018). Vercruysse, T. et al. The Second-Generation Exportin-1 Inhibitor KPT-8602 Demonstrates Potent Activity against Acute Lymphoblastic Leukemia. Clin. Cancer Res. 23 , 2528–2541. 10.1158/1078-0432.CCR-16-1580 (2017). Kuruvilla, J. et al. Selective inhibition of nuclear export with selinexor in patients with non-Hodgkin lymphoma. Blood 129 , 3175–3183. 10.1182/blood-2016-11-750174 (2017). Gravina, G. L. et al. Nucleo-cytoplasmic transport as a therapeutic target of cancer. J. Hematol. Oncol. 7 , 85. 10.1186/s13045-014-0085-1 (2014). Chari, A. et al. Oral Selinexor-Dexamethasone for Triple-Class Refractory Multiple Myeloma. N Engl. J. Med. 381 , 727–738. 10.1056/NEJMoa1903455 (2019). Wang, S. et al. Combining selective inhibitors of nuclear export (SINEs) with chimeric antigen receptor (CAR) T cells for CD19–positive malignancies. Oncol. Rep. 46 10.3892/or.2021.8121 (2021). Boyd, M. T., Vlatkovic, N. & Rubbi, C. P. The nucleolus directly regulates p53 export and degradation. J. Cell. Biol. 194 , 689–703. 10.1083/jcb.201105143 (2011). Moll, U. M., LaQuaglia, M., Benard, J. & Riou, G. Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc. Natl. Acad. Sci. U S A . 92 , 4407–4411. 10.1073/pnas.92.10.4407 (1995). O'Brate, A. & Giannakakou, P. The importance of p53 location: nuclear or cytoplasmic zip code? Drug Resist. Updat . 6 , 313–322. 10.1016/j.drup.2003.10.004 (2003). Xue, B. et al. CD19 CAR-T treatment shows limited efficacy in r/r DLBCL with double expression and TP53 alterations. Cytotherapy 10.1016/j.jcyt.2024.07.011 (2024). Shouval, R. et al. Impact of TP53 Genomic Alterations in Large B-Cell Lymphoma Treated With CD19-Chimeric Antigen Receptor T-Cell Therapy. J. Clin. Oncol. 40 , 369–381. 10.1200/JCO.21.02143 (2022). Nguyen, R. et al. Combining selinexor with alisertib to target the p53 pathway in neuroblastoma. Neoplasia 26 , 100776. 10.1016/j.neo.2022.100776 (2022). Deng, M. et al. The synergy of the XPO1 inhibitors combined with the BET inhibitor INCB057643 in high-grade B-cell lymphoma via downregulation of MYC expression. Sci. Rep. 13 , 18554. 10.1038/s41598-023-45721-z (2023). Fang, Y. et al. Human endogenous retroviruses as epigenetic therapeutic targets in TP53-mutated diffuse large B-cell lymphoma. Signal. Transduct. Target. Ther. 8 , 381. 10.1038/s41392-023-01626-x (2023). Stock, S. et al. Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients. Int. J. Cancer . 145 , 1312–1324. 10.1002/ijc.32201 (2019). Schubert, M. L., Hoffmann, J. M., Dreger, P., Muller-Tidow, C. & Schmitt, M. Chimeric antigen receptor transduced T cells: Tuning up for the next generation. Int. J. Cancer . 142 , 1738–1747. 10.1002/ijc.31147 (2018). Gargett, T. et al. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade. Mol. Ther. 24 , 1135–1149. 10.1038/mt.2016.63 (2016). Cao, Y. et al. Anti-CD19 Chimeric Antigen Receptor T Cells in Combination With Nivolumab Are Safe and Effective Against Relapsed/Refractory B-Cell Non-hodgkin Lymphoma. Front. Oncol. 9 , 767. 10.3389/fonc.2019.00767 (2019). Yoo, H. J. et al. Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies. Int. J. Mol. Sci. 20 10.3390/ijms20102469 (2019). Gauthier, J. et al. Feasibility and efficacy of CD19-targeted CAR T cells with concurrent ibrutinib for CLL after ibrutinib failure. Blood 135 , 1650–1660. 10.1182/blood.2019002936 (2020). Fraietta, J. A. et al. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia. Blood 127 , 1117–1127. 10.1182/blood-2015-11-679134 (2016). Ruella, M. et al. Kinase inhibitor ibrutinib to prevent cytokine-release syndrome after anti-CD19 chimeric antigen receptor T cells for B-cell neoplasms. Leukemia 31 , 246–248. 10.1038/leu.2016.262 (2017). Yoshimura, M. et al. Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma. Cancer Sci. 105 , 795–801. 10.1111/cas.12430 (2014). Additional Declarations No competing interests reported. Supplementary Files Fig5ACSupplementary.pdf WB.zip file.pdf informedconsent.pdf 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5946503","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":445345854,"identity":"f2c6a9e2-e9e9-4932-a1a6-79c875184cbb","order_by":0,"name":"Zhimin Bai","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Zhimin","middleName":"","lastName":"Bai","suffix":""},{"id":445345855,"identity":"8a0945c1-a493-4b5c-af0b-3389d2383f29","order_by":1,"name":"Xiaoyu Huang","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Huang","suffix":""},{"id":445345856,"identity":"73731686-1fd2-40b6-9080-2b7a3f9ae6a2","order_by":2,"name":"Xinfeng Wang","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Xinfeng","middleName":"","lastName":"Wang","suffix":""},{"id":445345857,"identity":"c62ab686-e922-4758-81d3-8a586f13d16d","order_by":3,"name":"Yong Zhou","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhou","suffix":""},{"id":445345858,"identity":"938c9426-8640-4525-9334-98105fbe7f46","order_by":4,"name":"Zenghua Lin","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Zenghua","middleName":"","lastName":"Lin","suffix":""},{"id":445345859,"identity":"4da47da3-d1c2-4209-97fc-7b5e54397768","order_by":5,"name":"Hong Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACAxDxwIBBTp69sfHBB6K1JBgwGBv2HG42nEG8FgaGRIYb6W3SHMRoMWfvPfwioeBwAuPMhw3SDAx2croNBLRY9pxLs0gwOJzHLp3YYFzAkGxsdoCQw27kmBkAtRQzzk5sSJ7BcCBxG7FaEhtuHmw4zEOkFuMHYC03GBubidNy5owZMJDTgYGc2Mw4w4AYvxzvMf7w4Y81MCqPP//xocJOjqAWIGCTQDKBsHIQYCYqmYyCUTAKRsEIBgADyUfjvxIQEAAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-02-02 15:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5946503/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5946503/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81060873,"identity":"b4d5eca4-7e6c-4838-9c53-2ef1ff0b51fe","added_by":"auto","created_at":"2025-04-21 18:55:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6797785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelated research of XPO1 in DLBCL. \u003c/strong\u003eA. Expression of XPO1 in tumors and normal tissues in the pan-cancer data of The Cancer Genome Atlas (TCGA). B. Correlation between XPO1 expression and prognosis of DLBCL was analyzed using the GEO database. C. Correlation between exportin-1 (XPO1) and mut-p53 was analyzed by relative PCR levels in clinical patients.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/60d210fa4ff02e5462606a16.png"},{"id":81060876,"identity":"cf52dd6f-75a8-41b2-a6a7-e2f3b76af3ee","added_by":"auto","created_at":"2025-04-21 18:55:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1334253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between XPO1 and mut-TP53 was verified at the transcriptional and translational levels.\u003c/strong\u003e A. Expression levels of XPO1 and mut-TP53 after plasmid transfection of OCI-LY19 cells. B. Expression levels of XPO1 and mut-TP53 after small interfering RNA transfection of OCI-LY19 cells. C. CCK8 assayed the inhibitory effect of XPO1 inhibitor KPT-330 on OCI-LY19 at different drug concentrations. The curve was drawn to calculate the IC50 (82.40Nm). D. Expression levels of XPO1 and mut-TP53 after plasmid-transfected OCI-LY19 cells were treated with XPO1 inhibitor KPT-330. Data are shown as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/71c61fcb57fab009857cc080.png"},{"id":81061680,"identity":"48ab7b56-924a-4d84-b78e-b9d146fad1b7","added_by":"auto","created_at":"2025-04-21 19:19:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5329277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of XPO1 expression level on proliferation, migration and apoptosis of OCI-LY19 cells. \u003c/strong\u003eA. CCK-8 assay was used to detect the proliferation ability of OCI-LY19 cells transfected with plasmids to overexpress XPO1, transfected with si-RNA to knock down XPO1, and treated with the inhibitor KPT-330. B. Migration assay was used to determine the number of OCI-LY19 cells that migrated to the lower chamber under different treatments. C. Flow cytometry was used to detect cell apoptosis after different treatments. Data are shown as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/3777e8ee26fa38d49ec1c3cc.png"},{"id":81060974,"identity":"d3516a3c-4eb2-4e39-9820-c3bc6a7b9e97","added_by":"auto","created_at":"2025-04-21 19:03:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7547885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation and enrichment analysis of XPO1.\u003c/strong\u003e A. The top 10 genes in the TCGA volcano plot (ranked by correlation) were positively correlated with XPO1. B. Gene ontology analysis showed that the most important gene ontology terms most positively correlated with XPO1 included biological process, cellular component, and molecular function.C. Display of genes corresponding to the first three KEGG pathways with the smallest p-value. D. Display diagram of genes corresponding to KEGG pathway. KEGG, Kyoto encyclopedia of genes and genomes; TCGA, the cancer genome atlas.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/feacaa3ec2908fb69461f8fc.png"},{"id":81061488,"identity":"5d9f2564-a877-44bc-bcf3-a121313817b0","added_by":"auto","created_at":"2025-04-21 19:11:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3621914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXPO1 expression level and the effect of XPO1 inhibitor (KPT-330) on p53 and signal transduction pathways.\u003c/strong\u003e A. Protein imprints of XPO1 and P53 after plasmid and si-RNA transfection of OCI-LY19 cells. B. Changes of XPO1 and P53 proteins over time after plasmid-transfected OCI-LY19 cells were treated with XPO1 inhibitor KPT-330. C. Protein imprints in the PI3K/AKT pathway after KPT-330 and si-XPO1 transfection. Data are shown as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/30fcd55a36c599907568da1d.png"},{"id":81061490,"identity":"095b094c-85c4-4499-a2e7-708987d3aaec","added_by":"auto","created_at":"2025-04-21 19:11:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3160161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePretreatment of tumor cells with XPO1 inhibitor (KPT-330) improved the killing ability of CD19 CAR T cells. \u003c/strong\u003eA. CCK8 was used to determine the killing rate of OCI-LY19 by KPT-330 combined with CAR-T at different E/T ratios (0.5:1, 1:1, 2:1). B. CD19CAR-T and target cells were co-incubated at E/T ratios (1:1) for 24 hours, and the cell apoptosis efficiency histogram was detected by flow cytometry. C. CD19CAR-T cells produce higher levels of cytokines. CAR-T cells and tumor cells were incubated together for 12 hours; Cytokine levels in the culture supernatant were determined by ELISA.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/6f0706d6c467c69fd2207206.png"},{"id":84295960,"identity":"ab0831d2-4443-4d34-98bf-39e4c31c5d80","added_by":"auto","created_at":"2025-06-10 09:32:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26983357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/77cd53fa-0fb8-4826-82c6-fe06b4572b1c.pdf"},{"id":81061679,"identity":"e977f6ea-2196-4ee2-bbd2-152aefdaa1e0","added_by":"auto","created_at":"2025-04-21 19:19:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":498432,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5ACSupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/64c9eb23969165cf89c30b4e.pdf"},{"id":81060979,"identity":"f43aa90f-f9ed-4590-b9f6-b6bef2d25755","added_by":"auto","created_at":"2025-04-21 19:03:38","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9721791,"visible":true,"origin":"","legend":"","description":"","filename":"WB.zip","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/654f23ee4175bfb1eb1edc31.zip"},{"id":81060969,"identity":"2b86d58e-fe8b-4848-ae4c-acd762fb2abc","added_by":"auto","created_at":"2025-04-21 19:03:37","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":398323,"visible":true,"origin":"","legend":"","description":"","filename":"file.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/f4dc6eeb4d84333cb48c8d41.pdf"},{"id":81060878,"identity":"1bf01713-853f-42c6-a4cc-0df0407ba016","added_by":"auto","created_at":"2025-04-21 18:55:37","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":95136,"visible":true,"origin":"","legend":"","description":"","filename":"informedconsent.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5946503/v1/5bf2645dbc4a0d11c6a9d4ac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic effects of XPO1 inhibitors combined with CD19 CAR-T cells in TP53-mutated DLBCL","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous type of aggressive non-Hodgkin lymphoma (NHL). About 60% of patients reported a complete and sustained response with traditional chemotherapy with rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). However, up to 40% of patients develop refractory or recurrent (R/R) after treatment\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the most common cause of which is gene mutation. Approximately 20\u0026ndash;30% of DLBCL have TP53 mutations\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, which leads to a worse prognosis of DLBCL\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. TP53 is a tumor suppressor gene that plays an important role in cell cycle and proliferation. One of its main functions is to induce apoptosis in cells induced by DNA damage. TP53 gene mutations can destroy genetic stability and lead to uncontrolled proliferation of cancer cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The role of TP53 mutations as a negative prognostic factor has been well established in many malignancies, including DLBCL. Therefore, the search for other effective treatment strategies is an unmet medical need for DLBCL patients with TP53 mutations.\u003c/p\u003e \u003cp\u003eXPO1 (exportin 1), also termed chromosome region maintenance 1 (CRM1), is a nuclear export receptor involved in the transportation of proteins such as histones, polymerases, transcription factors and/or RNA from the nucleus into the cytosol and responsible for the nuclear-cytoplasmic transport and cellular homeostasis of up to 220 cargoes, including the tumor suppressors p53 and I-κB\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. XPO1 over-expression has been observed in many malignancies, and elevated XPO1 levels have been associated with poor clinical prognosis\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. Thus, down-regulation of XPO1 constitutes an interesting therapeutic strategy. Notably, inhibiting XPO1 by selective inhibitors of nuclear export (SINEs) has been demonstrated in hematological malignancies including multiple myeloma (MM), ALL, NHL, acute myeloid leukemia (AML). The XPO1 inhibitor selinexor was approved for the treatment of adults with r/r MM by the U.S. Food and Drug Administration (FDA) in September 2019, and by the European Medicines Agency (EMA) in December 2019. Moreover, selinexor is currently under clinical evaluation for treatment of DLBCL, r/r AML as well as myelodysplastic syndrome (MDS)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Researches have found that p53 and XPO1 are inextricably linked. In some human tumor cells, wild-type p53 has been reported to be abnormally isolated in the cytoplasm. Nuclear export of p53 is mediated by both MDM2 and XPO1. MDM2 activates nuclear export signaling (NES) in p53 through its E3 ubiquitin ligase activity, causing conformational changes in p53 that expose the NES domain of p53. After ubiquitination, XPO1 recognizes p53\u0026rsquo;s NES and exports the protein from the nucleus to the cytoplasm, thereby limiting p53-mediated transcriptional activity and inhibiting the ability of p53 to trigger apoptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA promising treatment for relapsed/refractory (r/r) DLBCL patients is chimeric antigen receptor (CAR) T-cell therapy, which modifies the natural course of chemorefractory DLBCL. In the laboratory, T cells are extracted from the patient's blood, equipped with so-called CARs to help to recognize and destroy DLBCL cells, and then delivered back into the patient's blood. However, 25\u0026ndash;50% of patients with r/r DLBCL still do not achieve remission after CD19 CAR-T therapy and survive for less than one year\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Many factors influencing the poor prognosis of DLBCL have been published in the literature, among which TP53 alterations and double expression are the most factors associated with poor prognosis of the r/r DLBCL patients and have a lower likelihood of achieving complete remission after treatment with CD19 CAR-T\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that XPO1 blocking promotes p53 nuclear retention and can have a powerful anti-lymphoma effect on HGBCL-DH cells with or without TP53 mutations\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.The present study investigated the effectiveness of combining CD19-CAR-T cells with the XPO1 inhibitor KPT-330 administration. The results of this study suggest that this combinatorial strategy is superior to CAR-T monotherapy against DLBCL.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Cell lines and primary samples\u003c/h2\u003e \u003cp\u003eThe DLBCL cell lines OCI-Ly19 was generously gifted by Dr. GQ. Song (Affiliated Hospital of Nantong University, Jiangsu, China). OCI-Ly19 cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% heat-inactivated fetal bovine serum (Gibco), 100U/ml penicillin and 100ug/ml streptomycin (NCM Biotech) at 37℃ in a 5% CO2 incubator. The medium was changed every 2\u0026ndash;3 days to maintain the cell density between 2\u0026times;10^5 and 2\u0026times;10^6 cells/ml.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. CAR-T cells generation and manufacturing\u003c/h3\u003e\n\u003cp\u003ePeripheral blood lymphocytes were obtained and collected from patients by density gradient separation. T lymphocytes were further sorted with anti-CD3 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and activated with 5 ug/mL monoclonal anti-CD3/CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for 48 h. Then T cells were transfected with a lentivirus encoding CD19/22-4-1BB-CD3z transgene, and the T cells were treated in AIM-V medium supplemented with 10% autologous serum, 100 IU/ml IL-2, 5 ng/ml IL-7, and 5 ng/ml IL-15 (Gibco, New York, New York, USA) for 12 to 20 days until the number of cells reached the preset value. All of these CAR-T products were provided by the Taihe Chunyu biotechnology co. (Hebei, China). The study protocol was conducted in accordance with the amended Declaration of Helsinki and was approved by the Ethics Committee of Affiliated Hospital of Nantong University (2025-L092). All patients gave informed written consent for participation in the study.\u003c/p\u003e\n\u003ch3\u003e3. Flow cytometry\u003c/h3\u003e\n\u003cp\u003eThe treated OCI-LY19 cells were washed twice with PBS, and stained with the AnnexinV-PE/7AAD double staining kit (BD Biosciences) for 15\u0026ndash;20 min. After incubation in the dark for 15 minutes, 1X Binding Buffer was added, and the cells were resuspended and detected by flow cytometry (BD FACSCalibur). The proportion of early and late apoptotic cells was analyzed using FlowJoVX software.\u003c/p\u003e\n\u003ch3\u003e4. siRNA and plasmid transfection\u003c/h3\u003e\n\u003cp\u003esiRNA and the plasmid vector (pCMV) were purchased from Genepharma (Shanghai, China) and carried the target gene. The transfection steps were as follows: OCI-LY19 cells were seeded in a 6-well plate at a density of 5\u0026times;10^5 cells/mL, siRNA transfection was performed according to the steps of the Ribo kit, and the overexpression plasmid was transfected using Lipofectamine3000 reagent (Invitrogen, USA). After 48 hours, the cells were extracted for subsequent experimental analysis. The siRNA sequences were:\u003c/p\u003e \u003cp\u003eXPO1-homo-3285: forward primer, GGCUGUCAAUUCUCAUUGUTT.\u003c/p\u003e \u003cp\u003eReverse primer, ACAAUGAGAAUUGACAGCCTT.\u003c/p\u003e \u003cp\u003eXPO1-homi-1854: forward primer, GGCUGCUGAACUCUAUAGATT.\u003c/p\u003e \u003cp\u003eReverse primer, UCUAUAGAGUUCAGCAGCCTT.\u003c/p\u003e\n\u003ch3\u003e5. Determination of IC50 and cytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eKPT-330 (Selinexor) (Selleck, USA) OCI-LY19 cells were seeded in 96-well plates at 1\u0026times;10^5 cells/well and cultured for 48 hours using medium containing different concentrations (concentration range:0.00nM to 400nM) of KPT-330 (Selleck, USA). After culture, 10ul CCK-8 reagent (Dojindo) was added to each well and incubated for another 2 hours. The optical density (OD450) was measured at a wavelength of 450nm using a microplate reader (Bio-Rad). The half inhibitory concentration (IC50) was calculated based on the measured data to evaluate the cytotoxicity of KPT-330. The target cells, OCI-Ly19, were co-cultured with CAR-T cells at different E/T ratio (0.5:1, 1:1, 2:1). CCK8 was added to each well at different time points (0h.12h.24h.48h.72h), and the optical density (OD) (BioTek) was measured after 2 hours. The OD450 value was measured by a microplate reader to calculate the survival rate of tumor cells and the killing rate of CAR-T cells. The killing rate was calculated by the following equation: [1-(OD of the effector target cell well)-(OD of the effector cell well)/OD of the target cell well].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6. Cell invasion assay\u003c/h2\u003e \u003cp\u003eThe cell invasion assay was performed using a Transwell chamber (Corning). The pre-treated OCI-LY19 cell suspension (2\u0026times;10^5 cells/100\u0026micro;L) was added to the upper chamber, and RPMI-1640 medium containing 20% fetal bovine serum was added to the lower chamber, and incubated at 37\u0026deg;C for 24 hours. After the incubation, the non-invaded cells in the upper chamber were removed, and the lower chamber was photographed under an optical microscope. At the same time, the cells in the lower chamber were collected and counted using a cell counting plate, and the experiment was repeated 3 times.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e7. RT-qPCR detection of mRNA transcription levels\u003c/h3\u003e\n\u003cp\u003eOCI-LY19 cells were treated with KPT-330 (82.4 nmol) for OCI-LY19 cells, alone or in combination for 24h. Total RNA was extracted using TRIzol reagent (Invitrogen) as directed by the manufacturer. cDNA was then synthesized by reverse transcription using PrimeScript RT Reagent Kit (Takara, Dalian, China). qPCR SYBR Green PCR Master Mix (Vazyme) was used for fast and real-time quantification in TOUCH DEEP WELL with CFX96 PCR system (Bio-rad,USA). The relative expression of each gene was calculated using the 2^-ΔΔCt method. GAPDH was the internal reference gene, and mut-P53 and XPO1 were the target genes. The primer sequences were as follows:\u003c/p\u003e \u003cp\u003eXPO1: forward primer, TCTCATTGTTTCCCAGCATTCCTTG.\u003c/p\u003e \u003cp\u003ereverse primer: TAAGCCCGTATCTGCGACATTCC.\u003c/p\u003e\n\u003ch3\u003e8. Western Blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e8. Western Blot\u003c/div\u003e \u003cp\u003eCells were collected 72 hours after transfection, and proteins in the cells were isolated using RIPA lysis buffer (Beyotime), and protein concentrations were determined using the BCA assay (Vazyme). Next, proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane (Beyotime). Then, the membranes were blocked with a rapid blocking solution for 30 minutes at room temperature. Primary antibodies were incubated according to standard procedures, and incubated overnight at 4\u0026deg;C, and appropriate HRP-conjugated secondary antibodies were incubated. After washing, the membranes were developed using a luminescence imaging system (Tanon5200).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e9. CFDA-SE cell proliferation\u003c/h2\u003e \u003cp\u003eCFDA-SE (Invitrogen) dye was used to detect CAR-T cell proliferation. CAR-T cells were stained with CFDA-SE, and CAR-T cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/tube) were stained with CSFE (Beyotime, China) at 37\u0026deg;C. After washing, the same number of target cells were co-cultured with CAR-T cells, and CAR-T cells were collected after 5 days, and fluorescence was detected by flow cytometry. The changes in fluorescence intensity at different time points were detected by flow cytometry to analyze cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e10. Cytokine analysis\u003c/h2\u003e \u003cp\u003eCAR-T cells and OCI-LY19 cells were cultured at a 1:1 effector-target ratio for 24 hours, and the culture medium was collected by centrifugation. The levels of inflammatory factors (IL-2, IL-4, IL-6, IL-10, TNF-α, TNF-β) were quantitatively detected using an ELISA kit (eBioscience). Sample incubation, enzyme-labeled secondary antibody reaction, and color development were performed according to the kit instructions, and the absorbance was finally measured at a wavelength of 450 nm using an ELISA kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e11. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with GraphPad Prism 6 (GraphPad Software Inc.). P‑values were calculated using the parametric two‑way t‑test between two groups, and the one‑way analysis of variance (ANOVA) with Bonferroni's multiple comparison test for three or four groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. When not otherwise indicated, results were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). IC50s were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Graphs and tables were designed using GraphPad Prism 6.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e1. XPO1 expression levels in pan-cancer: Unpaired comparisons of the levels of XPO1 expression levels between tumor tissues from TCGA database and normal tissues from TCGA database (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns\u0026thinsp;=\u0026thinsp;no significance).\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eReported studies have reported increased expression of XPO1 in a variety of cancers. We use the TCGA pan-cancer database to evaluate the expression of XPO1 mRNA in various tumors. The analysis results suggest that XPO1 is highly expressed in 21 types of tumors, including adrenocortical carcinoma (ACC), bladder cancer (BLCA), breast cancer (BRCA), cervical cancer (CESC), cholangiocarcinoma (CHOL), and colon adenocarcinoma (COAD), diffuse large B-cell lymphoma (DLBC), esophageal cancer (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), renal chromophobe cell carcinoma (KICH), renal papillary cell cancer (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), rectal adenocarcinoma (READ), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), and thymoma (THYM) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, XPO1 was highly expressed in diffuse large B-cell lymphoma in TCGA unpaired samples \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. At the same time, we used the GEO database to analyze the correlation between XPO1 expression and DLBCL prognosis, and found that high XPO1 expression was significantly associated with poor prognosis and shortened overall survival in DLBCL patients \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. These indicate that XPO1 may play an important role in the development of this tumor and deserves further study.\u003c/p\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2. Correlation between XPO1 and mut-TP53 expression in DLBCL patients\u003c/h2\u003e\n \u003cp\u003eThe mutation rate of TP53 in DLBCL is 10%-20%\u003csup\u003e23\u003c/sup\u003e. Abnormal regulation of the TP53 pathway can make the DLBCL genome unstable, leading to resistance to the chemotherapy regimens and disease progression. Therefore, we further studied the correlation between XPO1 and mut-TP53. We collected peripheral blood form 61 patients with DLBCL in the Department of Hematology, Affiliated Hospital of Nantong University in the past 3 years for experimental analysis. We analyzed the relative expression levels of XPO1 and mut-TP53 in clinical patients by Polymerase Chain Reaction (PCR), and analyzed the correlation between XPO1 and mut-TP53. The results showed that the expression of XPO1 was closely related to the expression of mut-TP53, and the expression of mut-TP53 was positively correlated with the expression of XPO1 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. This shows that in DLBCL, a high mutation rate of TP53 will affect the high expression of XPO1, leading to poor prognosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3. Correlation between XPO1 and mut-TP53 expression in OCI-LY19 cells and the role of inhibitor KPT-330\u003c/h2\u003e\n \u003cp\u003eWe used XPO1 overexpression plasmid HE and si-RNA in OCI-LY cells to study the correlation between XPO1 expression level and mut-TP53 in diffuse large B-cell lymphoma cells. Fluorescence quantitative PCR detection showed that when XPO1 was highly expressed, it was positively correlated with mut-TP53 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e, and when XPO1 was lowly expressed, it was negatively correlated with mut-TP53 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. We set up a concentration gradient of KPT-330 to treat OCI-LY19 for 48 hours, calculated the half-inhibitory concentration by CCK8 detection, and used KPT-330 at a concentration of 82.4nm to kill tumor cells \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. The results showed that the use of XPO1 inhibitor KPT-330 in OCI-LY19 cells inhibited XPO1 expression, and mut-TP53 decreased accordingly, and the correlation decreased \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e4. KPT‑330 treatment reduces XPO1 levels and inhibits OCI-LY19 cell growth in vitro\u003c/h2\u003e\n \u003cp\u003eXPO1 is highly expressed after plasmid transfection, XPO1 is low-expressed after siRNA transfection, and XPO1 is highly expressed and treated with KPT-330. After culturing the cells for 24h, 48h and 72h, CCK-8 experiments showed that over-expression of XPO1 significantly promoted the proliferation of DLBCL cells, low expression of XPO1 inhibited the proliferation of tumor cells, and KPT-330 inhibited the over-expression of Proliferation of OCI-LY19 cells by XPO1 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. Transwell is used to detect the migration ability of OCI-LY19. Compared with the control group, when XPO1 is over-expressed, the migration ability of OCI-LY19 is strong, and when XPO1 is low expressed, the migration ability is weak. However, the use of KPT-330 significantly inhibited OCI-LY19 migration ability \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. At the same time, flow cytometry analysis showed that compared with the control group, the apoptosis rate of OCI-LY19 cells with XPO1 over-expression increased, and the apoptosis rate of cells with low XPO1 expression decreased. The use of KPT-330 indicated the apoptosis rate of tumor cells increased \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. The above results suggest that high expression of XPO1 will significantly promote the proliferation of OCI-LY19 cells and reduce cell apoptosis. However, the XPO1 inhibitor KPT-330 can inhibit the proliferation of tumor cells that highly express XPO1 and induce tumor cell apoptosis. KPT-330 inhibited the proliferation of OCI-LY19 cells over-expressing XPO1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e5. Correlation and enrichment analysis of XPO1\u003c/h2\u003e\n \u003cp\u003eTo further clarify the role of XPO1 in DLBCL, we analyzed the genes that were positively correlated with XPO1 expression in the TCGA database. We selected genes associated with XPO1 for enrichment analysis and displayed the top 30 genes (ranked by correlation) in a volcano plot \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. At the same time, the gene ontology (GO) enrichment and KEGG pathways analysis were performed on the top 30 genes associated with XPO1. GO BP analysis revealed that the associated genes were markedly enriched in regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-templated and protein phosphorylation. The top three significantly enriched CC terms included nucleoplasm, cytosol and nucleus. For GO MF analysis, the top significantly enriched term was the metal ion binding \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. Furthermore, and the gene display map corresponding to the KEGG pathway showed that XPO1, as an important transporter, is involved in regulating the transport of intracellular proteins and is essential for biological processes such as cell cycle and cellular gene expression. KEGG pathways analysis suggested that XPO1 causes nuclear retention and activation of tumor suppressor proteins and other growth regulatory proteins, while downregulating the levels of multiple tumor suppressor proteins in the cytoplasm through the PI3K-AKT pathway \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e6. XPO1 expression levels and the effect of KPT-330 on P53\u003c/h2\u003e\n \u003cp\u003eP53 is a carrier protein of XPO1, so we used western blot to analyze the changes of P53 protein levels to clarify the status of P53 under the treatment of KPT-330. The results showed that when XPO1 was highly expressed, the protein expression level of P53 decreased, and conversely, when it was lowly expressed, the expression level of P53 increased \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. When the XPO1 inhibitor KPT-330 was used, XPO1 was gradually inhibited as the inhibitor action time increased, and the expression level of P53 protein increased accordingly \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. The above suggests that XPO1 inhibitor KPT-330 promotes the accumulation of P53 in cells and reduces its degradation rate, activating the function of P53 to exert anti-cancer effects.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e7. Western blot analysis of the effect of XPO1 expression on the PI3K-AKT pathway\u003c/h2\u003e\n \u003cp\u003eThe PI3K-AKT pathway is a key pathway regulating cell proliferation and apoptosis, and is closely related to the occurrence, proliferation, transformation, apoptosis, and drug resistance of tumors. We performed KEGG enrichment analysis on the up-regulated genes that were positively correlated with XPO1, and found that XPO1-related genes were significantly correlated with the PI3K-AKT pathway. Therefore, we further investigated whether XPO1 is involved in regulating the PI3K-AKT pathway. We used western blot to detect that when si-RNA was transfected to reduce the expression of XPO1 and the XPO1 inhibitor KPT-330 was used, the protein levels of p-PI3K and P-AKT decreased, indicating that when XPO1 was inhibited, the activation of the PI3K-AKT pathway was inhibited \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. The inhibition of the PI3K-AKT pathway can also reduce the degradation of P53.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e8. Killing rate and cytokine release levels of CAR T cells treated with KPT-330\u003c/h2\u003e\n \u003cp\u003eWe evaluated the killing ability of CAR-T cell treated with KPT-330. We treated with tumor cells with the half maximal inhibitory concentration (IC50) of KPT-330, via CCK8 killing experiment to detect the killing of KPT-330 combined with CAR-T cells on OCI-LY19. The efficacy target ratio and co-culture time were selected, and finally we chose 24 hours of co-culture and 1:1 efficacy target ratio for subsequent experiments \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. And the results also showed that the killing rate of tumor cells was higher after pretreatment with KPT-330 and combined with CAR-T cells. We detected by flow cytometry that the use of KPT-330 treatment combined with CAR-T increased the apoptosis rate of tumors \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. To investigate the effector function of KPT-330 combined with CAR-T, a group of cytokines were detected in the in vitro cytotoxicity assay. Compared with the control group, KPT-330 combined with CD19CAR-T displayed higher cytokine secretion levels when co-cultured with OCI-LY19. We could observe increased secretion of effector cytokines and chemokines such as interferon-\u0026gamma; (IFN-\u0026gamma;), tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), and interleukin-2 (IL-2) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eP53 is a well-known transcription factor that participates in a variety of cellular processes including apoptosis, cell cycle, and DNA damage response, and plays an anti-cancer role in the cell nucleus. TP53 mutation is a poor prognostic factor for diffuse large B-cell lymphoma. Clinical studies have shown that salvage therapy and ASCT have limited efficacy in patients with TP53 mutations and have failed to improve the prognosis of patients with TP53 mutations. In patients with lymphoid malignancies, although CD19 CAR-T cell therapy is more effective than other second-line therapies or new drugs, relapse and resistance after CAR-T cell therapy still pose a challenge. Patients with TP53 mutations are less likely to achieve complete remission after CD19 CAR-T therapy, which still limits and hinders the efficacy and long-term survival of relapsed and refractory DLBCL\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To address this problem, several studies are being explored. In order to improve the persistence and efficacy of CAR-T cells, there are currently multiple researches to enhance the production of CAR-T cells. For example, PI3K δ inhibitors were found to enhance in vivo function of CAR-T cells made from T lymphocytes of CLL patients\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In addition, armored CAR-T cells have also been developed. Additional genetic modifications can be made to CAR-T cells to express additional costimulatory ligands or cytokines to enhance the response of CAR-T cells\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In addition, in order to reduce tumor escape during CAR-T cell therapy, a variety of studies have been discovered that can enhance the targeting of CAR-T cells to kill tumor cells. For example, the combination of CAR-T cells with PD-1/PD-L1 inhibitors can enhance the efficacy of CAR-T cells and improve the treatment effect of patients\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CAR-T cells combined with reactive oxygen species (ROS) accelerators can overcome the therapeutic resistance mediated by the tumor micro-environment\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Recent clinical studies have found that the combination of BDK inhibitors, such as ibrutinib and CAR-T cell therapy, has better clinical responses in CLL patients\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In human xenotransplantation models, the combination of ibrutinib and CAR-T cells can promote the proliferation and anti-tumor efficacy of CAR-T cells and reduce the incidence of cytokine release syndrome (CRS)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eXPO1 is the only export protein that mediates the transport of multiple proteins, including tumor suppressor, growth regulatory, and anti-apoptotic proteins as well as several mRNAs and ribosomal proteins essential for ribosome biogenesis. The nuclear export of p53 is mediated by XPO1, which recognizes p53\u0026rsquo;s NES and exports the protein from the nucleus to the cytoplasm, where it is unable to perform transcriptional activity to regulate cell fate\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Due to XPO1 inhibitor general anti-tumor effect, we tried to combine XPO1 inhibitors (KPT-330) to improve the efficacy of CAR-T cell therapy. In the present study, we observed that the XPO1 inhibitor (KPT-330), synergized with CAR-T to reduce the viability of DLBCL cells and enhance the killing effect of CAR-T cells.\u003c/p\u003e \u003cp\u003eIn the present study, we found that XPO1 is highly expressed in diffuse large B-cell lymphoma through TCGA database analysis. At the same time, we used the GEO database to analyze the correlation between XPO1 expression and the prognosis of DLBCL, and found that high XPO1 expression was significantly related with poor prognosis in DLBCL patients. Since XOP1 is the nuclear export protein of P53, it can transport P53 from the nucleus to the cytoplasm, thereby losing its transcriptional activity and being unable to regulate cell functions. Therefore, we envision combining XOP1 inhibitors for diffuse large B-cell lymphoma with P53 mutations. KPT330 inhibits the expression of XPO1 and reduces the nuclear export of p53, thereby participating in regulating various cellular processes such as apoptosis, cell cycle and DNA damage response, exerting anti-cancer effects in the nucleus, and enhancing the killing effect of CAR-T on DLBCL. We applied the XPO1 inhibitor KPT-330 to DLBCL cell lines, and detected the decrease in XPO1 expression through fluorescence quantitative PCR, and the expression of mut-P53 also decreased, indicating that high expression of XPO1 is positively correlated with mut-P53. Application of the XPO1 inhibitor can reduce the expression of mut-P53. Simultaneous application of the XPO1 inhibitor KPT-330 can inhibit the proliferation of DLBCL tumor cells that highly express XPO1 and induce their apoptosis. We also conducted in-depth research on the mechanism by which XPO1 inhibitors inhibit DLBCL cell proliferation and induce and promote tumor cell apoptosis. Through the TCGA database, we screened out differential genes that were positively correlated with XPO1, and performed GO and KEGG enrichment analysis on the differential genes. The results show that XPO1, as an important transporter, participates in mediating intracellular protein transport and is crucial to biological processes such as cell cycle and cellular gene expression. At the same time, there is a clear correlation between XPO1-related genes and the PI3K-AKT pathway. Inhibiting the expression of XPO1 can inhibit the activation of the PI3K-AKT pathway and simultaneously down-regulate the levels of multiple tumor suppressor proteins in the cytoplasm. In addition, for potential combination methods, we chose KPT-330 with a half-inhibitory concentration of 82.4 nmol to treat tumor cells, so that KPT-330 and CAR-T cells can achieve the best synergy, which should be able to effectively inhibit target tumor cells without affecting CAR-T cells. Our research data showed that pretreatment with KPT-330 enhanced the anti-tumor cytotoxicity of CAR-T cells and promoted tumor apoptosis. At the same time, the combined application of KPT-330 also increased the cytotoxicity of CAR-T cells and enhanced the cytokine release of CAR-T cells. Therefore, presensitizing tumor cells with KPT-330 may presensitize the tumor cells (without killing them completely) in preparation for treatment with CAR-T cells. All in all, this finding has clinical promise and supports the combined application of KPT-330 for TP53-mutated DLBCL, which may inhibit tumor proliferation by inhibiting the activation of the PI3K-AKT pathway, thereby enhancing the killing function of CAR-T cells.\u003c/p\u003e \u003cp\u003eIn summary, this study shows that the XPO1 inhibitor KPT-330 can overcome the resistance of TP53-mutated DLBCL to CAR-T therapy. The enhancement of the anti-tumor function of CAR-T cells is attributed to the fact that KPT-330 decreases the expression of XPO1 and inhibits the activation of the PI3K-AKT pathway, thereby enhancing the killing function of CAR-T cells and also enhancing the biological functions of CAR-T cells, significantly. It significantly improves the therapeutic effect of TP53-mutated DLBCL and provides new ideas and directions for future clinical research and practice of CAR-T.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment in this study was approved by the Ethical and Welfare Committee of the\u0026nbsp;Affiliated Hospital of Nantong University\u0026nbsp;(2024-L034)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the analysis:\u0026nbsp;Bai Z, Lin Z\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollected the data:\u0026nbsp;Huang X, Wang X \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContributed data or analysis tools:\u0026nbsp;Huang X\u003c/p\u003e\n\u003cp\u003ePerformed the analysis:\u0026nbsp;Huang X, Zhou Y,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWrote the paper:\u0026nbsp;Bai Z\u003c/p\u003e\n\u003cp\u003eFunding Acquisition:\u0026nbsp;Bai Z,Liu H\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest relevant to this article was not reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Jiangsu Funding Program for Excellent Postdoctoral Talent, 2022ZB894; Social and Livelihood Science and Technology Project of Nantong, MSZ2023057.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in The Cancer Genome Atlas(TCGA) database, and the survival datasets can be obtain at the GEO repository with the accession number GSE10846 and the link https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10846.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCrump, M. et al. Outcomes in refractory diffuse large B-cell lymphoma: results from the international SCHOLAR-1 study. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 1800\u0026ndash;1808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2017-03-769620\u003c/span\u003e\u003cspan address=\"10.1182/blood-2017-03-769620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao, Y., Medeiros, L. J., Li, Y., Li, J. \u0026amp; Young, K. H. Genetic alterations and their clinical implications in DLBCL. \u003cem\u003eNat. Rev. Clin. Oncol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 634\u0026ndash;652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41571-019-0225-1\u003c/span\u003e\u003cspan address=\"10.1038/s41571-019-0225-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, P. et al. [Prognostic evaluation of P53 and BCL2 proteins in MYC/BCL2 double expression DLBCL]. \u003cem\u003eZhonghua Xue Ye Xue Za Zhi\u003c/em\u003e. \u003cb\u003e40\u003c/b\u003e, 589\u0026ndash;593. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3760/cma.j.issn.0253-2727.2019.07.010\u003c/span\u003e\u003cspan address=\"10.3760/cma.j.issn.0253-2727.2019.07.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePascual, M. et al. PD-1/PD-L1 immune checkpoint and p53 loss facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e133\u003c/b\u003e, 2401\u0026ndash;2412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood.2018889931\u003c/span\u003e\u003cspan address=\"10.1182/blood.2018889931\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchiefer, A. I. et al. Impact of Single or Combined Genomic Alterations of TP53, MYC, and BCL2 on Survival of Patients With Diffuse Large B-Cell Lymphomas: A Retrospective Cohort Study. \u003cem\u003eMed. (Baltim).\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, e2388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/MD.0000000000002388\u003c/span\u003e\u003cspan address=\"10.1097/MD.0000000000002388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGebauer, N. et al. TP53 mutations are frequent events in double-hit B-cell lymphomas with MYC and BCL2 but not MYC and BCL6 translocations. \u003cem\u003eLeuk. Lymphoma\u003c/em\u003e. \u003cb\u003e56\u003c/b\u003e, 179\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/10428194.2014.907896\u003c/span\u003e\u003cspan address=\"10.3109/10428194.2014.907896\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng, M. et al. Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents. \u003cem\u003eMol. Cancer Res.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 249\u0026ndash;260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1541-7786.MCR-20-0466\u003c/span\u003e\u003cspan address=\"10.1158/1541-7786.MCR-20-0466\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernard, E. et al. Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 1549\u0026ndash;1556. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41591-020-1008-z\u003c/span\u003e\u003cspan address=\"10.1038/s41591-020-1008-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzizian, N. G. \u0026amp; Li, Y. XPO1-dependent nuclear export as a target for cancer therapy. \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-020-00903-4\u003c/span\u003e\u003cspan address=\"10.1186/s13045-020-00903-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahlis, N. J. et al. Selinexor plus low-dose bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e132\u003c/b\u003e, 2546\u0026ndash;2554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2018-06-858852\u003c/span\u003e\u003cspan address=\"10.1182/blood-2018-06-858852\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVercruysse, T. et al. The Second-Generation Exportin-1 Inhibitor KPT-8602 Demonstrates Potent Activity against Acute Lymphoblastic Leukemia. \u003cem\u003eClin. Cancer Res.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 2528\u0026ndash;2541. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.CCR-16-1580\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.CCR-16-1580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuruvilla, J. et al. Selective inhibition of nuclear export with selinexor in patients with non-Hodgkin lymphoma. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e129\u003c/b\u003e, 3175\u0026ndash;3183. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2016-11-750174\u003c/span\u003e\u003cspan address=\"10.1182/blood-2016-11-750174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGravina, G. L. et al. Nucleo-cytoplasmic transport as a therapeutic target of cancer. \u003cem\u003eJ. Hematol. Oncol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-014-0085-1\u003c/span\u003e\u003cspan address=\"10.1186/s13045-014-0085-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChari, A. et al. Oral Selinexor-Dexamethasone for Triple-Class Refractory Multiple Myeloma. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e381\u003c/b\u003e, 727\u0026ndash;738. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa1903455\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa1903455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, S. et al. Combining selective inhibitors of nuclear export (SINEs) with chimeric antigen receptor (CAR) T cells for CD19\u0026ndash;positive malignancies. \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/or.2021.8121\u003c/span\u003e\u003cspan address=\"10.3892/or.2021.8121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyd, M. T., Vlatkovic, N. \u0026amp; Rubbi, C. P. The nucleolus directly regulates p53 export and degradation. \u003cem\u003eJ. Cell. Biol.\u003c/em\u003e \u003cb\u003e194\u003c/b\u003e, 689\u0026ndash;703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1083/jcb.201105143\u003c/span\u003e\u003cspan address=\"10.1083/jcb.201105143\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoll, U. M., LaQuaglia, M., Benard, J. \u0026amp; Riou, G. Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e92\u003c/b\u003e, 4407\u0026ndash;4411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.92.10.4407\u003c/span\u003e\u003cspan address=\"10.1073/pnas.92.10.4407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Brate, A. \u0026amp; Giannakakou, P. The importance of p53 location: nuclear or cytoplasmic zip code? \u003cem\u003eDrug Resist. Updat\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 313\u0026ndash;322. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drup.2003.10.004\u003c/span\u003e\u003cspan address=\"10.1016/j.drup.2003.10.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue, B. et al. CD19 CAR-T treatment shows limited efficacy in r/r DLBCL with double expression and TP53 alterations. \u003cem\u003eCytotherapy\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2024.07.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2024.07.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShouval, R. et al. Impact of TP53 Genomic Alterations in Large B-Cell Lymphoma Treated With CD19-Chimeric Antigen Receptor T-Cell Therapy. \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 369\u0026ndash;381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.21.02143\u003c/span\u003e\u003cspan address=\"10.1200/JCO.21.02143\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, R. et al. Combining selinexor with alisertib to target the p53 pathway in neuroblastoma. \u003cem\u003eNeoplasia\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 100776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neo.2022.100776\u003c/span\u003e\u003cspan address=\"10.1016/j.neo.2022.100776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng, M. et al. The synergy of the XPO1 inhibitors combined with the BET inhibitor INCB057643 in high-grade B-cell lymphoma via downregulation of MYC expression. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 18554. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-023-45721-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-45721-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang, Y. et al. Human endogenous retroviruses as epigenetic therapeutic targets in TP53-mutated diffuse large B-cell lymphoma. \u003cem\u003eSignal. Transduct. Target. Ther.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-023-01626-x\u003c/span\u003e\u003cspan address=\"10.1038/s41392-023-01626-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStock, S. et al. Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients. \u003cem\u003eInt. J. Cancer\u003c/em\u003e. \u003cb\u003e145\u003c/b\u003e, 1312\u0026ndash;1324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijc.32201\u003c/span\u003e\u003cspan address=\"10.1002/ijc.32201\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchubert, M. L., Hoffmann, J. M., Dreger, P., Muller-Tidow, C. \u0026amp; Schmitt, M. Chimeric antigen receptor transduced T cells: Tuning up for the next generation. \u003cem\u003eInt. J. Cancer\u003c/em\u003e. \u003cb\u003e142\u003c/b\u003e, 1738\u0026ndash;1747. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijc.31147\u003c/span\u003e\u003cspan address=\"10.1002/ijc.31147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGargett, T. et al. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade. \u003cem\u003eMol. Ther.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 1135\u0026ndash;1149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/mt.2016.63\u003c/span\u003e\u003cspan address=\"10.1038/mt.2016.63\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, Y. et al. Anti-CD19 Chimeric Antigen Receptor T Cells in Combination With Nivolumab Are Safe and Effective Against Relapsed/Refractory B-Cell Non-hodgkin Lymphoma. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 767. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2019.00767\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2019.00767\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoo, H. J. et al. Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms20102469\u003c/span\u003e\u003cspan address=\"10.3390/ijms20102469\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGauthier, J. et al. Feasibility and efficacy of CD19-targeted CAR T cells with concurrent ibrutinib for CLL after ibrutinib failure. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, 1650\u0026ndash;1660. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood.2019002936\u003c/span\u003e\u003cspan address=\"10.1182/blood.2019002936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFraietta, J. A. et al. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 1117\u0026ndash;1127. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2015-11-679134\u003c/span\u003e\u003cspan address=\"10.1182/blood-2015-11-679134\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuella, M. et al. Kinase inhibitor ibrutinib to prevent cytokine-release syndrome after anti-CD19 chimeric antigen receptor T cells for B-cell neoplasms. \u003cem\u003eLeukemia\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 246\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/leu.2016.262\u003c/span\u003e\u003cspan address=\"10.1038/leu.2016.262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura, M. et al. Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma. \u003cem\u003eCancer Sci.\u003c/em\u003e \u003cb\u003e105\u003c/b\u003e, 795\u0026ndash;801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cas.12430\u003c/span\u003e\u003cspan address=\"10.1111/cas.12430\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"XPO1, DLBCL, p53, CAR-T, PI3K-AKT","lastPublishedDoi":"10.21203/rs.3.rs-5946503/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5946503/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eDiffuse large B-cell lymphoma (DLBCL) is mostly curable by chemotherapy, but p53 mutations limit the therapeutic effect of DLBCL. Although chimeric antigen receptor (CAR) T cells have made revolutionary progress in the treatment of DLBCL, p53 mutations still lead to drug resistance and/or relapse of DLBCL, affecting the prognosis of lymphoma. Therefore, the project aim to explore additional therapeutic strategies to improve the prognosis of DLBCL with p53 mutations.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eWe investigated the correlation between XPO1 and mut-P53 employing qRT-PCR, WB, CCK8 and flow cytometry. Then, we conduct XPO1 inhibitor (KPT-330) to explore the apoptotic effect on DLBCL. Through the TCGA database, there is a clear correlation between XPO1-related genes and the PI3K-AKT pathway. Finally, the effect of KPT-330 on the killing ability of CAR-T cells was evaluated by CCK8, flow cytometry and ELISA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we showed that XPO1 inhibitor (KPT-330) synergized with CAR-T to reduce the viability of DLBCL cells and enhance the killing effect of CAR-T cells. As expected, KPT-330 combined with CAR-T therapy slowed tumor growth and reduced tumor burden in DLBCL with p53 mutations. Mechanistically, XPO1 inhibitor KPT-330 can cooperate with CAR-T in the treatment of DLBCL by activating the PI3K pathway. Then, in vitro cytotoxicity assays revealed that the KPT-330 combined with CAR-T group significantly enhanced the secretion of effector cytokines IFN-γ, TNF-α, and IL-2, and activated the immune system.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe XPO1 inhibitor KPT-330 exerts anti-cancer effects through dual mechanisms (stabilizing p53 and inhibiting the PI3K-AKT pathway), providing a molecular basis for DLBCL treatment. We may provide a potential promising combination therapy for the treatment of DLBCL with p53 mutations.\u003c/p\u003e","manuscriptTitle":"Synergistic effects of XPO1 inhibitors combined with CD19 CAR-T cells in TP53-mutated DLBCL","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 18:55:32","doi":"10.21203/rs.3.rs-5946503/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":"c5122435-dba9-4fa0-9ae8-b0b45ec4a1ef","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47411805,"name":"Biological sciences/Immunology"},{"id":47411806,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-06-10T09:23:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 18:55:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5946503","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5946503","identity":"rs-5946503","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}