Role and mechanism of Pim-2 kinase inhibitor-induced immunogenic cell death in multiple myeloma

Role and mechanism of Pim-2 kinase inhibitor-induced immunogenic cell death in multiple myeloma | 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 Research Article Role and mechanism of Pim-2 kinase inhibitor-induced immunogenic cell death in multiple myeloma Zhaoyun Liu, Hongli Shen, Mengting Che, Xianghong Zhao, Hao Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5730658/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 Background : Immune dysfunction is a major component in the pathogenesis of multiple myeloma (MM), and restoring antimyeloma immunity has become a key research direction. Methods : This study demonstrates, through in vivo and in vitro experiments, whether and how Pim-2 kinase inhibitors induce immunogenic cell death in MM. Results : Pim-2 kinase inhibitors upregulated IRE1 phosphorylation and promoted XBP1 and CHOP transcription, thereby mediating endoplasmic reticulum (ER) stress in MM cells. ER stress and increased reactive oxygen species levels promoted damage-related molecular pattern expression and immunogenic cell death in MM cells. Furthermore, Pim-2 kinase inhibitor-treated MM cell lines upregulated the expression of activation molecules on the surface of dendritic cells (DCs) in patients with MM, stimulated T lymphocyte differentiation from naïve T cells to effector memory T cells, and promoted the expression of T lymphocyte functional molecules. In vivo , Pim-2 kinase inhibitors stimulated human DC maturation and activated functional T lymphocytes. Conclusion : These data contribute to our knowledge about how Pim-2 kinase inhibitors regulate antimyeloma immunity and provide justification for applying Pim-2 kinase inhibitors in MM treatment. adaptive immunity bone marrow effector memory T cells endoplasmic reticulum immunogenic cell death immunomodulators immune tolerance multiple myeloma plasma cell disease renal insufficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Multiple myeloma (MM) is a type of monoclonal plasma cell malignant proliferation that secretes numerous monoclonal immunoglobulins typical of malignant plasma cell disease, which are the main clinical manifestations of anemia, repeated infections, hypercalcemia, high viscosity syndrome, widespread bone destruction, and renal insufficiency; the incidence of MM is rapidly increasing 1 . MM has a complex pathogenesis. Along with genetic abnormalities, epigenetic abnormalities, and other factors, the change in the MM bone marrow microenvironment also plays an important role in the occurrence, development, and drug resistance of diseases 2 . Furthermore, immune dysfunction was observed in patients with MM 3 . Advances in immunotherapy, including immunomodulators, monoclonal antibodies, and engineered cell therapies, have substantially improved patient survival and provided new strategies for the treatment of relapsed or refractory MM. However, the dysfunction of innate and adaptive immunity hinders recovery in patients with MM. Therefore, a strategy for reversing this immune tolerance and restoring antimyeloma immunity is a major research focus 4 . Immunogenic cell death (ICD) is a form of programed cell death. The concept, first proposed in 2005, involves changes in the composition of the cell surface and the release of soluble mediators occurring in a defined time series. This signal acts on a range of receptors expressed by dendritic cells (DCs) to stimulate tumor antigen presentation to T cells. Studies have shown that MM therapeutic drugs, such as bortezomib and doxorubicin, can trigger specific antitumor immunity through “ICD” 5 , 6 . During ICD induction, tumor immunogenicity can be enhanced by increasing the local release and exposure of endogenous immune adjuvants, including damage-related molecular patterns (DAMPs), which are present in living cells and participate in cell structure and metabolism. Upon ICD activation, they can further activate pattern recognition receptors (PRRs) and lead to DC maturation to activate CD4 + and CD8 + T cells 7 . Previous studies have shown that endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production are significant components for initiating the intracellular danger signaling pathways that control ICD 8 . Pim kinase is a recently discovered and constitutively active serine/threonine kinase that plays a key role in controlling cell proliferation, apoptosis, and migration. Pim kinase is expressed as three different isoenzymes as follows: Pim-1, Pim-2, and Pim-3, which are overexpressed in several cancers. Among them, the excessive Pim-2 expression occurs in lymphoma, leukemia, and MM 9 . The mechanisms by which Pim kinases regulate the immune microenvironment and immune cells as well as the effects of Pim kinase inhibitors on immunity have not been demonstrated. However, previous studies have shown that Pim kinase inhibition induces mitochondrial hyperfission and substantial upregulation of mitochondrial superoxide, thereby increasing intracellular ROS levels 10 . These findings make Pim kinases a new target for MM. Benzofurans, indindes, oxadiazoles, pyrazines, pyrimidines, pyrroles, and quinolones are the main classifications of Pim kinase inhibitors. Newly developed pan-Pim kinase inhibitors are currently in clinical trials. SMI-16a belongs to the thiazolidane-2,4-diketide family, which has better Pim-2 inhibition and fewer side effects than other Pim kinase inhibitors, whereas the combination with proteasome inhibitors can enhance anti-MM effects 11 . This finding prompted us to investigate whether Pim-2 kinase inhibitors can enhance intracellular ROS levels, thereby resulting in ICD production. This study aimed to explore the role and mechanisms of Pim-2 kinase inhibitors in MM immunotherapy to provide a theoretical basis for MM immunotherapy. 2. Materials and Methods 2.1 Experimental participants The study participants were 30 patients with newly diagnosed MM admitted to the Hematology Department of Tianjin Medical University General Hospital from October 2022 to May 2023, based on the 2014 International Myeloma Working Group (IMWG) diagnostic criteria and the 2016 IMWG efficacy criteria 12 , 13 . Of the participants, 18 were men and 12 were women, with a median age of 70 (40–83) years. The study protocol was approved by the Hospital Ethics Committee of Tianjin Medical University, and informed consent was obtained from all participants. The baseline data of the patients with MM are presented in Table 1 . Bone marrow samples were collected from patients as previously described. To obtain the supernatant, patient bone marrow samples were centrifuged, and the samples were stored at − 80°C for subsequent LC-MS/MS analysis. To keep the concentration of metabolites consistent in each group, 200 µL of the supernatant was used from each group for the assay. Table 1 Baseline characteristics of the MM patients Primary MM patients (30 patients) Gender [example (%)] man 18(60.0) woman 12(40.0) Age [years, M (range)] < 65-year-old 8(26.7) ≥ 65-year-old 22(73.3) Median value (range) 70(40–83) The ISS staging [Example (%)] I designated time 4(13.3) II designated time 12(40.0) III designated time 14(46.7) M protein type [Example (%)] IgG mould 8(26.7) IgA mould 8(26.7) IgM mould 0(0.0) light chain type 12(40.0) Do not secrete type 2(6.6) 2.2 Cell culture Human myeloma cells (MM1. S, U266, and OPM2) were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Sciences (Beijing, China). The cell lines were cultured in RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Solarbio, Beijing, China) in an incubator at 37°C in a humidified atmosphere comprising 95% air and 5% CO 2 . The Pim kinase inhibitor, SMI-16a, was provided by MedChemExpress (Monmouth Junction, Middlesex County, NJ, USA) and was frozen as a stock solution at − 80°C. 2.3 MM intracellular ROS levels were measured using flow cytometry (FCM) The culture conditions were as follows: 37°C, 5% CO 2 incubator, MM cell line RPMI-8226 cultured with 20% fetal calf serum (FBS), penicillin 100 U/mL, and streptomycin 100 µg/mL, and MM cell lines OPM-2, U266 cultured in RPMI1640 complete medium containing 10% FBS, penicillin 100 U/mL, and streptomycin 100 µg/mL. Cells were resuspended in a serum-free medium in 12-well plates with 5 × 10 5 cells/well. Subsequently, the experimental groups treated with the negative control, positive control, and different SMI-16a concentrations were set. The cultures were grown for 24 h. The cells were collected and centrifuged at 800 g for 5 min, the supernatant was discarded, and a 2-mL-diluted DCFH-DA probe was added and incubated in an incubator at 37°C for 20 min. Next, the cells were collected and centrifuged at 800 g for 5 min, the supernatant was removed, and 2 mL of phosphate-buffered saline (PBS) was added for washing twice. Finally, the cells were mixed with 300 µL of PBS. The MM intracellular ROS levels were determined using CytoFLEX FCM. 2.4 FCM detected the expression rate of CALR on the surface of MM cells The negative control and experimental groups treated with different SMI-16a concentrations were set. The cultures were grown for 24 h. The cells were harvested and centrifuged at 800 g for 5 min, the supernatant was removed, and 2 mL of PBS was added for washing twice, centrifuged at 800 g for 5min, the supernatant was discarded, and subsequently mixed with 100 µL of PBS with shaking. An appropriate amount of mouse antihuman anti-CALR antibody was added, mixed, and incubated at room temperature (approximately 20°C–25°C) for 15 min, washed twice with 2 mL of PBS, centrifuged at 800 g for 5 min, and mixed with 300 µL of PBS for shaking. The expression rate of CALR on the surface of MM cells was determined using CytoFLEX FCM. 2.5 WB detection of DAMP (HMGB 1 and HSP 70) expression in the supernatant The cells from each group were treated under the previously described conditions for 24 h. Subsequently, the supernatant was collected and precooled centrifuged at 4℃ at 12,000 rpm for 10 min. Next, 200 uL of supernatant and 40 uL of 5 × loading buffer were absorbed in sediments. To mark the desired protein volume, 95℃ boiled for 10 min. Electrophoresis, membrane transfer solution (Tris, 3.03 g; glycine, 14.48 g; methanol, 200 mL; and double steaming to 1,000 mL), 1 × TBST buffer, blocking solution (5% skim milk), loading, electrophoresis, membrane transfer, sealing, wash membrane incubation, primary antibody, membrane washing, secondary antibody, wash membrane, and chemiluminescence to obtain protein bands. 2.6 In vitro induction of mDC in patients with MM Five milliliters of blood marrow from naïve patients with MM with heparin anticoagulation and mononuclear cells were isolated using lymphocyte separation solution density gradient centrifugation; cells were counted using cell counting plates, and the supernatant was removed at 37°C with 5% CO 2 incubator overnight at 2 × 10 5 /mL in 10% FBS, penicillin 100 U/mL, streptomycin 100 µg/mL, IL-4 containing 50- and 500-ng/mL GM-CSF, and changed until day 7 on the other day. 2.7 Pan-T lymphocytes were sorted by immunomagnetic bead sorting Mononuclear cells were isolated as previously described, and the cells were counted using cell counting plates under a microscope. The pan-T lymphocyte subset magnetic bead antibody was added according to the number of cells. The cells were sorted and collected under magnetic conditions, tested using FCM, and used for in vitro coculture experiments. 2.8 Coculture system construction mDCs of patients with MM with pan-T lymphocytes treated with SMI-16a and untreated MM cell lines were cocultured for 24 h at a 1:1:1 ratio for 72 h, and MM cell lines were subjected to cell membrane labeling with Dio dye. 2.9 FCM detected the expression of DC surface-activated molecules Cells in the coculture system were collected and centrifuged at 800 g for 5 min, the supernatant was removed, added with 2 mL of PBS, washed twice, and centrifuged at 800 g for 5min; the supernatant was discarded and mixed with 100 µL of PBS for shaking. The appropriate amounts of mouse antihuman anti-lineage, HLA-DR, CD80, CD86, CD40, and CD70 antibodies were mixed and incubated at room temperature (approximately 20°C–25°C) for 15 min. The expression rates of CD80, CD86, CD40, and CD70 on the surface of MM cells were determined using a CytoFLEX flow cytometer. 2.10 Differentiation of T lymphocyte subsets was detected using FCM Cells from the coculture system were collected. Subsequently, the sample was added to the 10-color T lymphocyte subset vacuum analysis tube. The sample was incubated for 15 min in the dark. T lymphocyte subset differentiation was determined using CytoFLEX FCM. 2.11 Activation molecules of NK cells was detected using FCM MM cell lines were treated with PBS, SMI-16a, IL-15 superagonist fusion protein, and SMI-16a + IL-15 superagonist fusion protein for 24 h. Subsequently, MM cells, patients with MM mDC, patients with MM NK cells, and pan-T lymphocytes were cocultured at a 1:1:1 ratio for 72 h. Cells from the coculture system were collected. The appropriate amounts of mouse antihuman CD107a, NKG2D, granzyme B, and perforin antibodies were added to each sample. The sample was incubated for 15 min in the dark. The CD107a, NKG2D, granzyme B, and perforin expression rates in the NK cells were determined using a CytoFLEX flow cytometer. 2.12 MM cell apoptosis was measured using FCM Cells in culture were harvested, washed with cold PBS, and resuspended in 400 µL of annexin binding buffer. Thereafter, 5 µL each of annexin V-PE and 7AAD (BD Pharmingen; BD Biosciences) were added to each sample; after mixing, the sample was incubated for 15 min in the dark. Stained samples were measured using FCM within 1 h. To analyze the FCM data, CytExpert software2.0 (Beckman CytoFLEX) was used. 2.13 Analysis of single-cell RNA sequencing (scRNA-seq) data in the Gene Expression Omnibus (GEO) database. We downloaded scRNA-seq data (number GSE193531) from the GEO database. To process the scRNA-seq data, the Seurat R package was used. Unified manifold approximation and projection (UMAP) was used for dimensionality reduction and cluster identification, and the Single R package was used for cell annotation. Differential expression analysis was performed using the DESeq Bioconductor software package and was adjusted using Benjamin and Hochberg’s method for controlling the error detection rate. The P-value of the gene was set to < 0.05, and the differentially expressed genes were statistically analyzed to meet a twofold difference. 2.14 qt-PCR for detecting ER stress-related gene expression Total RNA was extracted using one-step TRIzol (Invitrogen, Carlsbad, CA, USA), reverse transcribed for cDNA, and subjected to qRT-PCR (SYBR Green qPCR Master MTX, Affymetrix, Santa Clara, CA, USA). Relative amplifications of CHOP, IRE 1, XBP 1, and ATF 4 gene expression were calculated using 2 −ΔCT . 2.15 WB detected the expression of ER stress-related proteins Cells were lysed using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), and the supernatant was collected by centrifugation, followed by adding SDS loading buffer and boiling at 95°C for 15 min. The cooled protein samples were added to the polyacrylamide gels for electrophoretic separation. The separated protein samples in the gel were subsequently transferred to a polyvinylidene fluoride membrane, and the antibodies were added after blocking in 5% nonfat milk for 1 h. Subsequently, the membrane was washed thrice with TBST (Solarbio, Beijing, China) and incubated for 1 h with a secondary antibody at room temperature, followed by three TBST washes. The final results were observed by adding a luminescence solution and using an ECL chemiluminescence system. The primary antibodies used were CHOP (#2895), IRE 1 (#3294), BAX (#5023), Bcl-2 (#4223), and XBP 1 (#40435), which were purchased from Cell Signaling Technology (Danvers, MA, USA). Furthermore, p-IRE 1 (ab124945) was purchased from Abcam (Cambridge, UK). The secondary antibody (#7074#7076) was purchased from Cell Signaling Technology. 2.16 Statistical analysis The measurement data consistent with normal distribution and homogeneity of variance were expressed as x ± s, t-test for comparison between the two groups. Student’s t -test was performed using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was set at p < 0.05. 2.17 Animal model The animal studies were conducted in accordance with the guidelines of the Tianjin Experimental Animal Use and Care Committee, and the entire project protocol was approved by the Animal Ethics Committee of the Tianjin Medical University General Hospital. The Shanghai MODEL Organisms (Shanghai, China) provided 6-week-old female NOD-SCID IL-2 receptor gamma null (NSG) mice (NOD-Prkdcscid II2rgem1/Smoc) for this study. This study enrolled 10 patients with MM with a median age of 70 (range, 40 − 83) years. They were diagnosed according to the MM diagnostic criteria launched by the IMWG and were admitted in the Department of Hematology, General Hospital of Tianjin Medical University, from January 2023 to March 2023. The study protocol was approved by the ethics committee (ethical no. IRB2022-WZ-144), and informed consent was provided by all participants. 2.18 In vivo antitumor experiments The mouse model was developed according to that described in the literature 14 – 16 . First, each mouse was seeded with 10 7 RPMI-8226 cells (-13d) in the armpit; 13 days later, when the tumor size grew to approximately 100 mm 3 , tumor-bearing mice were randomly divided into three groups (n = 3 per group): PBS, SMI-16a, and SMI-16a + daratumumab. Next, 2 × 10 7 patient-derived lymphocytes (d0) were intravenously injected into mice from the tail vein, followed by different treatments: SMI-16a (25 mg kg − 1 ) was intraperitoneally injected once every other day until 18 days; daratumumab (8 mg mL − 1 ) were administered four times on days 0, 7, 14, and 21. Mouse tumor tissues (3 × 3 × 3 mm) were harvested and ground into single-cell suspensions (7,500 rpm, 20 s, and five repetitions), and the immune cells in the tumor tissue were analyzed using FCM. The other half of the mice in each group (n = 3 per group) were routinely fed, and their body weight and tumor volumes were measured daily. The mouse model demonstrated the metabolic, biological characteristics, toxic side effects, and tumor-suppressive effects in vivo , relatively closer to the effects in humans. 3. Results 3.1 Reanalysis of the single-cell sequencing results of MM and normal controls in the GEO database Bone marrow CD138 + cells were collected by Rebecca Boiarsky from 9 healthy individuals, 6 MGUS, 12 SMMs, and 8 patients with MM. Single-cell sequencing was performed (RNA data included in the GEO database, number GSE193531), revealing the transcriptional signature of early tumor origin (Fig. 1 A) 17 After reanalyzing the RNA data for dimensionality reduction and cluster identification with UMAP, cells from healthy control (NBM) samples clustered together, whereas the majority of cells from MGUS, SMMs, and patients with MM formed separate cell groups (Fig. 1 B). We obtained 20 cell clusters by applying Leiden clustering (Fig. 1 C). The expression of the Pim-2 gene was higher in population with MM (Figs. 1 D–E). Further analysis of ER stress and DAMP-related genes in different samples showed that CALR , HSP90B1 (DAMPs), and XBP 1 (ER stress-related genes) had the highest expression in the population with MM (Figs. 1 F–G). GO analysis results suggested that differential mRNAs were mostly involved in biological processes, including immune response regulation, antigen binding, ER stress, and the Fc receptor signaling pathway (Fig. 1 H). KEGG results showed that the differentially expressed mRNAs were mainly enriched in the ER stress signaling pathways (Fig. 1 I). 3.2 Pim-2 kinase inhibitors induced increased ROS levels and increased DAMP secretion in MM cells As shown in Figs. 2 A–B, SMI-16a shows a much higher intracellular ROS generation than the control group using DCFH-DA as the intracellular ROS indicator, making SMI-16a more promising for ICD. At the preapoptotic stage, the dying cells undergoing ICD translocated CALR from the perinuclear ER to the cellular surface, and such CALR is a hallmark of ICD. We first evaluated the expression of CALR from MM cells using FCM following treatment with SMI-16a (concentrations: 0, 25, 50, and 75 µM). As shown in Fig. 2 C, the amount of CALR-positive cells enhances with increasing Pim-2 kinase inhibitor concentration. Quantitatively, in OPM-2 and U266, the average number of CALR-positive cells from the groups treated with an arbitrary SMI-16a concentration was higher than that of the control group ( p < 0.001, p < 0.001). In RPMI-8226, nonsignificant CALR expression was observed in the 25- and 50-µM SMI-16a–treated groups; however, significant CALR expression was observed in the 75-µM SMI-16a–treated group (Fig. 2 D). We further evaluated the elicitation of other DAMPs, including HMGB1 and HSP70, by western blotting (Fig. 2 E). A moderately high SMI-16a concentration (50 µM) was required to induce the release of HMGB1 and HSP70 from RPMI-8226, OPM-2, and U266 MM cells. The massive ROS produced by SMI-16a generated focused oxidative stress and directly induced efficient ICD, thereby representing the ideal ICD inducer for antitumor immunotherapy. 3.3 MM cells treated with SMI-16a induced elevated expression of surface-activating molecules in the DC of patients with MM The abovementioned results suggested that the Pim-2 kinase inhibitor could induce increased ROS levels in MM cells and promote DAMP secretion, with a marked effect at the 50-µM Pim-2 kinase inhibitor concentration and the strongest effect at the 75-µM concentration. The induction of apoptosis by the Pim-2 kinase inhibitor in MM cell lines is shown in Figure S1 . When the Pim-2 kinase inhibitor reached a concentration of 50 µM in RPMI-8226, OPM-2, and U266, the total apoptosis was 53.74%, 57.74%, and 51.19%, respectively. However, when the Pim-2 kinase inhibitor reached a concentration of 75 µM in RPMI-8226, OPM-2 and U266, the total apoptosis was 82.53%, 83.26%, and 69.48%, respectively. Therefore, 50 µM was selected as the applied concentration of the Pim-2 kinase inhibitor in the coculture system. DAMPs can promote DC activation by binding to the PRR on the DC surface, and its production helps to predict the ability of the chemotherapeutic drug ICD induction 6 . To investigate whether increasing DAMP production in human MM cell line by SMI-16a could properly and efficiently prime the potent immune response, SMI-16a–treated MM cells were cocultured with DC and T lymphocytes from patients with MM for 72 h (Fig. 3 A-B). The representative marks of DC maturation, including CD80 and CD86 (co-stimulation molecules), CD40 and CD70 were first evaluated 18 . As shown in Figs. 4 B–C, SMI-16a treatment can increase DC marks to a certain degree in the three MM cell lines, indicating ICD-mediated DC maturation. In particular, the percentages of CD80 + DCs in the SMI-16a group (69.79% ± 0.89%, 30.46% ± 4.66%, and 44.59% ± 3.13% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (41.33% ± 4.49%, 17.82% ± 3.81%, and 24.61% ± 1.97% for RPMI-8226, OPM-2, and U266, respectively). Additionally, the percentages of CD70 + DCs in the SMI-16a group (62.81% ± 0.76%, 34.08% ± 4.5%, and 19.42% ± 1.87% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (53.88% ± 4.44%, 15.4% ± 1.71%, and 14.58% ± 0.06% for RPMI-8226, OPM-2, and U266, respectively). Meanwhile, the proportions of CD86 + and CD40 + DCs (fully activated DCs) in the SMI-16a group exhibited an increasing trend compared with those in the control groups (Figs. 3 C–D), suggesting that SMI-16a with promising ROS generation could significantly stimulate DC maturation. 3.4 Effects of SMI-16a–treated MM cells on T lymphocytes in the coculture system During the ICD process, the matured DC cells activated T cells by contacting T cell receptor (TCR) and CD28 on T cells, and these activated T lymphocytes acted as the “killer” by releasing cytokines to induce tumor cell death. CD3 + and CD8 + T lymphocytes were circled by CD3 and CD8 antigens, respectively. CD3 + and CD3 + CD8 + T lymphocyte subsets were classified by antigen markers, including CD57, CD45RA, and CCR7. A nonsignificant difference in the total number of CD3 + CD8 + T cells was observed between the SMI-16a–treated and control groups (Figure S2). However, the populations of different T cells varied among the SMI-16a–treated and control groups. Compared with the control group, the SMI-16a–treated group exhibited the lowest percentages of PD-1 + CD3 + T cells (Figs. 4 A–B) and PD-1 + CD8 + T cells (Figs. 4 C–D). SMI-16a–treated MM cells promoted the differentiation of the T lymphocytes of patients with MM from naïve T lymphocytes to effector memory T lymphocytes (TEM) in both CD3 + T cells (CCR7 − CD45RA − CD3 + ) and CD8 + T cells (CCR7 − CD45RA − CD8 + ), suggesting the possible long-lasting effect of antitumor immunity. Particularly, the percentages of CD8 + TEM in the SMI-16a group (51.54% ± 1.33%, 36% ± 5.87%, and 49.25% ± 4.96% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (34.68% ± 0.26%, 18.99% ± 2.56%, and 34.12% ± 2.01% for RPMI-8226, OPM-2, and U266, respectively). Moreover, the proportion of naïve CD8 + T lymphocytes in the SMI-16a–treated group (4.8% ± 0.28%, 12.79% ± 2.98%, and 6.38% ± 2.47% for RPMI-8226, OPM-2, and U266, respectively) was lower than those in the control group (8.48% ± 0.02%, 23.49% ± 1.11%, and 11.61% ± 0.91% for RPMI-8226, OPM-2, and U266, respectively). Intriguingly, the percentages of aging T lymphocytes (CD57 + CD8 + T and CD57 + CD3 + T cells) showed a decreasing trend in the SMI-16a–treated group. CD57 was present on both senescent CD3 + and CD8 + T cells in the late stages of differentiation, and these senescent T cells could not contribute to the immune response but could promote tumor development and suppress antitumor immunity 19 – 23 . Additionally, functional molecules released from lymphocytes were evaluated and showed increased granzyme B and perforin levels following SMI-16a treatment (Figs. 4 E–F). 3.5 Effects of SMI-16a on apoptosis in MM cells We next assessed the ability of Pim-2 kinase inhibitors to induce apoptosis in MM cells after coculture with DCs and T cells. We used 7AAD/annexin V to double-stain MM cells and analyzed the apoptosis rate using FCM. The percentages of apoptotic cells increased in the SMI-16a–treated group (55.32% ± 1.45%, 29.15% ± 0.37%, and 44.23% ± 0.39% for RPMI-8226, OPM-2, and U266, respectively) compared with those in the control group (1.58% ± 0.2%, 13.51% ± 0.37%, and 14.11% ± 0.2% for RPMI-8226, OPM-2, and U266, respectively) (Figs. 5 A–B). These results suggested that Pim-2 kinase inhibitors induce the killing effect of MM cells following immune system activation. 3.6 Mechanism of ICD production induced by Pim-2 kinase inhibitors ROS have been demonstrated to play an essential role in the ICD induced by anticancer drugs 24 . ROS are highly associated with damage to the mitochondria and ER 25 . In our study, the relative expression of the XBP 1 gene in SMI-16a–treated MM cells (4.66 ± 2.93, 3.22 ± 1.99, and 5.12 ± 1.23 for RPMI-8226, OPM-2, and U266, respectively) was significantly increased compared with those in the control group (1.04 ± 0.30, 1.02 ± 0.22, and 1.00 ± 0.13 for RPMI-8226, OPM-2, and U266, respectively). Meanwhile, compared with the control group, the relative expression of the CHOP, IRE1, and ATF-4 in the SMI-16a–treated group exhibited an increasing trend (Fig. 5 C). Previous studies have shown a significant decrease in phosphorylated IRE1-α and XBP1s protein coupled with a reduction in CRT exposure, extracellular ATP level, and HMGB1 release 26 . We further evaluated the expression of the proteins involved in the ER stress signaling pathway, including IRE1, p-IRE1, GRP78, XBP1, and CHOP, using western blotting (Fig. 5 D). Results showed that the p-IRE1, XBP1, CHOP, and BAX protein expression in SMI-16a–treated MM cells was significantly increased compared with that in the control group. In contrast, the GRP78 and Bcl-2 protein expression in SMI-16a–treated MM cells was significantly decreased compared with that in the control group. This finding showed that SMI-16a downregulates GRP78, leading to the upregulation of phosphorylated IRE1 and XBP1 and subsequently inducing CHOP transcription, which activates ER stress and promotes DAMP secretion. 3.7 Anti-MM performance and immune status in the NSG mouse model To evaluate the ICD induction and antitumor immune response of SMI-16a in vivo , an NSG mouse model without the immune system (T\B\NK cells) was developed (Fig. 6 A). To establish an MM tumor model, RPMI-8226 cells were subcutaneously injected into the NSG mice, and transfusion patient-derived lymphocytes from patients with MM were intravenously injected into RPMI-8226 tumor-bearing NSG mice to develop the human immune system. In this experiment, the tumor-bearing NSG mice with the immune system of patients with MM were randomly separated into the following two groups (n = 3 per group): PBS and SMI-16a. SMI-16a showed tumor inhibitory performance where the tumor volume reached 864 mm 3 , smaller than those in PBS group (1,200 mm 3 ) (Figs. 6 B–C). Furthermore, the SMI-16a group did not show significant weight suppression in mice (Fig. 6 D). SMI-16a can slow down tumor growth, but its antitumor ability is not satisfactory. Therefore, we combined SMI-16a with daratumumab to enhance its antitumor effect. SMI-16a combined with daratumumab showed a marked tumor inhibitory performance, wherein the tumor volume shrunk to 453.9 mm 3 . Moreover, PET-CT revealed that the SMI-16a combined with daratumumab group showed lower contrast agent uptake intensity than the PBS group (Fig. 6 E). Subsequently, the apoptosis of MM cells in the tumor tissues was accessed. The percentage of apoptotic cells increased in the SMI-16a and SMI-16a + daratumumab groups compared with that in the control group (Fig. 6 F). To further reveal the underlying reason for the best antitumor performance of SMI-16a and the contribution of ICD induction, the main antitumor immunity cells, such as DCs and T cells, in the peripheral blood of the mice was examined. The representative marks of DC maturation, including CD80 and CD86 (co-stimulation molecules), CD40, and CD70 were first evaluated. As displayed in Figs. 7 A–B, SMI-16a treatment can increase DC marks to a certain degree, and the combined application of daratumumab can significantly enhance the expression of DC-activated molecules. Similar to the in vitro experiments, SMI-16a–treated MM cells promoted the differentiation of the T lymphocytes of patients with MM from naïve T lymphocytes to TEM in both CD3 + T cells (CCR7 − CD45RA − CD3 + ) and CD8 + T cells (CCR7 − CD45RA − CD8 + ). The SMI-16a–treated group exhibited lower percentages of PD-1 + CD3 + T cells (Figs. 7 C–D) and PD-1 + CD8 + T cells (Figs. 7 E–F) than the control group. Intriguingly, the percentage of aging T lymphocytes (CD57 + CD8 + T and CD57 + CD3 + T cells) showed a decreasing trend in the SMI-16a–treated group. Moreover, the functional molecules released from the lymphocytes were evaluated and showed increased granzyme B and perforin levels following SMI-16a treatment (Figs. 7 G–H). The abovementioned trend was more significant after adding daratumumab. 4. Discussion MM is a hematological malignancy characterized by the proliferation of malignant plasma cells 27 . Pim-2 kinase is highly expressed in MM and is associated with poor prognosis 28 . Thus, Pim-2 kinase inhibitors have been develop rapidly, and some have entered clinical trials 29 – 31 . SMI-16a, as a Pim-2 kinase inhibitor, has achieved some success in MM treatment, especially in combination with other drugs 11 , 30 , 32 – 34 . However, the mechanisms of action of these treatments have not been fully elucidated, particularly the immune-related mechanisms. Immunotherapy, including the use of CAR-T cells, bispecific antibodies, and antibody-drug combinations, is revolutionizing MM treatment 35 . ICD has recently become a hot topic in cancer therapy 7 . ICD is a specific controlled cell death that occurs in dying tumor cells and triggers the production of DAMPs, including surface-exposed calreticulin, HSP 70, and HMGB 1 3 . Previous studies have shown that ER stress and ROS production are significant components of the intracellular danger signaling pathway that initiates and controls ICD 8 . Additionally, studies have reported that inhibiting Pim kinase leads to the significant upregulation of mitochondrial fission and mitochondrial superoxide, thereby increasing intracellular ROS levels 10 . Therefore, we propose the following: can Pim-2 kinase inhibitors induce an increase in intracellular ROS levels, mediate ICD production, and thus achieve immunomodulatory effects on MM? We reanalyzed the scRNA-seq data detected by Boiarsky et al. 17 in the GEO database to verify our hypothesis. KEGG analysis revealed that the differentially expressed mRNAs were enriched in the ER stress-related pathways. Previous studies have reported that MM cells secrete a large amount of monoclonal proteins, and ER stress, as a protective response, is expressed higher in MM cells than in normal cells. Conversely, studies have shown that inhibiting Pim kinase activity can exacerbate ER stress. ER stress overexpression causes terminal UPR and apoptosis through the overactivation of the same protein 36 . Based on this, we observed that ROS levels increased in MM cells, and DAMP secretion increased. Moreover, the increasing concentration of SMI-16a enhanced this effect. Previous studies have shown that ROS-triggered ER stress plays a key role in the cellular danger signaling pathway controlling ICD 37 – 39 , and ROS-based ER stress more effectively enhances ICD-associated immunogenic 40 . DAMPs can promote DC activation by binding to the PRR on the DC surface, and its production aids in predicting the ability of the chemotherapeutic drug ICD induction 6 . These results suggest that SMI-16a can induce ICD production. Furthermore, by cocultivating SMI-16a–treated MM cells with DC and T lymphocytes from patients with MM, FCM revealed that the SMI-16a–treated group had increased expression of the surface activation molecules, including CD80, CD86, CD40, and CD70, compared with the control group. Previous studies have observed that CD40 binding to CD40L on the surface of T lymphocytes triggers CD40 signaling, which increases the ability of DC antigen presentation and the expression of costimulatory ligands and cytokines 41 . CD80 and CD86 promote T cell activation 42 through their interaction with CD28 and cytotoxic T lymphocyte antigen 4, respectively. CD70 promotes T cell proliferation, differentiation, and survival through its interaction with CD27 43 . Specifically, CD80 and CD70 showed a significant difference between the SMI-16a and control groups in three cell lines, whereas RPMI-8226 did not significantly promote SMI-16a cell surface CD86 and CD40 expression in patients with MM. This finding may be related to the individual differences among patients with MM or to the smaller number of experiments. The SMI-16a–treated group tended to differentiate from naïve T lymphocytes to TEM compared with the control group. TEMs express homing receptors, promote migratory to nonlymphoid inflammatory sites, and produce multiple bactericidal cytokines within hours of TCR stimulation 44 . The differentiation of naïve T lymphocytes to TEM indirectly proved T lymphocyte activation following SMI-16a–treated MM cells and also confirmed ICD production. The higher proportion of TEM suggested that the antitumor immunity produced by SMI-16a can have a long-term effect 45 . Furthermore, our study showed that compared with the control group, the proportion of aging T lymphocytes (CD57 + T lymphocytes) and the expression of PD-1 molecules on the surface of T lymphocytes decreased in the SMI-16a–treated group. CD57 + is a marker of T lymphocyte dysfunction and aging 46 . PD-1 upregulation indicates that T cells tend to be depleted, no longer secrete IFN- γ and IL-2, and no longer proliferate and move toward apoptotic 47 . Downregulation of surface PD-1 and CD57 on T lymphocytes contributes to T lymphocyte function maintenance. Additionally, compared with the control group, perforin and granzyme B expression increased in the SMI-16a–treated group. T lymphocytes kill target cells by secreting perforin and granzyme B, and these particles with perforin do not harm their own 48 . Perforin and granzyme B upregulation suggests that SMI-16a can enhance T lymphocyte function. Finally, SMI-16a promoted IRE 1 phosphorylation as well as XBP 1 and CHOP transcription through IRE 1 and GRP 78 dissociation. The abovementioned pathway-mediated ER stress and promoted DAMP release in MM cells. Previous studies have shown that IRE 1 dimerizes and autophosphorylates, which subsequently activates and splices the target XBP 1, ultimately enhancing the ICD effect 49 . In addition, CHOP, as a characteristic protein for ER-promoting apoptosis, is also the intersection point 50 of three signaling pathways following ER stress. The upregulation of CHOP protein expression levels promotes apoptosis in MM cells. In vivo , SMI-16a showed good inhibitory effects on tumor growth and immune response activation. However, owing to the inhibitory nature of the tumor microenvironment, the efficiency of the immune response generated is insufficient 51 . To further activate the immune response, we attempted to combine SMI-16a with daratumumab. Interestingly, SMI-16a combined with daratumumab significantly decreased in tumor load and uptake in mice and led to a further activation of DC and T lymphocytes. This study demonstrated that Pim-2 kinase inhibitors can induce ICD through the excessive activation of ER stress and ROS production, which provides a new direction for MM immunotherapy. However, some limitations exist in this study. For example, the ER stress pathway of induced ICD was insufficiently explored. The immunological mechanism of the effect of Pim-2 kinase inhibitors on MM is highly complex, and further investigation and optimization are required. 5. Conclusion Pim-2 kinase inhibitors promote the efflux of CALR proteins and the secretion of DAMPs (HMGB1 and HSP70) by causing an increase in ROS levels in MM cells. Following treatment with Pim-2 kinase inhibitors, MM cell lines can upregulate the expression of activation molecules on the surface of DCs from patients with MM, promote T lymphocyte differentiation from naïve T cells to effector memory T cells, and promote the expression of T lymphocyte functional molecules. In vivo , the combination of SMI-16a and daratumumab significantly decreased in tumor load and uptake in mice and led to further DC and T lymphocyte activation. Mechanistically, Pim-2 kinase inhibitors upregulate IRE1 phosphorylation and promote XBP1 and CHOP transcription, thereby mediating ER stress in MM cells and promoting DAMP release in MM cells. Declarations Resource availability Data available on request from the authors Funding Declaration This work was supported by the National Natural Science Foundation of China Youth Project (grant no. 81900131), the Tianjin Municipal Natural Science Foundation (grant no. 18JCQNJC80400), the Tianjin Education Commission Research Project (grant no. 2018KJ043), the Tianjin Education Commission Research Project (grant no. 2018KJ045), the Tianjin Science and Technology Planning Project (no. 20YFZCSY00060), the Tianjin Municipal Health Commission Youth Project（grant no. TJWJ2021QN001), the Medjaden Academy & Research Foundation for Young Scientists (Grant No. MJR20221011), the Tianjin Key Medical Discipline(Specialty) Construction project, Grant Number: XZDXK-028A, and Tianjin Municipal Health Health Science and Technology Project(grant no. TJWJ2023XK003). Author contributions ZYL, 1,2,3 HLS, 1,2,3 and MTC 1,2,3 contributed equally to this work. 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(\u003cstrong\u003eA\u003c/strong\u003e) Schematic diagram of RNA sequencing. (\u003cstrong\u003eB\u003c/strong\u003e) Cluster analysis of CD138+ cells identified according to the disease status of the sample. (\u003cstrong\u003eC\u003c/strong\u003e) Leiden clustering results for all cells. (\u003cstrong\u003eD\u003c/strong\u003e) Unified manifold approximation and projection visualization of Pim-2 gene expression. (\u003cstrong\u003eE\u003c/strong\u003e) The expression of the Pim-2 gene in CD138+ cells in different disease states. (\u003cstrong\u003eF\u003c/strong\u003e) The expression of the damage-related molecular pattern (DAMP)-related gene in CD138+ cells in different disease states. (\u003cstrong\u003eG\u003c/strong\u003e) The expression of the ER stress-related genes in CD138+ cells in different disease states. (\u003cstrong\u003eH\u003c/strong\u003e) Scatter plot of the differentially expressed mRNAs GO enrichment. (\u003cstrong\u003eI\u003c/strong\u003e) Scatter plot of the differentially expressed mRNAs KEGG enrichment (point sizes and colors represent gene counts and adjusted p-values in the selected pathways.).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/2a835f238a814e9b5d43bc32.png"},{"id":72740315,"identity":"6afcb076-0d4f-45af-ab0a-65e985469875","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":374814,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitors induce increased reactive oxygen species (ROS) levels and increase DAMP secretion in multiple myeloma (MM) cells. (\u003cstrong\u003eA\u003c/strong\u003e) The intracellular ROS levels in MM are determined using flow cytometry (FCM). (\u003cstrong\u003eB\u003c/strong\u003e) Ratio of the mean fluorescence intensity of the DCFH-DA probe. (\u003cstrong\u003eC\u003c/strong\u003e) CALR expression on the cell surface of MM cells. (\u003cstrong\u003eD\u003c/strong\u003e) Statistics of CALR expression on the MM cell surface. (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001) (\u003cstrong\u003eE\u003c/strong\u003e). Pim-2 kinase inhibitors induce increased DAMP secretion in MM cells.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/e44ec629b68cc8026009c0d5.png"},{"id":72740318,"identity":"758b39ba-de9b-43a8-a6fa-b0f86c547dfd","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":779845,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitors promote dendritic cell (DC) activation \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) SMI-16a–treated MM cells form a coculture system with DC and T lymphocytes from patients with MM. (\u003cstrong\u003eB\u003c/strong\u003e) Morphology of DC under microscope and gate labeling in flow cytometry. (\u003cstrong\u003eC\u003c/strong\u003e) SMI-16a–treated MM cells induce the expression of DC surface-activating molecules in patients with MM. (\u003cstrong\u003eD\u003c/strong\u003e) Statistics of surface-activating molecule expression on the DC surface (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/dce8065e1f72c46ec4bf26ab.png"},{"id":72740330,"identity":"8626dbe1-d2bf-4ed8-952b-621a17f17ba5","added_by":"auto","created_at":"2025-01-01 09:30:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":993198,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitors promote T lymphocyte activation \u003cem\u003ein vitro\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) CD3\u003csup\u003e+\u003c/sup\u003e T lymphocyte differentiation and surface PD-1 expression of CD3\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the coculture system. (\u003cstrong\u003eB\u003c/strong\u003e) Statistics of CD3\u003csup\u003e+\u003c/sup\u003e T lymphocyte differentiation and surface PD-1 expression of CD3\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the coculture system. (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001). (\u003cstrong\u003eC\u003c/strong\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte differentiation and surface PD-1 expression of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the coculture system. (\u003cstrong\u003eD\u003c/strong\u003e) Statistics of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte differentiation and surface PD-1 expression of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the coculture system. (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001). (\u003cstrong\u003eE\u003c/strong\u003e) Perforin and granzyme B expression in CD3\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the coculture system. (\u003cstrong\u003eF\u003c/strong\u003e) Statistics of perforin and granzyme B expression in CD3\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes. (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/f8b78cafd43014153c1b8066.png"},{"id":72740323,"identity":"9e42b3ec-a691-4733-b83e-fdce84700101","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":639374,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitors promote apoptosis of MM cells and activate ER stress in MM cells. (\u003cstrong\u003eA\u003c/strong\u003e) Flow pattern of apoptotic MM cells in the coculture system. (\u003cstrong\u003eB\u003c/strong\u003e) Statistical plot of MM cell apoptosis in the coculture system. (\u003cstrong\u003eC\u003c/strong\u003e) Effect of Pim-2 kinase inhibitors on the expression of genes involved in ER stress in MM cells. (\u003cstrong\u003eD\u003c/strong\u003e) ER stress-related protein expression in MM cells following SMI-16a treatment. (\u003cstrong\u003eE\u003c/strong\u003e) Statistical plot of ER stress-related protein expression in MM cells following SMI-16a treatment. (* Compared with normal control, p \u0026lt; 0.05; * * compared with normal control, p \u0026lt; 0.01; * * * compared with normal control, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/dad18cb82629821848de4a4d.png"},{"id":72740325,"identity":"5725b177-66ce-4b8f-92f6-36978db7689a","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":758378,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitors enhance antitumor effects in NSG mouse model. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the NSG model development. (\u003cstrong\u003eB\u003c/strong\u003e) Physical image of mouse tumors in different groups. (\u003cstrong\u003eC\u003c/strong\u003e) Tumor growth curves in different groups. (\u003cstrong\u003eD\u003c/strong\u003e) Mice body weight changes after different treatments. (\u003cstrong\u003eE\u003c/strong\u003e) PET-CT following different treatments. (\u003cstrong\u003eF\u003c/strong\u003e) The apoptotic levels of RPMI-8226 cells in mouse tumor tissues following different treatments. (Error bars, mean ± standard deviation [SD], n = 3, significance is presented compared with the BSA/TPA-Erdn group, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001*).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/ccf6cda89d892aa9c295f66a.png"},{"id":72740324,"identity":"52b9230f-c735-43af-85f8-a50ab32a9f59","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":839451,"visible":true,"origin":"","legend":"\u003cp\u003ePim-2 kinase inhibitor activates the immune response in the NSG mice model. (\u003cstrong\u003eA and B\u003c/strong\u003e) The surface expression of functional molecules on mice peripheral blood DCs measured using FCM. (\u003cstrong\u003eC and D\u003c/strong\u003e) Distribution of different CD3\u003csup\u003e+\u003c/sup\u003e T cells in the mice peripheral blood in these groups. (\u003cstrong\u003eE and F\u003c/strong\u003e) Distribution of different CD8\u003csup\u003e+\u003c/sup\u003e T cells in the mice peripheral blood in these groups. (\u003cstrong\u003eG and H\u003c/strong\u003e) Granzyme B and perforin levels in mice peripheral sera in these groups measured using FCM. (Error bars, mean ± SD, n = 3, significance is presented compared with the BSA/TPA-Erdn group, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001*).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/fdf35f54c61004d8fa7bf8a4.png"},{"id":73271330,"identity":"34a3337d-d0be-4133-a4e9-6f1afa043835","added_by":"auto","created_at":"2025-01-08 10:54:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5551408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/18e2d4aa-d499-4005-941f-cc893ec23f28.pdf"},{"id":72740321,"identity":"8ad8ba5d-1eaa-422f-91bc-0900dd23c8f0","added_by":"auto","created_at":"2025-01-01 09:30:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":806953,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5730658/v1/bd1e864f5334fb711d737b27.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role and mechanism of Pim-2 kinase inhibitor-induced immunogenic cell death in multiple myeloma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMultiple myeloma (MM) is a type of monoclonal plasma cell malignant proliferation that secretes numerous monoclonal immunoglobulins typical of malignant plasma cell disease, which are the main clinical manifestations of anemia, repeated infections, hypercalcemia, high viscosity syndrome, widespread bone destruction, and renal insufficiency; the incidence of MM is rapidly increasing\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. MM has a complex pathogenesis. Along with genetic abnormalities, epigenetic abnormalities, and other factors, the change in the MM bone marrow microenvironment also plays an important role in the occurrence, development, and drug resistance of diseases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Furthermore, immune dysfunction was observed in patients with MM\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Advances in immunotherapy, including immunomodulators, monoclonal antibodies, and engineered cell therapies, have substantially improved patient survival and provided new strategies for the treatment of relapsed or refractory MM. However, the dysfunction of innate and adaptive immunity hinders recovery in patients with MM. Therefore, a strategy for reversing this immune tolerance and restoring antimyeloma immunity is a major research focus\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImmunogenic cell death (ICD) is a form of programed cell death. The concept, first proposed in 2005, involves changes in the composition of the cell surface and the release of soluble mediators occurring in a defined time series. This signal acts on a range of receptors expressed by dendritic cells (DCs) to stimulate tumor antigen presentation to T cells. Studies have shown that MM therapeutic drugs, such as bortezomib and doxorubicin, can trigger specific antitumor immunity through \u0026ldquo;ICD\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. During ICD induction, tumor immunogenicity can be enhanced by increasing the local release and exposure of endogenous immune adjuvants, including damage-related molecular patterns (DAMPs), which are present in living cells and participate in cell structure and metabolism. Upon ICD activation, they can further activate pattern recognition receptors (PRRs) and lead to DC maturation to activate CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production are significant components for initiating the intracellular danger signaling pathways that control ICD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePim kinase is a recently discovered and constitutively active serine/threonine kinase that plays a key role in controlling cell proliferation, apoptosis, and migration. Pim kinase is expressed as three different isoenzymes as follows: Pim-1, Pim-2, and Pim-3, which are overexpressed in several cancers. Among them, the excessive Pim-2 expression occurs in lymphoma, leukemia, and MM\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The mechanisms by which Pim kinases regulate the immune microenvironment and immune cells as well as the effects of Pim kinase inhibitors on immunity have not been demonstrated. However, previous studies have shown that Pim kinase inhibition induces mitochondrial hyperfission and substantial upregulation of mitochondrial superoxide, thereby increasing intracellular ROS levels\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These findings make Pim kinases a new target for MM. Benzofurans, indindes, oxadiazoles, pyrazines, pyrimidines, pyrroles, and quinolones are the main classifications of Pim kinase inhibitors. Newly developed pan-Pim kinase inhibitors are currently in clinical trials. SMI-16a belongs to the thiazolidane-2,4-diketide family, which has better Pim-2 inhibition and fewer side effects than other Pim kinase inhibitors, whereas the combination with proteasome inhibitors can enhance anti-MM effects\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This finding prompted us to investigate whether Pim-2 kinase inhibitors can enhance intracellular ROS levels, thereby resulting in ICD production.\u003c/p\u003e \u003cp\u003eThis study aimed to explore the role and mechanisms of Pim-2 kinase inhibitors in MM immunotherapy to provide a theoretical basis for MM immunotherapy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental participants\u003c/h2\u003e \u003cp\u003eThe study participants were 30 patients with newly diagnosed MM admitted to the Hematology Department of Tianjin Medical University General Hospital from October 2022 to May 2023, based on the 2014 International Myeloma Working Group (IMWG) diagnostic criteria and the 2016 IMWG efficacy criteria\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Of the participants, 18 were men and 12 were women, with a median age of 70 (40\u0026ndash;83) years. The study protocol was approved by the Hospital Ethics Committee of Tianjin Medical University, and informed consent was obtained from all participants. The baseline data of the patients with MM are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Bone marrow samples were collected from patients as previously described. To obtain the supernatant, patient bone marrow samples were centrifuged, and the samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent LC-MS/MS analysis. To keep the concentration of metabolites consistent in each group, 200 \u0026micro;L of the supernatant was used from each group for the assay.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of the MM patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary MM patients (30 patients)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender [example (%)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18(60.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewoman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge [years, M (range)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;65-year-old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(26.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;65-year-old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22(73.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian value (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70(40\u0026ndash;83)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe ISS staging [Example (%)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI designated time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(13.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII designated time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII designated time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(46.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM protein type [Example (%)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG mould\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(26.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgA mould\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(26.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgM mould\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elight chain type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDo not secrete type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(6.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eHuman myeloma cells (MM1. S, U266, and OPM2) were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Sciences (Beijing, China). The cell lines were cultured in RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin\u0026ndash;streptomycin (Solarbio, Beijing, China) in an incubator at 37\u0026deg;C in a humidified atmosphere comprising 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e. The Pim kinase inhibitor, SMI-16a, was provided by MedChemExpress (Monmouth Junction, Middlesex County, NJ, USA) and was frozen as a stock solution at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MM intracellular ROS levels were measured using flow cytometry (FCM)\u003c/h2\u003e \u003cp\u003eThe culture conditions were as follows: 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, MM cell line RPMI-8226 cultured with 20% fetal calf serum (FBS), penicillin 100 U/mL, and streptomycin 100 \u0026micro;g/mL, and MM cell lines OPM-2, U266 cultured in RPMI1640 complete medium containing 10% FBS, penicillin 100 U/mL, and streptomycin 100 \u0026micro;g/mL. Cells were resuspended in a serum-free medium in 12-well plates with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. Subsequently, the experimental groups treated with the negative control, positive control, and different SMI-16a concentrations were set. The cultures were grown for 24 h. The cells were collected and centrifuged at 800 g for 5 min, the supernatant was discarded, and a 2-mL-diluted DCFH-DA probe was added and incubated in an incubator at 37\u0026deg;C for 20 min. Next, the cells were collected and centrifuged at 800 g for 5 min, the supernatant was removed, and 2 mL of phosphate-buffered saline (PBS) was added for washing twice. Finally, the cells were mixed with 300 \u0026micro;L of PBS. The MM intracellular ROS levels were determined using CytoFLEX FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 FCM detected the expression rate of CALR on the surface of MM cells\u003c/h2\u003e \u003cp\u003eThe negative control and experimental groups treated with different SMI-16a concentrations were set. The cultures were grown for 24 h. The cells were harvested and centrifuged at 800 g for 5 min, the supernatant was removed, and 2 mL of PBS was added for washing twice, centrifuged at 800 g for 5min, the supernatant was discarded, and subsequently mixed with 100 \u0026micro;L of PBS with shaking. An appropriate amount of mouse antihuman anti-CALR antibody was added, mixed, and incubated at room temperature (approximately 20\u0026deg;C\u0026ndash;25\u0026deg;C) for 15 min, washed twice with 2 mL of PBS, centrifuged at 800 g for 5 min, and mixed with 300 \u0026micro;L of PBS for shaking. The expression rate of CALR on the surface of MM cells was determined using CytoFLEX FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 WB detection of DAMP (HMGB 1 and HSP 70) expression in the supernatant\u003c/h2\u003e \u003cp\u003eThe cells from each group were treated under the previously described conditions for 24 h. Subsequently, the supernatant was collected and precooled centrifuged at 4℃ at 12,000 rpm for 10 min. Next, 200 uL of supernatant and 40 uL of 5 \u0026times; loading buffer were absorbed in sediments. To mark the desired protein volume, 95℃ boiled for 10 min. Electrophoresis, membrane transfer solution (Tris, 3.03 g; glycine, 14.48 g; methanol, 200 mL; and double steaming to 1,000 mL), 1 \u0026times; TBST buffer, blocking solution (5% skim milk), loading, electrophoresis, membrane transfer, sealing, wash membrane incubation, primary antibody, membrane washing, secondary antibody, wash membrane, and chemiluminescence to obtain protein bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 \u003cem\u003eIn vitro\u003c/em\u003e induction of mDC in patients with MM\u003c/h2\u003e \u003cp\u003eFive milliliters of blood marrow from na\u0026iuml;ve patients with MM with heparin anticoagulation and mononuclear cells were isolated using lymphocyte separation solution density gradient centrifugation; cells were counted using cell counting plates, and the supernatant was removed at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e incubator overnight at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/mL in 10% FBS, penicillin 100 U/mL, streptomycin 100 \u0026micro;g/mL, IL-4 containing 50- and 500-ng/mL GM-CSF, and changed until day 7 on the other day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Pan-T lymphocytes were sorted by immunomagnetic bead sorting\u003c/h2\u003e \u003cp\u003eMononuclear cells were isolated as previously described, and the cells were counted using cell counting plates under a microscope. The pan-T lymphocyte subset magnetic bead antibody was added according to the number of cells. The cells were sorted and collected under magnetic conditions, tested using FCM, and used for \u003cem\u003ein vitro\u003c/em\u003e coculture experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Coculture system construction\u003c/h2\u003e \u003cp\u003emDCs of patients with MM with pan-T lymphocytes treated with SMI-16a and untreated MM cell lines were cocultured for 24 h at a 1:1:1 ratio for 72 h, and MM cell lines were subjected to cell membrane labeling with Dio dye.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 FCM detected the expression of DC surface-activated molecules\u003c/h2\u003e \u003cp\u003eCells in the coculture system were collected and centrifuged at 800 g for 5 min, the supernatant was removed, added with 2 mL of PBS, washed twice, and centrifuged at 800 g for 5min; the supernatant was discarded and mixed with 100 \u0026micro;L of PBS for shaking. The appropriate amounts of mouse antihuman anti-lineage, HLA-DR, CD80, CD86, CD40, and CD70 antibodies were mixed and incubated at room temperature (approximately 20\u0026deg;C\u0026ndash;25\u0026deg;C) for 15 min. The expression rates of CD80, CD86, CD40, and CD70 on the surface of MM cells were determined using a CytoFLEX flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Differentiation of T lymphocyte subsets was detected using FCM\u003c/h2\u003e \u003cp\u003eCells from the coculture system were collected. Subsequently, the sample was added to the 10-color T lymphocyte subset vacuum analysis tube. The sample was incubated for 15 min in the dark. T lymphocyte subset differentiation was determined using CytoFLEX FCM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Activation molecules of NK cells was detected using FCM\u003c/h2\u003e \u003cp\u003eMM cell lines were treated with PBS, SMI-16a, IL-15 superagonist fusion protein, and SMI-16a\u0026thinsp;+\u0026thinsp;IL-15 superagonist fusion protein for 24 h. Subsequently, MM cells, patients with MM mDC, patients with MM NK cells, and pan-T lymphocytes were cocultured at a 1:1:1 ratio for 72 h. Cells from the coculture system were collected. The appropriate amounts of mouse antihuman CD107a, NKG2D, granzyme B, and perforin antibodies were added to each sample. The sample was incubated for 15 min in the dark. The CD107a, NKG2D, granzyme B, and perforin expression rates in the NK cells were determined using a CytoFLEX flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 MM cell apoptosis was measured using FCM\u003c/h2\u003e \u003cp\u003eCells in culture were harvested, washed with cold PBS, and resuspended in 400 \u0026micro;L of annexin binding buffer. Thereafter, 5 \u0026micro;L each of annexin V-PE and 7AAD (BD Pharmingen; BD Biosciences) were added to each sample; after mixing, the sample was incubated for 15 min in the dark. Stained samples were measured using FCM within 1 h. To analyze the FCM data, CytExpert software2.0 (Beckman CytoFLEX) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Analysis of single-cell RNA sequencing (scRNA-seq) data in the Gene Expression Omnibus (GEO) database.\u003c/h2\u003e \u003cp\u003eWe downloaded scRNA-seq data (number GSE193531) from the GEO database. To process the scRNA-seq data, the Seurat R package was used. Unified manifold approximation and projection (UMAP) was used for dimensionality reduction and cluster identification, and the Single R package was used for cell annotation. Differential expression analysis was performed using the DESeq Bioconductor software package and was adjusted using Benjamin and Hochberg\u0026rsquo;s method for controlling the error detection rate. The P-value of the gene was set to \u0026lt;\u0026thinsp;0.05, and the differentially expressed genes were statistically analyzed to meet a twofold difference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 qt-PCR for detecting ER stress-related gene expression\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using one-step TRIzol (Invitrogen, Carlsbad, CA, USA), reverse transcribed for cDNA, and subjected to qRT-PCR (SYBR Green qPCR Master MTX, Affymetrix, Santa Clara, CA, USA). Relative amplifications of CHOP, IRE 1, XBP 1, and ATF 4 gene expression were calculated using 2\u003csup\u003e\u0026minus;ΔCT\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 WB detected the expression of ER stress-related proteins\u003c/h2\u003e \u003cp\u003eCells were lysed using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), and the supernatant was collected by centrifugation, followed by adding SDS loading buffer and boiling at 95\u0026deg;C for 15 min. The cooled protein samples were added to the polyacrylamide gels for electrophoretic separation. The separated protein samples in the gel were subsequently transferred to a polyvinylidene fluoride membrane, and the antibodies were added after blocking in 5% nonfat milk for 1 h. Subsequently, the membrane was washed thrice with TBST (Solarbio, Beijing, China) and incubated for 1 h with a secondary antibody at room temperature, followed by three TBST washes. The final results were observed by adding a luminescence solution and using an ECL chemiluminescence system.\u003c/p\u003e \u003cp\u003eThe primary antibodies used were CHOP (#2895), IRE 1 (#3294), BAX (#5023), Bcl-2 (#4223), and XBP 1 (#40435), which were purchased from Cell Signaling Technology (Danvers, MA, USA). Furthermore, p-IRE 1 (ab124945) was purchased from Abcam (Cambridge, UK). The secondary antibody (#7074#7076) was purchased from Cell Signaling Technology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe measurement data consistent with normal distribution and homogeneity of variance were expressed as x\u0026thinsp;\u0026plusmn;\u0026thinsp;s, t-test for comparison between the two groups. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was performed using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Animal model\u003c/h2\u003e \u003cp\u003eThe animal studies were conducted in accordance with the guidelines of the Tianjin Experimental Animal Use and Care Committee, and the entire project protocol was approved by the Animal Ethics Committee of the Tianjin Medical University General Hospital. The Shanghai MODEL Organisms (Shanghai, China) provided 6-week-old female NOD-SCID IL-2 receptor gamma null (NSG) mice (NOD-Prkdcscid II2rgem1/Smoc) for this study. This study enrolled 10 patients with MM with a median age of 70 (range, 40\u0026thinsp;\u0026minus;\u0026thinsp;83) years. They were diagnosed according to the MM diagnostic criteria launched by the IMWG and were admitted in the Department of Hematology, General Hospital of Tianjin Medical University, from January 2023 to March 2023. The study protocol was approved by the ethics committee (ethical no. IRB2022-WZ-144), and informed consent was provided by all participants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 \u003cem\u003eIn vivo\u003c/em\u003e antitumor experiments\u003c/h2\u003e \u003cp\u003eThe mouse model was developed according to that described in the literature\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. First, each mouse was seeded with 10\u003csup\u003e7\u003c/sup\u003e RPMI-8226 cells (-13d) in the armpit; 13 days later, when the tumor size grew to approximately 100 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, tumor-bearing mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;3 per group): PBS, SMI-16a, and SMI-16a\u0026thinsp;+\u0026thinsp;daratumumab. Next, 2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e patient-derived lymphocytes (d0) were intravenously injected into mice from the tail vein, followed by different treatments: SMI-16a (25 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was intraperitoneally injected once every other day until 18 days; daratumumab (8 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were administered four times on days 0, 7, 14, and 21. Mouse tumor tissues (3 \u0026times; 3 \u0026times; 3 mm) were harvested and ground into single-cell suspensions (7,500 rpm, 20 s, and five repetitions), and the immune cells in the tumor tissue were analyzed using FCM. The other half of the mice in each group (n\u0026thinsp;=\u0026thinsp;3 per group) were routinely fed, and their body weight and tumor volumes were measured daily. The mouse model demonstrated the metabolic, biological characteristics, toxic side effects, and tumor-suppressive effects \u003cem\u003ein vivo\u003c/em\u003e, relatively closer to the effects in humans.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Reanalysis of the single-cell sequencing results of MM and normal controls in the GEO database\u003c/h2\u003e \u003cp\u003eBone marrow CD138\u003csup\u003e+\u003c/sup\u003e cells were collected by Rebecca Boiarsky from 9 healthy individuals, 6 MGUS, 12 SMMs, and 8 patients with MM. Single-cell sequencing was performed (RNA data included in the GEO database, number GSE193531), revealing the transcriptional signature of early tumor origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003eAfter reanalyzing the RNA data for dimensionality reduction and cluster identification with UMAP, cells from healthy control (NBM) samples clustered together, whereas the majority of cells from MGUS, SMMs, and patients with MM formed separate cell groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We obtained 20 cell clusters by applying Leiden clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The expression of the Pim-2 gene was higher in population with MM (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;E). Further analysis of ER stress and DAMP-related genes in different samples showed that \u003cem\u003eCALR\u003c/em\u003e, \u003cem\u003eHSP90B1\u003c/em\u003e (DAMPs), and \u003cem\u003eXBP 1\u003c/em\u003e (ER stress-related genes) had the highest expression in the population with MM (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;G). GO analysis results suggested that differential mRNAs were mostly involved in biological processes, including immune response regulation, antigen binding, ER stress, and the Fc receptor signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). KEGG results showed that the differentially expressed mRNAs were mainly enriched in the ER stress signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Pim-2 kinase inhibitors induced increased ROS levels and increased DAMP secretion in MM cells\u003c/h2\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;B, SMI-16a shows a much higher intracellular ROS generation than the control group using DCFH-DA as the intracellular ROS indicator, making SMI-16a more promising for ICD. At the preapoptotic stage, the dying cells undergoing ICD translocated CALR from the perinuclear ER to the cellular surface, and such CALR is a hallmark of ICD. We first evaluated the expression of CALR from MM cells using FCM following treatment with SMI-16a (concentrations: 0, 25, 50, and 75 \u0026micro;M). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the amount of CALR-positive cells enhances with increasing Pim-2 kinase inhibitor concentration. Quantitatively, in OPM-2 and U266, the average number of CALR-positive cells from the groups treated with an arbitrary SMI-16a concentration was higher than that of the control group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). In RPMI-8226, nonsignificant CALR expression was observed in the 25- and 50-\u0026micro;M SMI-16a\u0026ndash;treated groups; however, significant CALR expression was observed in the 75-\u0026micro;M SMI-16a\u0026ndash;treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We further evaluated the elicitation of other DAMPs, including HMGB1 and HSP70, by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). A moderately high SMI-16a concentration (50 \u0026micro;M) was required to induce the release of HMGB1 and HSP70 from RPMI-8226, OPM-2, and U266 MM cells. The massive ROS produced by SMI-16a generated focused oxidative stress and directly induced efficient ICD, thereby representing the ideal ICD inducer for antitumor immunotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.3 MM cells treated with SMI-16a induced elevated expression of surface-activating molecules in the DC of patients with MM\u003c/p\u003e \u003cp\u003eThe abovementioned results suggested that the Pim-2 kinase inhibitor could induce increased ROS levels in MM cells and promote DAMP secretion, with a marked effect at the 50-\u0026micro;M Pim-2 kinase inhibitor concentration and the strongest effect at the 75-\u0026micro;M concentration. The induction of apoptosis by the Pim-2 kinase inhibitor in MM cell lines is shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. When the Pim-2 kinase inhibitor reached a concentration of 50 \u0026micro;M in RPMI-8226, OPM-2, and U266, the total apoptosis was 53.74%, 57.74%, and 51.19%, respectively. However, when the Pim-2 kinase inhibitor reached a concentration of 75 \u0026micro;M in RPMI-8226, OPM-2 and U266, the total apoptosis was 82.53%, 83.26%, and 69.48%, respectively. Therefore, 50 \u0026micro;M was selected as the applied concentration of the Pim-2 kinase inhibitor in the coculture system.\u003c/p\u003e \u003cp\u003eDAMPs can promote DC activation by binding to the PRR on the DC surface, and its production helps to predict the ability of the chemotherapeutic drug ICD induction\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To investigate whether increasing DAMP production in human MM cell line by SMI-16a could properly and efficiently prime the potent immune response, SMI-16a\u0026ndash;treated MM cells were cocultured with DC and T lymphocytes from patients with MM for 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The representative marks of DC maturation, including CD80 and CD86 (co-stimulation molecules), CD40 and CD70 were first evaluated\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;C, SMI-16a treatment can increase DC marks to a certain degree in the three MM cell lines, indicating ICD-mediated DC maturation. In particular, the percentages of CD80\u003csup\u003e+\u003c/sup\u003e DCs in the SMI-16a group (69.79% \u0026plusmn; 0.89%, 30.46% \u0026plusmn; 4.66%, and 44.59% \u0026plusmn; 3.13% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (41.33% \u0026plusmn; 4.49%, 17.82% \u0026plusmn; 3.81%, and 24.61% \u0026plusmn; 1.97% for RPMI-8226, OPM-2, and U266, respectively). Additionally, the percentages of CD70\u003csup\u003e+\u003c/sup\u003e DCs in the SMI-16a group (62.81% \u0026plusmn; 0.76%, 34.08% \u0026plusmn; 4.5%, and 19.42% \u0026plusmn; 1.87% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (53.88% \u0026plusmn; 4.44%, 15.4% \u0026plusmn; 1.71%, and 14.58% \u0026plusmn; 0.06% for RPMI-8226, OPM-2, and U266, respectively). Meanwhile, the proportions of CD86\u003csup\u003e+\u003c/sup\u003e and CD40\u003csup\u003e+\u003c/sup\u003e DCs (fully activated DCs) in the SMI-16a group exhibited an increasing trend compared with those in the control groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;D), suggesting that SMI-16a with promising ROS generation could significantly stimulate DC maturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effects of SMI-16a\u0026ndash;treated MM cells on T lymphocytes in the coculture system\u003c/h2\u003e \u003cp\u003eDuring the ICD process, the matured DC cells activated T cells by contacting T cell receptor (TCR) and CD28 on T cells, and these activated T lymphocytes acted as the \u0026ldquo;killer\u0026rdquo; by releasing cytokines to induce tumor cell death. CD3\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes were circled by CD3 and CD8 antigens, respectively. CD3\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte subsets were classified by antigen markers, including CD57, CD45RA, and CCR7. A nonsignificant difference in the total number of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells was observed between the SMI-16a\u0026ndash;treated and control groups (Figure S2). However, the populations of different T cells varied among the SMI-16a\u0026ndash;treated and control groups. Compared with the control group, the SMI-16a\u0026ndash;treated group exhibited the lowest percentages of PD-1\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B) and PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;D). SMI-16a\u0026ndash;treated MM cells promoted the differentiation of the T lymphocytes of patients with MM from na\u0026iuml;ve T lymphocytes to effector memory T lymphocytes (TEM) in both CD3\u003csup\u003e+\u003c/sup\u003e T cells (CCR7\u003csup\u003e\u0026minus;\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e) and CD8\u003csup\u003e+\u003c/sup\u003eT cells (CCR7\u003csup\u003e\u0026minus;\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e), suggesting the possible long-lasting effect of antitumor immunity. Particularly, the percentages of CD8\u003csup\u003e+\u003c/sup\u003eTEM in the SMI-16a group (51.54% \u0026plusmn; 1.33%, 36% \u0026plusmn; 5.87%, and 49.25% \u0026plusmn; 4.96% for RPMI-8226, OPM-2, and U266, respectively) were significantly higher than those in the control groups (34.68% \u0026plusmn; 0.26%, 18.99% \u0026plusmn; 2.56%, and 34.12% \u0026plusmn; 2.01% for RPMI-8226, OPM-2, and U266, respectively). Moreover, the proportion of na\u0026iuml;ve CD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes in the SMI-16a\u0026ndash;treated group (4.8% \u0026plusmn; 0.28%, 12.79% \u0026plusmn; 2.98%, and 6.38% \u0026plusmn; 2.47% for RPMI-8226, OPM-2, and U266, respectively) was lower than those in the control group (8.48% \u0026plusmn; 0.02%, 23.49% \u0026plusmn; 1.11%, and 11.61% \u0026plusmn; 0.91% for RPMI-8226, OPM-2, and U266, respectively). Intriguingly, the percentages of aging T lymphocytes (CD57\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT and CD57\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eT cells) showed a decreasing trend in the SMI-16a\u0026ndash;treated group. CD57 was present on both senescent CD3\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the late stages of differentiation, and these senescent T cells could not contribute to the immune response but could promote tumor development and suppress antitumor immunity\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, functional molecules released from lymphocytes were evaluated and showed increased granzyme B and perforin levels following SMI-16a treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effects of SMI-16a on apoptosis in MM cells\u003c/h2\u003e \u003cp\u003eWe next assessed the ability of Pim-2 kinase inhibitors to induce apoptosis in MM cells after coculture with DCs and T cells. We used 7AAD/annexin V to double-stain MM cells and analyzed the apoptosis rate using FCM. The percentages of apoptotic cells increased in the SMI-16a\u0026ndash;treated group (55.32% \u0026plusmn; 1.45%, 29.15% \u0026plusmn; 0.37%, and 44.23% \u0026plusmn; 0.39% for RPMI-8226, OPM-2, and U266, respectively) compared with those in the control group (1.58% \u0026plusmn; 0.2%, 13.51% \u0026plusmn; 0.37%, and 14.11% \u0026plusmn; 0.2% for RPMI-8226, OPM-2, and U266, respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B). These results suggested that Pim-2 kinase inhibitors induce the killing effect of MM cells following immune system activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Mechanism of ICD production induced by Pim-2 kinase inhibitors\u003c/h2\u003e \u003cp\u003eROS have been demonstrated to play an essential role in the ICD induced by anticancer drugs\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. ROS are highly associated with damage to the mitochondria and ER\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In our study, the relative expression of the XBP 1 gene in SMI-16a\u0026ndash;treated MM cells (4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93, 3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99, and 5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 for RPMI-8226, OPM-2, and U266, respectively) was significantly increased compared with those in the control group (1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30, 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22, and 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 for RPMI-8226, OPM-2, and U266, respectively). Meanwhile, compared with the control group, the relative expression of the CHOP, IRE1, and ATF-4 in the SMI-16a\u0026ndash;treated group exhibited an increasing trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Previous studies have shown a significant decrease in phosphorylated IRE1-α and XBP1s protein coupled with a reduction in CRT exposure, extracellular ATP level, and HMGB1 release\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We further evaluated the expression of the proteins involved in the ER stress signaling pathway, including IRE1, p-IRE1, GRP78, XBP1, and CHOP, using western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Results showed that the p-IRE1, XBP1, CHOP, and BAX protein expression in SMI-16a\u0026ndash;treated MM cells was significantly increased compared with that in the control group. In contrast, the GRP78 and Bcl-2 protein expression in SMI-16a\u0026ndash;treated MM cells was significantly decreased compared with that in the control group. This finding showed that SMI-16a downregulates GRP78, leading to the upregulation of phosphorylated IRE1 and XBP1 and subsequently inducing CHOP transcription, which activates ER stress and promotes DAMP secretion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Anti-MM performance and immune status in the NSG mouse model\u003c/h2\u003e \u003cp\u003eTo evaluate the ICD induction and antitumor immune response of SMI-16a \u003cem\u003ein vivo\u003c/em\u003e, an NSG mouse model without the immune system (T\\B\\NK cells) was developed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To establish an MM tumor model, RPMI-8226 cells were subcutaneously injected into the NSG mice, and transfusion patient-derived lymphocytes from patients with MM were intravenously injected into RPMI-8226 tumor-bearing NSG mice to develop the human immune system. In this experiment, the tumor-bearing NSG mice with the immune system of patients with MM were randomly separated into the following two groups (n\u0026thinsp;=\u0026thinsp;3 per group): PBS and SMI-16a. SMI-16a showed tumor inhibitory performance where the tumor volume reached 864 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, smaller than those in PBS group (1,200 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;C). Furthermore, the SMI-16a group did not show significant weight suppression in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). SMI-16a can slow down tumor growth, but its antitumor ability is not satisfactory. Therefore, we combined SMI-16a with daratumumab to enhance its antitumor effect. SMI-16a combined with daratumumab showed a marked tumor inhibitory performance, wherein the tumor volume shrunk to 453.9 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, PET-CT revealed that the SMI-16a combined with daratumumab group showed lower contrast agent uptake intensity than the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Subsequently, the apoptosis of MM cells in the tumor tissues was accessed. The percentage of apoptotic cells increased in the SMI-16a and SMI-16a\u0026thinsp;+\u0026thinsp;daratumumab groups compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further reveal the underlying reason for the best antitumor performance of SMI-16a and the contribution of ICD induction, the main antitumor immunity cells, such as DCs and T cells, in the peripheral blood of the mice was examined. The representative marks of DC maturation, including CD80 and CD86 (co-stimulation molecules), CD40, and CD70 were first evaluated. As displayed in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B, SMI-16a treatment can increase DC marks to a certain degree, and the combined application of daratumumab can significantly enhance the expression of DC-activated molecules. Similar to the \u003cem\u003ein vitro\u003c/em\u003e experiments, SMI-16a\u0026ndash;treated MM cells promoted the differentiation of the T lymphocytes of patients with MM from na\u0026iuml;ve T lymphocytes to TEM in both CD3\u003csup\u003e+\u003c/sup\u003e T cells (CCR7\u003csup\u003e\u0026minus;\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e) and CD8\u003csup\u003e+\u003c/sup\u003eT cells (CCR7\u003csup\u003e\u0026minus;\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e). The SMI-16a\u0026ndash;treated group exhibited lower percentages of PD-1\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;D) and PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;F) than the control group. Intriguingly, the percentage of aging T lymphocytes (CD57\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT and CD57\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eT cells) showed a decreasing trend in the SMI-16a\u0026ndash;treated group. Moreover, the functional molecules released from the lymphocytes were evaluated and showed increased granzyme B and perforin levels following SMI-16a treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG\u0026ndash;H). The abovementioned trend was more significant after adding daratumumab.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMM is a hematological malignancy characterized by the proliferation of malignant plasma cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Pim-2 kinase is highly expressed in MM and is associated with poor prognosis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Thus, Pim-2 kinase inhibitors have been develop rapidly, and some have entered clinical trials\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. SMI-16a, as a Pim-2 kinase inhibitor, has achieved some success in MM treatment, especially in combination with other drugs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms of action of these treatments have not been fully elucidated, particularly the immune-related mechanisms.\u003c/p\u003e \u003cp\u003eImmunotherapy, including the use of CAR-T cells, bispecific antibodies, and antibody-drug combinations, is revolutionizing MM treatment\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. ICD has recently become a hot topic in cancer therapy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. ICD is a specific controlled cell death that occurs in dying tumor cells and triggers the production of DAMPs, including surface-exposed calreticulin, HSP 70, and HMGB 1\u003csup\u003e3\u003c/sup\u003e. Previous studies have shown that ER stress and ROS production are significant components of the intracellular danger signaling pathway that initiates and controls ICD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Additionally, studies have reported that inhibiting Pim kinase leads to the significant upregulation of mitochondrial fission and mitochondrial superoxide, thereby increasing intracellular ROS levels\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, we propose the following: can Pim-2 kinase inhibitors induce an increase in intracellular ROS levels, mediate ICD production, and thus achieve immunomodulatory effects on MM?\u003c/p\u003e \u003cp\u003eWe reanalyzed the scRNA-seq data detected by Boiarsky et al.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e in the GEO database to verify our hypothesis. KEGG analysis revealed that the differentially expressed mRNAs were enriched in the ER stress-related pathways. Previous studies have reported that MM cells secrete a large amount of monoclonal proteins, and ER stress, as a protective response, is expressed higher in MM cells than in normal cells. Conversely, studies have shown that inhibiting Pim kinase activity can exacerbate ER stress. ER stress overexpression causes terminal UPR and apoptosis through the overactivation of the same protein\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on this, we observed that ROS levels increased in MM cells, and DAMP secretion increased. Moreover, the increasing concentration of SMI-16a enhanced this effect. Previous studies have shown that ROS-triggered ER stress plays a key role in the cellular danger signaling pathway controlling ICD\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and ROS-based ER stress more effectively enhances ICD-associated immunogenic\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. DAMPs can promote DC activation by binding to the PRR on the DC surface, and its production aids in predicting the ability of the chemotherapeutic drug ICD induction\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These results suggest that SMI-16a can induce ICD production.\u003c/p\u003e \u003cp\u003eFurthermore, by cocultivating SMI-16a\u0026ndash;treated MM cells with DC and T lymphocytes from patients with MM, FCM revealed that the SMI-16a\u0026ndash;treated group had increased expression of the surface activation molecules, including CD80, CD86, CD40, and CD70, compared with the control group. Previous studies have observed that CD40 binding to CD40L on the surface of T lymphocytes triggers CD40 signaling, which increases the ability of DC antigen presentation and the expression of costimulatory ligands and cytokines\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. CD80 and CD86 promote T cell activation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e through their interaction with CD28 and cytotoxic T lymphocyte antigen 4, respectively. CD70 promotes T cell proliferation, differentiation, and survival through its interaction with CD27\u003csup\u003e43\u003c/sup\u003e. Specifically, CD80 and CD70 showed a significant difference between the SMI-16a and control groups in three cell lines, whereas RPMI-8226 did not significantly promote SMI-16a cell surface CD86 and CD40 expression in patients with MM. This finding may be related to the individual differences among patients with MM or to the smaller number of experiments.\u003c/p\u003e \u003cp\u003eThe SMI-16a\u0026ndash;treated group tended to differentiate from na\u0026iuml;ve T lymphocytes to TEM compared with the control group. TEMs express homing receptors, promote migratory to nonlymphoid inflammatory sites, and produce multiple bactericidal cytokines within hours of TCR stimulation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The differentiation of na\u0026iuml;ve T lymphocytes to TEM indirectly proved T lymphocyte activation following SMI-16a\u0026ndash;treated MM cells and also confirmed ICD production. The higher proportion of TEM suggested that the antitumor immunity produced by SMI-16a can have a long-term effect\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Furthermore, our study showed that compared with the control group, the proportion of aging T lymphocytes (CD57\u003csup\u003e+\u003c/sup\u003e T lymphocytes) and the expression of PD-1 molecules on the surface of T lymphocytes decreased in the SMI-16a\u0026ndash;treated group. CD57\u003csup\u003e+\u003c/sup\u003e is a marker of T lymphocyte dysfunction and aging\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. PD-1 upregulation indicates that T cells tend to be depleted, no longer secrete IFN- γ and IL-2, and no longer proliferate and move toward apoptotic\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Downregulation of surface PD-1 and CD57 on T lymphocytes contributes to T lymphocyte function maintenance. Additionally, compared with the control group, perforin and granzyme B expression increased in the SMI-16a\u0026ndash;treated group. T lymphocytes kill target cells by secreting perforin and granzyme B, and these particles with perforin do not harm their own\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Perforin and granzyme B upregulation suggests that SMI-16a can enhance T lymphocyte function.\u003c/p\u003e \u003cp\u003eFinally, SMI-16a promoted IRE 1 phosphorylation as well as XBP 1 and CHOP transcription through IRE 1 and GRP 78 dissociation. The abovementioned pathway-mediated ER stress and promoted DAMP release in MM cells. Previous studies have shown that IRE 1 dimerizes and autophosphorylates, which subsequently activates and splices the target XBP 1, ultimately enhancing the ICD effect\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In addition, CHOP, as a characteristic protein for ER-promoting apoptosis, is also the intersection point\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e of three signaling pathways following ER stress. The upregulation of CHOP protein expression levels promotes apoptosis in MM cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, SMI-16a showed good inhibitory effects on tumor growth and immune response activation. However, owing to the inhibitory nature of the tumor microenvironment, the efficiency of the immune response generated is insufficient\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. To further activate the immune response, we attempted to combine SMI-16a with daratumumab. Interestingly, SMI-16a combined with daratumumab significantly decreased in tumor load and uptake in mice and led to a further activation of DC and T lymphocytes.\u003c/p\u003e \u003cp\u003eThis study demonstrated that Pim-2 kinase inhibitors can induce ICD through the excessive activation of ER stress and ROS production, which provides a new direction for MM immunotherapy. However, some limitations exist in this study. For example, the ER stress pathway of induced ICD was insufficiently explored. The immunological mechanism of the effect of Pim-2 kinase inhibitors on MM is highly complex, and further investigation and optimization are required.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003ePim-2 kinase inhibitors promote the efflux of CALR proteins and the secretion of DAMPs (HMGB1 and HSP70) by causing an increase in ROS levels in MM cells. Following treatment with Pim-2 kinase inhibitors, MM cell lines can upregulate the expression of activation molecules on the surface of DCs from patients with MM, promote T lymphocyte differentiation from na\u0026iuml;ve T cells to effector memory T cells, and promote the expression of T lymphocyte functional molecules. \u003cem\u003eIn vivo\u003c/em\u003e, the combination of SMI-16a and daratumumab significantly decreased in tumor load and uptake in mice and led to further DC and T lymphocyte activation. Mechanistically, Pim-2 kinase inhibitors upregulate IRE1 phosphorylation and promote XBP1 and CHOP transcription, thereby mediating ER stress in MM cells and promoting DAMP release in MM cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eResource availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request from the authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China Youth Project (grant no. 81900131), the Tianjin Municipal Natural Science Foundation (grant no. 18JCQNJC80400), the Tianjin Education Commission Research Project (grant no. 2018KJ043), the Tianjin Education Commission Research Project (grant no. 2018KJ045), the Tianjin Science and Technology Planning Project (no. 20YFZCSY00060), the Tianjin Municipal Health Commission Youth Project（grant no. TJWJ2021QN001), the Medjaden Academy \u0026amp; Research Foundation for Young Scientists (Grant No. MJR20221011), the Tianjin Key Medical Discipline(Specialty) Construction project, Grant Number: XZDXK-028A, and Tianjin Municipal Health Health Science and Technology Project(grant no. TJWJ2023XK003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZYL,\u003csup\u003e1,2,3\u003c/sup\u003e HLS,\u003csup\u003e1,2,3\u003c/sup\u003e and MTC\u003csup\u003e1,2,3\u003c/sup\u003e contributed equally to this work. ZYL, \u0026nbsp;HLS, \u0026nbsp;and MTC: conceptualization, data curation and writing-original draft. XHZ, HW, and CY: writing-review and editing. RF:resources, supervision, writing-review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu ZY, Xiang CH, Han M, Meng NH, Luo JY, Fu R. Study on Tim3 Regulation of Multiple Myeloma Cell Proliferation\u0026thinsp;\u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;via\u0026thinsp;NF-κB Signal Pathways. 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A pH/ROS dual-responsive system for effective chemoimmunotherapy against melanoma via remodeling tumor immune microenvironment.\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":"adaptive immunity, bone marrow, effector memory T cells, endoplasmic reticulum, immunogenic cell death, immunomodulators, immune tolerance, multiple myeloma, plasma cell disease, renal insufficiency","lastPublishedDoi":"10.21203/rs.3.rs-5730658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5730658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Immune dysfunction is a major component in the pathogenesis of multiple myeloma (MM), and restoring antimyeloma immunity has become a key research direction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This study demonstrates, through \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, whether and how Pim-2 kinase inhibitors induce immunogenic cell death in MM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Pim-2 kinase inhibitors upregulated IRE1 phosphorylation and promoted XBP1 and CHOP transcription, thereby mediating endoplasmic reticulum (ER) stress in MM cells. ER stress and increased reactive oxygen species levels promoted damage-related molecular pattern expression and immunogenic cell death in MM cells. Furthermore, Pim-2 kinase inhibitor-treated MM cell lines upregulated the expression of activation molecules on the surface of dendritic cells (DCs) in patients with MM, stimulated T lymphocyte differentiation from naïve T cells to effector memory T cells, and promoted the expression of T lymphocyte functional molecules. \u003cem\u003eIn vivo\u003c/em\u003e, Pim-2 kinase inhibitors stimulated human DC maturation and activated functional T lymphocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: These data contribute to our knowledge about how Pim-2 kinase inhibitors regulate antimyeloma immunity and provide justification for applying Pim-2 kinase inhibitors in MM treatment.\u003c/p\u003e","manuscriptTitle":"Role and mechanism of Pim-2 kinase inhibitor-induced immunogenic cell death in multiple myeloma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-01 09:30:28","doi":"10.21203/rs.3.rs-5730658/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":"0e52fd77-485a-48dc-b56d-ae6d83e9b626","owner":[],"postedDate":"January 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-08T10:53:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-01 09:30:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5730658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5730658","identity":"rs-5730658","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}