Multiple Myeloma Cell Lines U266 Research Articles

Expression and Function of FAM72A Gene in Multiple myelomaFAM72A.

This study aims to comprehensively investigate the role of Family Member A with sequence similarity 72-A (FAM72A) in multiple myeloma. Multiple myeloma poses significant challenges. This study delves into FAM72A's impact on key cellular processes, shedding light on potential therapeutic targets and enhancing our understanding of multiple myeloma progression. Investigate the impact of FAM72A on the proliferation, apoptosis, and bortezomib sensitivity of multiple myeloma cell line U266. qRT-PCR analyzed FAM72A expression levels in bone marrow samples from 30 patients with multiple myeloma and 10 healthy donors at the Second Hospital of Shanxi Medical University. Cell lines overexpressing FAM72A were constructed, and Cell Counting Kit 8 (CCK-8) and flow cytometry were used to assess U266 cell proliferation, apoptosis, and sensitivity to bortezomib. Biological predictions for FAM72A were performed to find transcription factors binding to the FAM72A promoter region, verified using a luciferase assay. U266 cells were transfected with si-POU2F2 (POU class 2 homeobox 2), and the impact on cell proliferation was validated. Western blot analysis detected the expression of downstream proteins in the p53 signaling pathway. In vivo, experiments established a xenograft mouse model further to study the role of FAM72A in multiple myeloma. FAM72A was upregulated in multiple myeloma bone marrow tissues. Compared to the OE-NC group, the OE-FAM72A group showed increased Mouse Double Minute 2 homolog (MDM2) expression, decreased p53 expression, increased cell proliferation, and decreased apoptosis. POU2F2 was identified as the upstream transcription factor for FAM72A. Compared to the si-NC group, the si-POU2F2 group exhibited decreased MDM2 expression, increased p53 expression, slowed cell proliferation, and increased apoptosis. Silencing POU2F2 could reverse the pro-proliferative effect of over-expressing FAM72A in U266 cells. In vivo experiments in a xenograft mouse model further studied the role of FAM72A in multiple myeloma. Overexpression of FAM72A promotes U266 cell proliferation, inhibits apoptosis, and reduces sensitivity to bortezomib by regulating the POU2F2/FAM72A/p53 signaling pathway.

Possible mechanisms underlying the regulation of postmenopausal osteoporosis by follicle-stimulating hormone

ObjectiveTo explore the possible mechanisms by which follicle-stimulating hormone (FSH) regulates postmenopausal osteoporosis through the FSH/FSH receptor (FSHr)/G protein/C/EBPβ/heat shock protein 90 alpha (HSP90α) signalling pathways. MethodsWe measured serum FSH, luteinising hormone (LH), and HSP90α levels in the serum and adipose tissue of women of childbearing age and menopausal status. In the in vivo studies, 12 B57CL female mice were divided equally into Sham, OVX, and OVX + FSHr Blocker groups. Serum levels of alkaline phosphatase, FSH, and HSP90α, along with StRACP vitality, were determined, and femur micro-computed tomography was performed. Additionally, FSH, FSHr, G protein, C/EBPβ, and HSP90α levels were assessed using quantitative polymerase chain reaction. Finally, we divided the human multiple myeloma cell line U266 into three groups. The activity of tartrate-resistant acid phosphatase (TRAP) in the supernatant at different stages was detected, and myeloma cells were stained with TRAP. ResultsHSP90α levels in adipose tissue supernatant and serum were lower in women of childbearing age than in menopausal women (P < 0.05). Serum FSH and HSP90α levels demonstrated a strong correlation. Treatment with FSHr blockers resulted in decreased FSH, FSHr, G protein, C/EBPβ, and HSP90α levels in mice. TRAP staining of osteoclast-like cells exhibited a significantly higher intensity in the M-CSF + RANKL + recombinant HSP90α group than in the M-CSF + RANKL and blank control groups (P < 0.05). ConclusionsOur results indicate that FSH promotes HSP90α secretion by adipocytes via the FSHr/G protein/C/EBPβ pathway. This mechanism affects osteoclast activity and exacerbates osteoporosis.

Targeting Loss of the Tumor Suppressor TENT5C in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which multiple genomic events lead to tumor development and progression. Loss of TENT5C through mutation or deletion on chromosome 1p occurs in more than 20% of newly diagnosed MM patients and is associated with a poor progression free and overall survival. Although, many studies have identified TENT5C as a tumor suppressor gene in MM, the impact of gene loss on MM physiopathology remain to be elucidated. Here we sought to identify drugs which may specifically target TENT5C-null MM cells using targeted and high-throughput drug screens (HTS). Methods: We generated CRISPR/Cas9 knockout (ko) of TENT5C in wild type (wt) U266 and KMS-11 MM cell lines and over-expressed (oe) TENT5C in MM cell lines with biallelic inactivation (H929 -/- and LP-1 -/-). Modified cell lines were compared to their original counterparts for gene expression, proliferation, and cell cycle differences. Targeted and HTS were performed to identify synthetic lethal pathways related to loss of TENT5C in MM that will result in specific cell death. Results: In TENT5C ko cells the RNA poly(A) polymerase activity was decreased at two TENT5C top targeted substrates, immunoglobulin kappa light chain and signal sequence receptor. Consistent with TENT5C tumor suppressor function, cell proliferation analysis showed that TENT5C ko cells had a higher growth rate than TENT5C wt cells. Cell cycle analysis, after nocodazole synchronization, showed TENT5C-null cells had an increased G2/M population compared to TENT5C-positive cells (U266 TENT5C ko 19.86% vs. TENT5C wt 2.6%, p&amp;lt;0.001; H929 TENT5C -/- 8.22% vs. TENT5C oe 1.18%, p&amp;lt;0.001), indicating an increase in cell cycle progression in TENT5C-null cells. Given our prior data indicating an increase in splicing abnormalities and non-homologous end joining (NHEJ) activation in TENT5C -/- patient samples, we performed growth inhibition curves using appropriate drugs. The spliceosome inhibitors pladienolide B and isoginkgetin affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=0.58 vs. TENT5C wt GI 50=1.43 nM, U266 TENT5C ko GI 50=7.03 vs. TENT5C wt GI 50=13.72 µM, p&amp;lt;0.001, respectively) as did NHEJ inhibitors STL127705 and SCR7 (U266 TENT5C ko GI 50=22.5 vs. TENT5C wt GI 50=62.55 µM, U266 TENT5C ko GI 50=41.93 vs. TENT5C wt GI 50=117.38 µM, p&amp;lt;0.01). These results were confirmed on the other modified cells to ensure TENT5C specificity. A HTS using the LOPAC1280 library also identified several hits specifically affecting TENT5C null cells. The hits were categorized according to their targets to identify pathway enrichment. Inhibitors targeting the cell cycle (roscovitine, apigenin, SP60012) and angiogenesis (amsacrine) were selected for validation and further investigation. The differential effect observed in the HTS data was confirmed with full dose kill-curves on the same cell line (U266) and validated in another TENT5C ko cell line (KMS-11) to ensure target specificity. For example, amsacrine, an angiogenesis inhibitor, exhibited differential activity onT ENT5C ko and TENT5C wt cells (U266 TENT5C ko GI 50=5.63 µM vs. U266 TENT5C wt GI 50=9.19, p&amp;lt;0.001; KMS-11 TENT5C ko GI 50=9.26 µM vs. KMS-11 TENT5C wt GI 50=14.38, p&amp;lt;0.001). Similarly, roscovitine, a CDK inhibitor, affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=23.33 vs. U266 TENT5C wt GI 50=34.91 µM; KMS-11 TENT5C ko GI 50=13.47 vs. KMS-11 TENT5C wt GI 50=29.33 µM, p&amp;lt;0.001). Additionally, cell cycle analysis showed that roscovitine induced a G 0/G 1 arrest in a dose-response manner in U266 and KMS-11 cells. Interestingly, TENT5C ko cells were more sensitive to the CDK inhibitor, and the arrested KMS-11 G 0/G 1 populations increased from 3.35 in TENT5C wt to 6.46 % in TENT5C ko and from 15.51 in TENT5C wt to 21.15 % in TENT5C ko (p&amp;lt;0.05), after treatment with 12.5 and 25 µM of roscovitine, respectively. The same trend was also validated in U266 TENT5C ko cells to ensure target specificity. Conclusion: Our results indicate a tumor suppressor role of TENT5C in MM, with antiproliferative properties and a susceptibility to spliceosome and NHEJ inhibitors. The HTP screen identified other targets that are currently undergoing validation leading to the identification of the molecular mechanisms underlying this function.

Chimeric Antigen Receptor T Cells Targeting LILRB4, an Immunoreceptor Mediating T-Cell Suppression, Are Potently Effective in Multiple Myeloma

Significant progress has been made in the treatment of multiple myeloma (MM) however, no strategy to date has proven curative and relapse invariably occurs. We recently developed an integrated pipeline to identify biologically and therapeutically relevant targets for MM immunotherapy by using Mass-Spectrometry analyses from seven MM cell lines and RNA-seq data from 900+ patients. Starting from 4,000+ candidates, ILT3 aka LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor emerged as a promising candidate target: it has absent/low expression in most normal tissues and high and frequent expression in many primary patient samples we used for validation, similarly to BCMA (Di Meo et al., Cell Reports Medicine 2023). We sought to examine its functional role in MM and develop Chimeric Antigen Receptor (CAR) T-cells targeting LILRB4. Interrogating RNA-seq data from MM patients, LILRB4 expression results higher in patients with high-risk cytogenetics such as t:(14;16), t:(14;20), and t:(8;14) translocations compared to patients with no genomic abnormalities. Investigating function, we find no difference between cell proliferation of U266/KMS11/OPM2 genetically depleted of LILRB4 (KO) compared to controls by measuring cell growth in vitro for 120 hours and MTS assays. Similarly, when KO and control cells are seeded in plastic chambers containing a porous membrane and serum-free medium, both KO and control cells are attracted to the lower chambers containing FBS, suggesting that LILRB4 is also not required for MM cell migration. Despite these initial observations, RNAseq data from LILRB4 KO and overexpressing (OE) U266 MM cells identify ~3,000 differentially expressed genes compared to controls. Thus, we next investigate potential extrinsic effects on T-cell proliferation by co-culturing T-cells from healthy donors with LILRB4 KO or OE U266 MM cells (T:E ratio 1:2). T cells are previously activated with anti-CD3/CD28-coated beads and IL-2. 5 days after activation, T cells and LILRB4 KO U266 MM are seeded in the same well plate. After 48h of co-culture, T-cell proliferation is decreased (-30%) in the presence of LILRB4 U266 OE MM cells, whereas it is increased (+50%) when the co-culture includes LILRB4 U266 KO cells. CD25 expression is also reduced by 20% on T cells co-cultured with LILRB4 OE cells and increased by the same extent when T cells are co-cultured with LILRB4 KO cells, suggesting a role as a negative immune receptor. This effect seems dependent on the direct cell-cell interaction given that when T and MM cells are separated by a transwell in co-culture, no effect on the T cell proliferation is observed. Finally, we develop multiple Chimeric Antigen Receptor (CAR) constructs targeting LILRB4, by using different single-chain variable fragments (scFVs) and rearranging the variable regions of the heavy (VH) and light (VL) regions. We used lentiviral LILRB4 scFV-41BB-CD3zeta vectors. LILRB4 CAR T-cells specifically kill U266, H929 and KMS11 MM cell lines in vitro with cytotoxicity ranging from 30 to 80% depending on the construct (T:E ratio 1:10). Equal efficacy is observed in U266 cells resistant to Bortezomib and BCMA KO U266cells. No effect on LILRB4 null cells is noted. These data suggest that LILRB4 is a negative immune receptor that is active on malignant plasma cells mediating T-cell suppression, similar to previously described data in leukemia (Deng et al., Nature 2018). Our work supports the rationale development of ILT3 CAR-T cell therapy as a viable therapeutic approach for MM patients especially high-risk patients. Ongoing studies are investigating the functional role of LILRB4 in mediating the cross-talk between MM ad immune cells.

Establishment of A Three-Dimensional Culture Condition for The U266 Cell Line Based on Peripheral Blood Plasma-Derived Fibrin Gels.

The study of pathophysiology as well as cellular and molecular aspects of diseases, especially cancer, requires appropriate disease models. In vitro three-dimensional (3D) structures attracted more attention to recapitulate diseases rather than in vitro two-dimensional (2D) cell culture conditions because they generated more similar physiological and structural properties. Accordingly, in the case of multiple myeloma (MM), the generation of 3D structures has attracted a lot of attention. However, the availability and cost of most of these structures can restrict their use. Therefore, in this study, we aimed to generate an affordable and suitable 3D culture condition for the U266 MM cell line. In this experimental study, peripheral blood-derived plasma was used to generate fibrin gels for the culture of U266 cells. Moreover, different factors affecting the formation and stability of gels were evaluated. Furthermore, the proliferation rate and cell distribution of cultured U266 cells in fibrin gels were assessed. The optimal calcium chloride and tranexamic acid concentrations were 1 mg/ml and 5 mg/ml for gel formation and stability, respectively. Moreover, the usage of frozen plasma samples did not significantly affect gel formation and stability, which makes it possible to generate reproducible and available culture conditions. Furthermore, U266 cells could distribute and proliferate inside the gel. This available and simple fibrin gel-based 3D structure can be used for the culture of U266 MM cells in a condition similar to the disease microenvironment.

Identification of long noncoding RNA NEAT1 as a key gene involved in the extramedullary disease of multiple myeloma by bioinformatics analysis

ABSTRACTObjectiveLong non-coding RNAs (lncRNAs) are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in extramedullary disease of multiple myeloma (EMD), we analyzed the expression profile of lncRNAs in EMD.MethodsThree pairs of EMD patients and their intramedullary MM cells were screened by microarray first. We extracted data from gene chips and made an identification of lncRNAs and mRNAs with significant differences between EMD group and non EMD group. WGCNA confirmed the EMD related gene module and drew a heat map to further determine the key gene lncRNA-NEAT1. In the meantime, bone marrow and extramedullary samples (hydrothorax and ascites) were collected from 2 MM patients and subjected to single-cell RNA-seq. Single cell Transcriptome analysis was conducted to verify the gene expression difference of malignant plasma cells derived from intramedullary and extramedullary. Then we verified high expression level of lncRNA-NEAT1 in EMD patients by using quantitative real-time PCR (qRT-PCR) and analyzed the correlation between expression patterns and survival and molecular genetics analysis of the LncRNA (NEAT1) involved in MM patients. At last, cell experiments were conducted to observe the effects of down-regulation of NEAT1on the proliferation, cell cycle and PTEN pathway related proteins of multiple myeloma cell lines U266 and RPMI8226.ResultsWe identified one of the EMD related key gene is lncRNA-NEAT1. Compared with patients without extramedullary lesions, intramedullary MM cells in EMD patients expressed NEAT1 highly. The outcome of parallel single-cell RNA sequencing (RNA-seq) revealed NEAT1 level of plasma cells came from pleural effusion /ascites increased significantly compared with myeloma-stricken bone marrow. By survival and molecular genetic analysis, NEAT1 gene expression was not associated with OS and PFS in MM patients. However, the expression of NEAT1 is related to adverse therapeutic reactions and the progression of MM. We found that the expressions of NEAT1 were negatively associated with albumin levels and were positively associated with gain of chromosome 1q, IGH-CCND1, IGH@-FGFR3/WHSC1,and IGH-MAF gene fusion, respectively. At the level of cell experiment, CCK-8, soft agar clone formation experiment and CFSE staining showed that down regulating NEAT1 could inhibit the proliferation of U266 and RPMI8226 cells; Cell cycle detection showed that down-regulation of NEAT1 would interfere with the cell cycle process, and RPMI 8226 cells were blocked in G1 phase. Western blot analysis showed that when the expression of NEAT1 was down regulated in U266 and RPMI 8226 cells, the expression of PTEN and p-PTEN (phosphorylated phosphatase and tensin homologue) was up-regulated, and the expression of PI3K, p-PI3K (human phosphorylated inositol 3 kinase), Akt, p-Akt (phosphorylated protein kinase B).Discuccion and conclusionThis study provides novel insights into the lncRNA-NEAT1 and reveals that NEAT1 maybe a potential lncRNA biomarkers in EMD.

The Evidence That 25(OH)D3 and VK2 MK-7 Vitamins Influence the Proliferative Potential and Gene Expression Profiles of Multiple Myeloma Cells and the Development of Resistance to Bortezomib.

Multiple myeloma (MM) remains an incurable hematological malignancy. Bortezomib (BTZ) is a proteasome inhibitor widely used in MM therapy whose potent activity is often hampered by the development of resistance. The immune system is vital in the pathophysiology of BTZ resistance. Vitamins D (VD) and K (VK) modulate the immune system; therefore, they are potentially beneficial in MM. The aim of the study was to evaluate the effect of BTZ therapy and VD and VK supplementation on the proliferation potential and gene expression profiles of MM cells in terms of the development of BTZ resistance. The U266 MM cell line was incubated three times with BTZ, VD and VK at different timepoints. Then, proliferation assays, RNA sequencing and bioinformatics analysis were performed. We showed BTZ resistance to be mediated by processes related to ATP metabolism and oxidative phosphorylation. The upregulation of genes from the SNORDs family suggests the involvement of epigenetic mechanisms. Supplementation with VD and VK reduced the proliferation of MM cells in both the non-BTZ-resistant and BTZ-resistant phenotypes. VD and VK, by restoring proper metabolism, may have overcome resistance to BTZ in vitro. This observation forms the basis for further clinical trials evaluating VD and VK as potential adjuvant therapies for MM patients.

The JAK Inhibitor Pacritinib Suppresses the Checkpoint Proteins Pvrig and Tigit Expression on T Cells in Multiple Myeloma Patients

Introduction: Immunotherapies blocking immune checkpoints have been effective in cancer treatment, but these drugs have significant toxicities. Previously, we have shown the JAK1/2 inhibitor ruxolitinib has beneficial effects for treating multiple myeloma (MM) patients. We have also found that it reduces expression of two immune checkpoint proteins, PD-L1 and B7-H3, in MM bone marrow (BM). Pacritinib is another JAK inhibitor that specifically targets JAK2, but not JAK1. We hypothesized pacritinib may inhibit other immune checkpoints in MM. The immune checkpoint proteins TIGIT and PVRIG have been shown to be over-expressed in MM BM, and we evaluated whether pacritinib could reduce expression of these checkpoint inhibitors and the effect of this JAK inhibitor on MM growth. Methods: The human MM cell lines U266, RPMI8226 and MM1s were obtained from ATCC. Mononuclear cells (MCs) were isolated from MM patients' BM aspirates. Cell viability was quantified using the MTS cell proliferation assay. We determined the IC curves for the MM cell lines RPMI8226, U266, and MM1S and fresh BMMCs from the MM patients using this JAK inhibitor and other anti-MM drugs. Specifically, we examined the anti-proliferative effects of pacritinib alone and in combination with dexamethasone, pomalidomide or bortezomib using these 3 MM cell lines and 1 patient's fresh MM BMMCs. In addition, 5 fresh MMBMMCs (3 patients with progressive disease [PD] and 2 patients in complete remission [CR]) were treated with pacritinib alone for 48 hours in vitro, followed by flow cytometric analysis to determine TIGIT and PVRIG expression using an Attune flow cytometer. Results: MM tumor cells from the 3 human myeloma cell lines were exposed to increasing concentrations of pacritinib from 0.1 to 100 µM. The results of anti-MM effects on cell viability showed that pacritinib alone inhibited MM tumor cell proliferation in all three MM cell lines (RPMI8226, U266 and MM1S) in a concentration dependent manner. We also found the viability in one PD patient was significantly decreased when pacritinib was combined with dexamethasone or bortezomib compared to these drugs alone, but there was no effect when this JAK inhibitor was combined with pomalidomide. Next, flow cytometric analysis showed PVRIG and TIGIT expression in MM BMMCs were significantly decreased after pacritinib treatment in vitro for 48 hours in both PD and CR patients, demonstrating that pacritinib inhibited PVRIG and TIGIT in MMBM. Specifically, pacritinib reduced the expression of both PVRIG and TIGIT on CD8+ T cells. Thus, blockade of PVRIG and TIGIT on CD8+ T cells with pacritinib may restore T cell anti-tumor activity. Summary: We demonstrated that pacritinib can inhibit expression of PVRIG and TIGIT in MM BM, and specifically blocks PVRIG and TIGIT expression on CD8+ T cells. We also show the anti-MM effects of pacritinib both alone and in combination with other anti-MM drugs. Pacritinib can be potentially beneficial for treating myeloma patients due to its inhibitory role on PVRIG and TIGIT expression which may enhance tumor cell killing by CD8+ T cells.

Evaluation of Repotrectinib's Anti-MM Effects in Combination with Bortezomib, Carfilzomib, Cyclophosphamide, and Dexamethasone in Vitro and Ex Vivo

Introduction: The proteasome inhibitors bortezomib and carfilzomib are anticancer drugs which are indicated for the treatment of multiple myeloma (MM). The alkylating agent cyclophosphamide is also an effective drug against MM. Glucocorticoids including dexamethasone induce MM cell apoptosis and are effective in MM. However, the disease remains incurable with currently available therapies. Repotrectinib (Turning Point Therapeutics, San Diego, CA) is a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI). Repotrectinib also inhibits the Janus kinase (JAK) 2 and indirectly down-regulates STAT5 phosphorylation in the SET2 cancer cell line harboring a JAK2 V617F tyrosine kinase mutation (Drilon et al 2018). We have previously shown the preclinical anti-MM effects of JAK1/2 inhibitors in combination with corticosteroids and other anti-MM drugs in vitro and in vivo. Although activation of JAK2 and PI3K signaling is often detected in hematological malignancies, there is no data regarding the potential anti-MM effects of repotrectinib alone or in combination with other anti-MM drugs. Thus, we evaluated the anti-myeloma effects of repotrectinib alone and in combination with bortezomib, carfilzomib, cyclophosphamide, and dexamethasone in vitro using MM cell lines and ex vivo using the human MM xenograft model LAG- λ1. Methods: The human MM cell lines U266, RPMI8226 and MM1S were obtained from ATCC. MCs were isolated from MM patients' BM aspirates. Cell viability was quantified using the MTS assay. For the ex vivo studies, cells were isolated from the MM xenograft LAG-λ1 after it was established in SCID mice. We determined the IC curves using the MM cell lines and fresh BMMCs from 6 MM patients. Next, we combined repotrectinib with other active anti-MM drugs (carfilzomib, bortezomib, cyclophosphamide, and dexamethasone) using these same three MM cell lines and BMMCs from 6 MM patients to evaluate the anti-proliferative effects of these combinations. Results: We first evaluated the anti-MM effects of repotrectinib in combination with bortezomib or carfilzomib by using the MM1S, RPMI-8226, and U266 MM cell lines. Specifically, the results with repotrectinib in combination with carfilzomib showed enhanced anti-MM effects when compared to repotrectinib alone or carfilzomib alone. We also determined the anti-MM effects of repotrectinib alone and in combination with carfilzomib by using isolated LAG- λ1 MM xenograft cells. Repotrectinib alone inhibited tumor cell proliferation in a concentration dependent manner (IC50 4.5 µM) and in combination with carfilzomib synergistically inhibited LAG- λ1 MM tumor cell proliferation. These results were not seen when it was combined with bortezomib. We studied BMMCs from 3 PD and 3 CR patients using a cell proliferation assay. The results showed that repotrectinib alone minimally inhibited MM tumor cell proliferation among patients with PD. Repotrectinib combined with carfilzomib showed enhanced anti-proliferative effects compared with either drug alone among BMMCs from PD patients but not those in CR. In contrast, bortezomib did not show this enhanced anti-proliferative effect. We also determined the anti-MM effects of repotrectinib in combination with cyclophosphamide using ex vivo and in vivo studies. The results showed that repotrectinib in combination with cyclophosphamide enhanced the inhibition of LAG- λ1 MM tumor cell and fresh MM BMMC proliferation compared with repotrectinib or cyclophosphamide alone. We further determined the anti-MM effects of repotrectinib in combination with dexamethasone. The results showed that repotrectinib in combination with dexamethasone reduced LAG- λ1 MM cell proliferation in a concentration dependent manner and enhanced inhibition in 2 PD patients' samples but not the other PD patient or 3 CR patients' samples. Summary Single agent repotrectinib decreased MM tumor cell proliferation in all 3 MM cell lines and reduced LAG- λ1 tumor cell proliferation ex vivo. Repotrectinib also reduced proliferation of BMMCs from MM patients with PD but not those in CR. Repotrectinib in combination with carfilzomib, cyclophosphamide or dexamethasone showed enhanced anti-proliferative effects compared with the drugs alone. The results suggest that this kinase inhibitor may have activity in treating MM patients in combination with other active anti-MM agents. Further evaluation is warranted.

Identification and in-vitro analysis of potential proteasome inhibitors targeting PSMβ5 for multiple myeloma

The proteasome subunit β5 (PSMβ5) is a chief target of proteasome inhibitors (PIs) for treatment of multiple myeloma (MM). The relevance of PSMβ5 mutations and their functional impact on the development of resistance to PIs have been demonstrated recently. Therefore, this present study deals with an in-depth E-pharmacophore based screening and repurposing of FDA-approved drugs that could target PSMβ5 for MM. Our molecular docking-based investigation revealed risedronate and zoledronate as potential alternative therapeutic molecules for targeting the PSMβ5 gene. Risedronate and zoledronate displayed high binding affinity (−9.51 and −8.56 kcal/mol respectively) to PSMβ5. Moreover, 100 ns molecular dynamics simulation analysis of docking complexes revealed risedronate and zoledronate with a superior binding free energies and stable interactions with PSMβ5. The RMSD plot shows that the risedronate-PSMβ5 (mean: 0.24 nm) and zoledronate-PSMβ5 (mean: 0.25 nm) complexes are identical and stays stable until 100 ns. We further validated the activity of zoledronate in MM cell lines RPMI8226 and U266 where zoledronate showed significant anti-proliferative and apoptotic activity. Importantly, zoledronate showed an enhanced anti-proliferative activity when combined with bortezomib in MM cell lines. Thus, this study demonstrates that combining bortezomib with zoledronate could have a significant impact on reducing MM cell growth and can be an alternative strategy for treating MM.

Anti-Myeloma Effects of Panobinostat in Combination with Duvelisib

Introduction: Panobinostat (Secura Bio) is a non-selective histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. Duvelisib (Secura Bio) is an FDA-approved oral drug and a potent small molecule inhibitor of the delta (δ) and gamma (γ) isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. There is no data regarding in combination of an HDACi with duvelisib for multiple myeloma (MM) treatment. We evaluated the anti-myeloma effects of the panobinostat in combination with duvelisib.Methods: The human MM cell lines U266, RPMI 8226 and MM1s were obtained from ATCC. Primary MM tumor cells were isolated from MM patients' bone marrow (BM) aspirates and mononuclear cells (MCs) were isolated. The cells were seeded at 10 5 cells/well in 96-well plates and incubated for 24 h in the presence of vehicle, panobinostat or duvelisib alone, or the two drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. For the apoptosis assay, U266, RPMI 8226, MM1s, or primary MM tumor cells were cultured at 1 x 10 6 cells per well for 48 hours at 37°C in 5% CO2 with or without the addition of panobinostat or duvelisib. Next, the cells were washed twice with PBS, re-suspended in binding buffer, and stained with FITC-conjugated annexin V and fluorescent dye propidium iodide (PrI) following annexin V assay standard protocol (BioVision, USA). For each drug treatment, 1 x 10 5 gated events were recorded.Results and Discussion: The MTS cell proliferation assay showed that panobinostat alone inhibited cell proliferation of the MM cell lines U266 and MM1s but not RPMI8226. Panobinostat alone induced concentration-dependent inhibition of BMMCs. Panobinostat (20nM) in combination with duvelisib markedly increased inhibition of primary MM cell proliferation. Panobinostat alone induced MM cell apoptosis in all three MM cell lines. Panobinostat in combination with duvelisib enhanced MM cell apoptosis in MM1s but not U266 and RPMI 8226 cells. We further examined apoptosis of primary MM cell treated with panobinostat alone and in combination with duvelisib. The results showed panobinostat alone induced primary MM cell apoptosis and in combination with duvelisib markedly increased the induction of apoptosis. This study illustrates that the combination of panobinostat and duvelisib shows potent anti-MM effects in vitro and we are currently evaluating the anti-MM effects of this combination in vivo using our human MM xenograft models.*The study was partially funded by Secura Bio, Inc DisclosuresNo relevant conflicts of interest to declare.

Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

CD200 Expression on Multiple Myeloma Cells Induces Attenuation of T Cell-Mediated Cytotoxicity Via DOK2

IntroductionThe CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape.MethodsPrimary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed.ResultsOf n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function.ConclusionOur study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. DisclosuresEinsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

OAB-017: Attenuation of T cell cytotoxicity mediated by CD200 expression on multiple myeloma cells

<h3>Background</h3> The CD200/CD200 receptor (CD200R) axis is known to entail an immunoregulatory function in myeloid derived cells. The ligand, CD200, is implicated in solid and hematological malignancies to be associated with poor prognosis. Multiple myeloma (MM), a plasma cell malignancy, shows strong expression of CD200 in the majority of patient derived primary cells. However, the downstream mechanism upon CD200 ligand binding to the CD200R in T cells is not well understood. In this study, we evaluate the role of CD200 as a potential immune checkpoint in MM and seek to unravel the mechanism of immune escape mediated by CD200. <h3>Methods</h3> CD200 expression on patient-derived primary MM cells and MM cell lines was tested using flow cytometry. A Sleeping Beauty Transposon vector system was used to overexpress CD200 on MM cell lines. CD3/CD28-activated healthy donor T cells were co-cultured with CD200+/- MM cell lines L363, U266 and MM.1s. Using flow cytometry or luciferase assay, the co-culture was analyzed to assess cytotoxicity. To study downstream signaling effects of CD200R activation, CD3/CD28-activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody. Western blotting was performed to analyze potential downstream effector signaling. <h3>Results</h3> Approximately three-quarters of patient-derived primary MM cells (n=120) expressed CD200. In MM cell lines (n=9), no surface or cytoplasmic expression of CD200 was observed. Hence, we stably expressed CD200 on MM cell lines using a Sleeping Beauty transposon vector system. In both, flow cytometric analysis and luciferase assay, we observed up to 50% decrease in CD3+ T cell-mediated cytotoxicity in the presence of CD200-expressing MM cells. Possible differences in proliferation rates of MM cell lines due to CD200 overexpression were ruled out by performing an Alamar blue assay. In myeloid derived cells, docking protein-2 (DOK2) is known to directly interact with CD200R. DOK2 was phosphorylated upon CD200 binding in activated T cells when treated with rhCD200 in a time and concentration-dependent manner. This effect could be reversed with an anti-CD200 blocking antibody, providing a mechanistic explanation for the observed attenuation of T cell function. <h3>Conclusions</h3> CD200 expression on MM cell lines leads to attenuated cytotoxicity from primary healthy donor CD3+ T cells. Here, we provide evidence that this inhibitory mechanism in CD3+ T cells is mediated via DOK2.

In vitro comparison of the cytotoxic effects of lenalidomide alone and in combination with verapamil on myeloma cell line

Introduction Multiple myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells. High-dose chemotherapy with novel agents and autologous stem cell transplantation are options for treatment. However, MM treatment generally results in failure. The most important reason for this failure is the resistance to chemotherapeutic drugs. Various studies have been tried to combine chemosensitizer agents that increase the cytotoxic effects of the chemotherapeutics to eliminate the drug resistance. In our study, we aimed to evaluate the effect of verapamil on the cytotoxic effect of lenalidomide on the myeloma cell line. Materials and methods Verapamil is a chemosensitizer that suppresses the P-glycoprotein. In our study, lenalidomide, an immunomodulatory agent, was compared alone and in combination with verapamil for cytotoxic effects. U266 MM cell line was used in the study. At the concentrations of 0.001, 0.01, 0.1, 1, 10, 50, and 100 µM, lenalidomide alone and the combination of lenalidomide at the same concentrations with 2.5 µg/ml of verapamil were compared in terms of possible cytotoxic properties. Cell viability was measured by XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test. Results A statistically significant decrease in the inhibitor concentration, causing 50% decrease in cell proliferation (IC50) of lenalidomide, was provided via verapamil administration. Our study revealed that the cytotoxic effect of lenalidomide increases when combined with verapamil. Conclusion We aimed to understand whether the cytotoxic effect of lenalidomide, which has an important place in the treatment of MM, can be increased with an easily available drug such as verapamil. We think that more studies and meta-analyses are needed owing to the different results related to the subject in the literature, and we hope to set an example for new studies.

Apobec Family of Proteins Mediate Genomic Changes in Multiple Myeloma By Inducing DNA Damage and C>T Mutations

Multiple myeloma (MM) is a heterogeneous disease characterized by unstable genome. Spontaneous DNA damage coupled with dysregulated repair accentuates the genomic instability seen in MM. The resulting clonal heterogeneity plays a critical role in resistance to treatment and relapse. Understanding the molecular factors that contribute to genomic instability in myeloma is therefore of great importance in developing better treatment strategies. Recently, a role for the AID/APOBEC family of cytidine deaminases in generating somatic mutations has been described with in MM as well as number of other cancers. We and others have previously shown that significant number of mutations bear APOBEC signature and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in MM. We have now further explored role of APOBEC in MM with special focus on its ability to induce clonal diversity.We observed that APOBECs 3A, 3B, 3C, and 3G are highly expressed in MM. Using a fluorescent oligonucleotide based APOBEC deaminase assay to measure deaminase activity, we also observed significantly increased APOBEC activity in MM cell lines as well as primary patient MM cells. Using shRNA-mediated knockdown, we report that depletion of APOBECs in MM cell lines results in decreased DNA double strand breaks. We next investigated the impact of overexpression of APOBEC genes on genome integrity in MM cells. Over expression of APOBECs 3B or 3G in U266 and LP1 MM cell lines led to 3-5 fold increase in deaminase activity in MM cell extracts measured by APOBEC deaminase assay. Furthermore, APOBEC overexpression resulted in elevated double strand breaks as evidenced by increased gH2AX nuclear foci and by western blot. In order to determine the functional impact of APOBEC-induced mutations, we co-transfected EGFP plasmid along with APOBEC expression vectors in myeloma cells. 72-hrs after transfection, we isolated the EGFP plasmid, transformed bacteria, and sequenced the plasmid DNA isolated from 5 colonies from each group. Overexpression of APOBECs resulted in increased number of C>T mutations (3-5 fold) in the EGFP plasmid co-transfected into these cells. Interestingly, APOBEC over expression also resulted in the loss of GFP fluorescence in 20-30% of the cells compared to control cells, suggesting that overexpression of APOBECs can cause deleterious mutations leading to loss of function.APOBEC-mediated deamination of cytosine to uracil leads to generation of abasic sites that are cleaved by abasic endonuclease (APE1), a regulator of homologous recombination pathway dysregulated in MM contributing to genomic instability. Therefore, we investigated how modulating APOBEC activity affects downstream DNA damage repair pathways in the gain-of and loss-of-function cells. Using in vitro activity assays, we measured APE1 and HR activity in extracts collected from APOBEC overexpression and knockdown cells. Extracts from APOBEC overexpressing cells showed elevated APE1 and HR activity (>50%), whereas depletion of APOBEC proteins resulted in up to 80% reduction in HR and APE1 activity in the extracts. Interestingly, using a small molecule HR inhibitor we found that inhibiting HR pathway can reduce the DNA breaks induced by APOBEC overexpression. Furthermore, we investigated the acquisition of genomic changes such as amplifications, deletions, and SNVs over time, in control and APOBEC knockdown cells, using SNP arrays and whole genome sequencing (WGS). Our results show that knockdown of APOBECs reduces the accumulation of copy number changes, structural variants and mutations over time. This suggests that elevated expression of APOBECs may be contributing to genomic instability in MM cells.In summary, our results suggest that APOBEC mediated DNA damage could contribute to increased dysregulation of these important repair pathways and genome stability in myeloma. Inhibiting APOBEC activity could reduce the rate of accumulation of the ongoing genomic changes. DisclosuresNo relevant conflicts of interest to declare.

Everolimus Shows Synergistic Anti-Myeloma Effects with Bortezomib Via the AKT/mTOR Pathway

Introduction: Multiple myeloma (MM) is characterized by the proliferation of malignant plasma cells and a subsequent overabundance of monoclonal paraprotein (M protein). Everolimus ,as an oral mammalian target of rapamycin (mTOR) inhibitor, plays an important role in multiple myeloma. In the phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients ,the median time to progression was 90 days (range, 13 to 278 days),and no dose-limiting toxicity was observed at daily dose of 10 mg. At the same time, the observed responses are promising in some testing combination strategies. The pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with everolimus caused potent G0/G1 arrest and induced pronounced apoptosis, the combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. Everolimus enhances the cytotoxicity of bendamustine in multiple myeloma cells through a network of pro-apoptotic and cell-cycle-progression regulatory proteins.Down regulation of vascular endothelial cell growth factor and everolimus synergistic antitumor activity in multiple myeloma cells. Furthermore, In a phase I study of everolimus in combination with lenalidomide in relapsed or refractory multiple myeloma patients, the overall response rate was 65% and median progression-free survival was 5.5 months. Bortezomib was the first therapeutic proteasome inhibitor to be tested in humans with MM. However, the combination of bortezomib and everolimus for the treatment of MM is still less reported.Methods: To evaluate the in vitro and in vivo anti-tumor activity of the combination of everolimus with bortezomib.CCK-8 was used to explore the proliferation of MM cells. Flow cytometry was used to determine the effect on apoptosis of cells.Western blot and Immunohistochemical staining was used to determine the epression of proteins.Results: In our study, the experimental results demonstrated that everolimus combined with bortezomib suppressed cell proliferation. Treatment with 20 nM everolimus in combination with 10 nM bortezomib for 72h achieved the lowest cell survival rate in both U266 and LP-1 MM cell lines. The combination of everolimus and bortezomib was synergistic in both cell lines indicated by combination index (CI) values of <0.8.Flow cytometry was used to determine the effect on apoptosis of bortezomib in combination with everolimus. everolimus alone and bortezomib alone induced approximately 22.5% and 17.2% apoptosis, combination treatment induced28.8% apoptosis in U266 cells, and the results were 15.6%,10.0% and 16.7% in LP-1 cells. Western blot was used to explore the mechanisms. The expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated while the expression of Caspase-3was significantly up regulated in combination group than other alone group. Furthermore, Synergistic antitumor effect of everolimus and bortezomib was observed in the animal model carrying the myeloma xenograft on the 25th day after tumor implantation . The results of immunohistochemical staining further demonstrated that the expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated and the expression of Caspase-3 was significantly up regulated in combination group.Conclusion:Our results shows synergistic antitumor effects of the combination therapy of bortezomib and everolimus induced myeloma cell apoptosis in vitro and in vivo xenograft mouse model ,which mainly through the inhibition of the AKT/mTOR pathway. Our results provided provide a rationale application for the combinatorial treatment of MM with proteasome inhibitor agents and mTOR inhibitors in future clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Targeted Remodeling of the Tumor Microenvironment Via Mechanical Stimulation Defines a Novel Therapeutic Strategy in Myeloma Bone Disease

Multiple myeloma (MM) patients develop devastating osteolytic bone disease causing non-healing bone fractures and pain. MM cells in the bone marrow microenvironment stimulate osteoclast activity and inhibit osteoblast and osteocyte function leading to massive bone resorption and therefore destruction. Bones have the capacity to locally adapt to changing loads and for self-repair and remodeling as dramatically seen in athletes during training. In this study we determined if mechanical loading can counterbalance osteolytic bone disease in the presence of established MM. We hypothesized that mechanical stimulation increases bone formation and decreases bone resorption by activating known bone (re)modeling pathways, which are deleteriously altered by MM cells in bone. We performed in vivo cyclic axial compressive tibial loading (Fig. 1) to MOPC315.BM BALB/c injected mice (n=19) with established MM bone disease (Fig. 2, bioluminescence imaging). The bone (re)modeling response to mechanical loading (5d/wk, over 3 wks) was investigated by longitudinal in vivo microCT imaging at days 14, 18, 23, 28 and 33 (Fig. 3, 4). Loading was administered to the left tibia starting 14 days after i.t. injection of left limbs of MOPC315.BM (n=10) and PBS (n=7) mice (left limb: εmax=2000µε determined by strain gauging, right limb was nonloaded). 15 mice served as nonloaded control mice (Fig. 3). We also analyzed a group of loaded, but not injected mice (n=10). MicroCT data (Fig. 3), such as cortical thickness (Ct.Th), cortical bone area (Ct.Ar) of loaded left limbs demonstrate that metaphyseal cortical bone formation was significantly greater and cortical porosity (Ct.Po) was lower compared to nonloaded control limbs. Our data provide for the first time an indication that mechanical loading counteracts MM induced bone loss over time. To further study mechanisms of mechanotransduction we used a small scale cell culture bioreactor system to analyze mechanical stimulation of human mesenchymal stem cells (hMSCs) and the translation of mechanical forces into biochemical signals. The bioreactor system allows for analysis of molecular mechanisms of mechanotransduction using a transcription factor activator protein (AP1) luciferase reporter gene construct. Cyclic stretching of telomerase immortalized hMSCs (hMSC-TERT cells) which were stably transfected by an AP1 construct resulted in stimulation of AP1 activity as measured by luciferase gene expression. We analyzed how MM cells interfere with mechanotransduction when co-cultured with hMSC-TERT cells. Our data show that in presence of MOPC315.BM cells and 2 (MM1S, INA-6) out of 4 human MM cell lines hMSCs can be mechanically stimulated and have osteogenic activity. In contrast the human MM cell lines Amo-1 and U266 inhibit the hMSC-TERT response to mechanical stimulation under these conditions. Biochemical and molecular genetic studies are underway to determine how MM cells in bone alter known bone (re)modeling pathways and which yet unanticipated factors regulate the anabolic response to mechanical loading in the MOPC315.BM model. Our study provides a first fundamental understanding of the mechano-biological mechanisms of anabolic bone adaption during MM bone disease in response to mechanical stimuli. We conclude that mechanical loading in the form of exercise has the potential to be a potent novel anabolic strategy for primary and secondary prevention of MM bone disease. Moreover dissection of the underlying molecular pathways will identify new targets for innovative antimyeloma strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assessing CCR1 Antagonists for Chemotaxis Inhibition in a Multiple Myeloma in vitro Model

Multiple myeloma (MM) is a plasma B-cell malignancy characterized by osteolytic bone lesions. MM cells secrete and express CCL3/MIP1α which upregulates osteoclastogenesis. Elevated CCL3 levels display a chemotactic ability on isolated osteoclast precursors. Increased levels of CCL3 in MM patients correlates with a greater disease burden, due to the increase of bone resorption, and is indicative of a worse prognosis when compared to MM patients exhibiting lower CCL3 levels. CCR1, a GPCR chemokine receptor, is endogenously expressed on MM cells, and can bind with CCL3. In a previous study, the RPMI8226 MM cell line, CCL3-mediated CCR1 chemotaxis was inhibited in a dose-dependent manner by six CCR1 antagonists (AZD4818, BX471, CCX354, CP481715, MLN3897, PS031291). In this study, we assessed the MM cell line U266 as well as a transfected cell line, U266_CCR1. While U266 cells express lower levels of CCR1 than RPMI8226, U266_CCR1 express higher levels of CCR1. Cells were treated with a serial dilution of the same six CCR1 antagonists and chemotaxis to either supernatant from osteoclast precursor RAW 264.7 cells or Fetal Bovine Serum (FBS) in which a multitude of growth factors and chemokines are present was examined. We hypothesized the six CCR1 antagonists would result in a dose-dependent inhibition of chemotaxis similar to that seen with RPMI8226. Instead, we found only two of the compounds (AZD4818 and BX471) inhibited chemotaxis of the U266 and U266_CCR1 MM cell lines towards RAW 264.7 supernatant or FBS. For both AZD4818 and BX471 there were differences between the chemotactic response of the U266 and U266_CCR1 cell lines, with the U266_CCR1 cell line having a greater degree of inhibition, suggesting the inhibition is driven by CCR1.

Transcription factor EB related autophagy in the treatment of multiple myeloma and its mechanism

目的明确硼替佐米和（或）西拉美新作用于多发性骨髓瘤（MM）细胞株后细胞增殖、转录因子EB（TFEB）核转位表达变化及自噬水平，为进一步探讨TFEB对自噬的调控机制提供依据。方法体外培养MM细胞株RPMI8226及U266，并以一定浓度的硼替佐米和西拉美新处理MM细胞，CCK-8法检测细胞增殖，实时定量PCR和Western blot法检测TFEB、自噬相关因子LC3B、Beclin1、p62、LAMP1的mRNA和蛋白相对表达量。结果随着硼替佐米浓度增加及作用时间延长，两个细胞系的增殖抑制率增高（P<0.05）。硼替佐米和西拉美新联用对上述MM细胞株的增殖有协同抑制作用（P<0.05）。空白对照组、单药组、联合用药组处理MM细胞株后，细胞质中TFEB的mRNA和蛋白相对表达量依次下降（P<0.05），细胞核中TFEB的mRNA和蛋白相对表达量依次上升（P<0.05），自噬相关因子LC3B、Beclin1、LAMP1的mRNA和蛋白相对表达量依次上升，p62的mRNA和蛋白相对表达量依次下降（P<0.05）。结论硼替佐米和西拉美新具有协同抑制MM细胞增殖作用，与其诱导MM细胞株自噬表达增强相关，发生核转位的TFEB表达亦增强。