RPMI-8226 Multiple Myeloma Cells Research Articles

Lactoferrin-Derived Peptide Chimera Induces Caspase-Independent Cell Death in Multiple Myeloma

Lactoferrin-derived peptide chimera is a synthetic peptide that mimics the functional unit of lactoferrin with antibacterial activity. Although LF has anticancer effects, to the best of our knowledge, its effects on multiple myeloma have not yet been studied. We explored the potential of a lactoferrin-derived chimera for multiple myeloma treatment, a malignant clonal plasma cell bone marrow disease. The lactoferrin-derived chimera effectively inhibited MM1S, MM1R, and RPMI8226 multiple myeloma cell growth, and induced the early and late phases of apoptosis, but not in normal peripheral blood mononuclear cells. Furthermore, the lactoferrin-derived chimera modulates the relative expression of genes involved in survival, apoptosis, and mitochondrial dysfunction at the transcriptional level. Mitochondrial analysis revealed that lactoferrin-derived chimera triggered oxidative stress in multiple myeloma cells, leading to reactive oxygen species generation and a decline in mitochondrial membrane potential, resulting in mitochondrial dysfunction. Although lactoferrin-derived chimera did not cause caspase-dependent cell death, it induced nuclear translocation of apoptosis-inducing factor and endonuclease G, indicating the initiation of caspase-independent apoptosis. Overall, the lactoferrin-derived chimera induces caspase-independent programmed cell death in multiple myeloma cell lines by increasing the nuclear translocation of apoptosis-inducing factor/endonuclease G. Therefore, it has potential for multiple myeloma cancer therapies.

MiR‑1343‑3p inhibits autophagy by directly targeting ATG7 in multiple myeloma cells.

Multiple myeloma (MM) is the second most common type of hematological malignancy globally. Despite application of several new drugs, such as daratumumab, bortezomib/lenalidomide/dexamethasone, in combination with hematopoietic stem cell transplantation, overall prognosis remains poor and the pathological mechanism of MM is still unknown. The present study used TargetScan to predict autophagy-related 7 (ATG7) as a candidate target gene of microRNA (miR)-1343-3p and confirmed the interaction between miR-1343-3p and the ATG7 3' untranslated region (3'UTR) using a dual-luciferase reporter assay. In U266 and RPMI-8226 MM cell lines, miR-1343-3p mimic transfection decreased mRNA and protein levels of ATG7, while miR-1343-3p inhibition increased ATG7 expression levels using reverse transcription-qPCR and western blot analysis. miR-1343-3p mimic transfection inhibited U266 and RPMI-8226 cell survival. Finally, miR-1343-3p regulated ATG7 and autophagy in MM cells using western blot analysis. The present findings suggested that miR-1343-3p may regulate ATG7 and autophagy by directly targeting the 3'UTR of ATG7. To the best of our knowledge, there are no direct data showing the roles of miR-1343-3p in development of MM; however, miR-1343-3p may be considered a potential target for MM treatment.

Ultradiluted Homeopathic Medicines Cause Apoptosis in RPMI-8226 Multiple Myeloma Cells in vitro: a Pilot Study.

Multiple myeloma (MM) is the second most common type of cancer among hematological malignancies and is difficult to treat. Although controversial in nature, homeopathy's effects have been tested on a wide range of cancer cell types in vitro, as well as clinically. However, homeopathic medicines have yet to be tested in MM cells. In this preliminary study, we investigated the effects of Arsenicum album, Hecla lava, Carcinosinum and Carboneum sulphuratum 200C on a human MM cell line. The RPMI-8226 MM cell line was cultured in vitro for up to 96 hours and treated with each of four homeopathic preparations. The spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric Annexin V-PE/7-actinomycin D (7-AAD) and propidium iodide (PI) staining were each used to examine cell viability, apoptosis and cell cycle, respectively. The MTT assay showed that all four homeopathic preparations reduced cell viability over time when compared to the control group cells, especially at 72 and 96 hours whereby only 50% of cells remained viable. Similarly, after 96 hours of treatment, the proportion of viable cells was significantly decreased and the proportion of early apoptotic (Annexin-V-PE +/7AAD-) cells was significantly increased for all four homeopathic preparations. Based on the PI-staining cell cycle data, cells treated with Hecla lava and Carboneum sulphuratum showed a statistically significant accumulation in the sub-G0/G1 phase of the cell cycle (p < 0.05). This is the first study to demonstrate that each of four homeopathic medicines causes apoptosis in a MM cell line. Further exploration of the potential of Arsenicum album, Hecla lava, Carcinosinum and Carboneum sulphuratum as a complementary therapeutic option in MM is warranted.

MiR-26 inhibits proliferation and promotes apoptosis of multiple myeloma cells by targeting BNIP3.

This study was carried out to investigate the molecular mechanism of microRNA-26 (miR-26) targeting BNIP3 to mediate proliferation and apoptosis of multiple myeloma (MM) cells. The expression of miR-26 and BNIP3 in MM and normal tissues was detected by qRT-PCR and Western blot. According to the average expression of miR-26 and BNIP3, the patients were divided into 12 cases with high miR-26 expression group, 18 cases with low miR-26 expression group, 20 cases with BNIP3 high expression group, and 10 cases with BNIP3 low expression group. The correlation between the expression of miR-26 and BNIP3 and the clinicopathological characteristics of MM patients was compared and analyzed. The effect of up-regulation of miR-26 expression and BNIP3 overexpression on the proliferation of multiple myeloma cells RPMI8226 was examined by MTT assay. Flow cytometry was used to detect the effect of miR-26 expression and BNIP3 overexpression on the apoptosis of RPMI8226 cells. The dual luciferase reporter assay validated the targeted regulation of miR-26 on BNIP3. The expression level of miR-26 in MM tissues was lower than that in normal tissues (P<0.05), and the expression level of BNIP3 in MM tissues was higher than that in normal tissues (P<0.05). miR-26 was closely related to clinical stage, M protein type and light chain type (P<0.05), while BNIP3 was closely related to M protein type and light chain type (P<0.05). After up-regulating miR-26 expression, cell viability was significantly decreased (P<0.05), apoptosis rate was significantly increased (P<0.05) Dual luciferase reporter experiments confirmed that miR-26 could target BNIP3 and negatively regulate the expression of BNIP3 (P<0.05). Overexpression of BNIP3 reversed the effect of up-regulation of miR-26 expression on proliferation and apoptosis of RPMI8226 cells. Up-regulation of miR-26 expression inhibits MM cell proliferation and promotes apoptosis by targeting BNIP3.

Melphalan-Induced Multiple Myeloma Cells Exhibit a Senescent-like Dormant Phenotype

Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. One mechanism that may contribute to MM relapse is tumor dormancy. Dormant MM cells may persist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells may then exit dormancy following treatment to drive disease relapse. Dormancy shares many features with cellular senescence, a state of stable growth arrest induced by cell stress. Chemotherapies that produce genotoxic stress have been associated with therapy-induced senescence (TIS). One such genotoxic agent, melphalan (Mel), is routinely administered to MM patients for myeloablation prior to autologous bone marrow transplant. We hypothesized that High Dose Mel (HDM) induces a senescent-like dormant phenotype in surviving MM cells. To test our hypothesis, we developed an in vitro model for HDM. We identified 10µM Mel as a concentration to induce &amp;gt;90% cell toxicity (HDM) in 5TGM1 mouse MM cells over 10 days. 5TGM1-GFP MM cells were cultured with HDM or vehicle (Veh) for 6 hours; cells were then cultured in normal growth medium in the presence of primary mouse (C57BL/6) bone marrow stromal cells (BMSCs). Co-cultures were imaged by fluorescent microscopy every other day to quantify cumulative population doublings (CPD) and cell morphology over the following 10 days. The effect of HDM on CPDs was also evaluated in the human RPMI-8226 MM cell line. 5TGM1-GFP cells were isolated by FACS on day 10 for evaluation of senescence histological characteristics and gene expression. All studies represent n=3 independent experiments unless otherwise indicated. HDM-5TGM1 cells exhibited a significant reduction in mean CPDs over 10 days compared to Veh (HDM 0.32±0.31, Veh 6.72±0.69, p&amp;lt;0.0001), indicating stable growth arrest in HDM treated cells consistent with senescence. HDM-5TGM1 cells were also significantly larger versus Veh (median size in pixels units: HDM 413.2±99.98, Veh 207±8.88, p&amp;lt;0.05), consistent with classical enlarged morphology in senescent cells. Preliminary assessment (n=1) of HDM-RPMI-8226 cells showed similar growth arrest (mean cumulative doublings: HDM 0.058±0.101, Veh 4.185±0.358) and enlargement (mean size in pixel units: HDM 804.1±6.048, Veh 526.9±2.115), suggesting a translatable mechanism in humans. FACS-sorted 5TGM1-GFP cells were stained for telomere associated foci (TAF), a marker of persistent DNA damage characteristic of senescent cells. HDM-5TGM1 cells showed a higher percentage of TAF+ cells (Percent TAF+ cells: HDM 39.7%±9.1%, Veh 10.6%±0.8%, n=2). Sorted HDM-5TGM1 cells also exhibited significant increases over Veh (p&amp;lt;0.001) in senescence markers ( Cdkn1a, Cdkn1c, Glb1), senescence cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), and senescence associated secretory phenotype (SASP) genes ( Ccl5, Icam1, Mmp13). In addition, HDM-5TGM1 cells showed significant increases in myeloid markers that have previously been shown to be increased in dormant 5TGM1 ( Axl, Fcer1g, Mpeg1). Single cell RNA-sequencing was used to further evaluate senescence gene expression changes in growth arrested HDM-5TGM1 cells. Gene set enrichment analysis (GSEA) of genes differentially expressed in HDM-5TGM1 versus Veh cells was performed for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescence Growth Arrest, SenGA; Inflammatory SASP, iSASP; SCAPs; and Plasma Cell Senescence; PCSen) as well as mouse ortholog versions of a published senescence and biological aging gene set (SenMayo) and KEGG curated gene sets. GSEA of HDM-5TGM1 versus Veh cells confirmed significant (FDR&amp;lt;0.25) upregulation of iSASP, MayoSen, PCSen, SenUp, SCAPs, and SenGA gene sets. Concurrently, GSEA revealed significant downregulation of KEGG DNA replication, cell cycle, and nucleotide excision repair gene sets, supporting our observations of proliferative arrest and persistent DNA damage TAF. Our findings demonstrate that HDM-MM cells exhibit multiple features of senescence, suggesting a potential mechanistic role for TIS in MM dormancy and highlight a novel therapeutic target that may be present in patients post-HDM. Thus, targeting senescence may be a novel approach to eliminate dormant MM cells and prevent relapse.

Apoptotic effect of selenium mushroom extract from Qinba on multiple myeloma cells.

Qinba selenium mushroom is a mushroom belonging to the Basidiomycetes family, which is believed to have anti- oxidant, anti-tumoral and anti-mutagenic activities. However, the efficacy of Qinba selenium mushroom against multiple myeloma has not been confirmed. The present study aimed to investigate the apoptotic effect of FA-2-b-β, the selenium mushroom extract from Qinba on multiple myeloma (MM) cells. The MM RPMI-8226 cells were treated with FA-2-b-β at different concentrations and time points. MM RPMI-8226 cell proliferation and apoptosis were detected by the Cell Counting Kit-8 (CCK-8) assay and Annexin V/propidium iodide (PI) assay, RT-QPCR and western blotting analyses were performed to determine the proteins and pathways involved. The results of the present study demonstrated that FA-2-b-β has high anti-proliferative activities and strong pro-apoptotic effects on MM RPMI-8226 cells, and its pharmacological effects on proliferation changes occurred in a dose- and time-dependent manner. In addition, we found that FA-2-b-β was able to induce cell apoptosis and promote cell cycle arrest at G0/G1 phase. In summary, the results illustrate the involvement of FA-2-b-β in mediating G0/G1 cell cycle arrest and apoptosis in MM RPMI-8226 cells, which suggested that FA-2-b-β might have therapeutic potential against multiple myeloma as an effective compound, and may provide useful information for the development of a novel therapeutic target in this area.

Aquaporin 3 inhibition suppresses the mitochondrial respiration rate and viability of multiple myeloma cells

Aquaporin 3 (AQP3) is a member of the aquaporin water channel family expressed by numerous cell types, including some cancer cells. Accumulating evidence suggests that AQP3 inhibition may impede cancer progression, but drugs targeting AQP3 are still in the early pre-clinical stage of development. Here, we examined the effect of AQP3 inhibition on multiple myeloma (MM), an incurable plasma cell malignancy. Four MM cell lines were cultured in the presence of an anti-AQP3 monoclonal antibody (mAb), the AQP3 inhibitor DFP00173, or corresponding controls, and the effects on cell viability, proliferation, apoptosis, and mitochondrial respiration capacity were compared. Both anti-AQP3 mAb and DFP00173 reduced cell growth, mitochondrial respiration rate, and electron transport chain complex I activity. Both agents also potentiated the antiproliferative efficacy of the anticancer drug venetoclax. Administration of the anti-AQP3 mAb to immunodeficient mice inoculated with RPMI8226 or KMS-11 MM cells significantly suppressed tumor growth. These data provide evidence that AQP3 blockade can suppress MM cell growth in vitro and tumor growth in mice. Thus, AQP3 inhibition may be an effective therapeutic strategy for MM.

Abstract 2916: CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells

Abstract NK (natural killer) cells have several advantages compared to T cells such as no cytokine release syndrome (CRS), neurotoxicity and GVHD in allogenic setting to kill tumors. We developed CAR-NK platform using RNA-LNP (lipid-nanoparticles) technology. High expansion of NK cells (up to 2000-fold) was achieved using K562 feeder cells with stable expression of 4-1BBL and IL-21. We showed efficient encapsulation of BCMA and CD19-CAR RNA into LNP that resulted in up to 99% expression of CAR+ CD56+ NK cells after transfection. The BCMA-CAR-NK cells more effectively than NK cells killed MM1S, H929 and RPMI8226 multiple myeloma cells and secreted IFN-gamma in a dose-dependent manner. In addition, frozen BCMA-CAR-NK cells were viable and expressed similar cytotoxic activity to not frozen expanded CAR-NK cells. In addition, frozen-thawed BCMA-CAR-NK cells showed 93% CAR expression at 24 hours post-thaw which remained &amp;gt;70% at 48 hours post-thaw. In a similar way, CD19-CAR-NK cells generated with CD19-CAR-LNP killed CD19-positive Raji and Daudi target cells and secreted high level of IFN-gamma. This, frozen/thawed CAR-NK cells demonstrated high in vitro cytotoxic activity against target cells. Manufacturing of NK cells using G-Rex system resulted in 1862-fold expansion at day 13 and 2450-fold expansion of 97% CD56+ NK cells at day 14. Transfection of BCMA-CAR RNA-LNP into 13 day expanded NK cells resulted in 95% BCMA-CAR-NK positive cells. Thus, CAR-NK cells generated with RNA-LNP technology can be used in future pre-clinical studies. Citation Format: Vita M. Golubovskaya, Jinying Sun, John Sienkiewicz, Yanwei Huang, Hua Zhou, Gongping Deng, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2916.

Abstract 6033: RA123, a new GLS1 allosteric inhibitor demonstrates in vitro and in vivo activity in multiple myeloma models

Abstract The conditional essential amino acid glutamine is utilized by tumors to sustain bioenergetic requirements in a nutrient-poor microenvironment. Based on this specific tumor need, efforts have been made to target the glutamine metabolism using glutaminase inhibitors. In this study, we present in vitro and in vivo studies demonstrating activities and target engagement of a new specific GLS1 allosteric inhibitor (RA123). In vitro, RA123 was tested in imid-resistant, CD38 low RPMI8226 multiple myeloma cell line using the ATP Cell titer Glo assay over a 72h treatment period. In vivo, RA123 was tested in mice xenografted with RPMI8226 multiple myeloma cell line. Compound was given at three different under a BID (twice a day) regimen. Tumor growth under treatment was monitored by caliper measurement for 20 days. A terminal PK/PD study on the same model was setup measuring impact of the drug on the glutamine and glutamate tumor content post 24 hours after the last administration. Over the same period, PK measurements were planned to document drug exposure in the plasma and in the tumor. Finally, in a stand-alone study, target engagement was documented using imaging modality. [18F]FSPG PET radiotracer was used to visualize and quantify impact of RA123 on tumor (RPMI8226) glutamate pool. RA123 was given at three doses under a BID regimen. Treatment was administered for 3 days, and PET signal was recorded at baseline and at end of day 3. In vitro study demonstrated that RA123 was able to induce cell killing of RPMI8226 after 72 hours of treatment. These data translated in vivo by a dose-dependent effect on cell growth. In a terminal PK/PD study, RA123 confirmed a dose-dependent impact on glutamine and glutamate tumor levels which correlated with both plasma and tumor exposure. Further confirmation of target engagement was observed with reduced PET uptake post- RA123 treatment at the three doses tested. In conclusion, this study demonstrated that RA123, a new specific GLS1 allosteric inhibitor was able to impact multiple myeloma tumor cell growth both in vitro and in vivo. This effect was associated with reduced glutamate pools and accumulation of glutamine levels within the tumor cells. Citation Format: Christophe Henry, Dimitri Gorge-Bernat, Pascal Pannier, Isabelle Meaux, Jane Cheng, Fangxian Sun, Olivier Pasquier, Philippe Lienard, Erwan Jouannot, Thierry Gouyon, Geneviève Estenne-Bouhtou, Bailin Zhang, Bérangère Thiers, Laurent Debussche, David Machnik. RA123, a new GLS1 allosteric inhibitor demonstrates in vitro and in vivo activity in multiple myeloma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6033.

Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neurotoxicity.

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2mg/kg, i.v.) on Day1 and Day4 each week for continuous 4weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

Molecular Mechanism of Busulfan Inhibiting the Malignant Biological Characteristics of Multiple Myeloma Cells

To investigate the effect of Busulfan on the malignant biological characteristics of multiple myeloma cells and explore the molecular mechanism. Multiple myeloma RPMI8226 cells were treated with Busulfan at different concentrations. Cell proliferation activity was detected by MMT assay, and cell apoptosis was detected by flow cytometry with Annexin V/PI double staining. Real-time quantitative PCR was used to detect the mRNA expression of apoptotic regulatory molecules Bax、Bcl-2 and Wnt3a/β-catenin pathway, and Western blot was used to detect the expression changes of Bax, Bcl-2 and Wnt3a/β-catenin pathway protein. Busulfan inhibited cell proliferation and induced apoptosis of myeloma RPMI8226 cells (P<0.05). After treatment with Busulfan at different concentrations for 48 h, the expression of anti-apoptotic protein Bcl-2 was decreased, the expression of pro-apoptotic protein Bax was up-regulated, and the activation of Wnt3a/β-catenin signaling pathway was inhibited to induce programmed death of RPMI8226 cells (P<0.05). Busulfan can inhibit the malignant biological characteristics of myeloma RPMI 8226 cells, and its mechanism may be related to regulating the expression of Bcl-2 family proteins and inhibiting the activation of Wnt3a/β-catenin signaling pathway.

Retracted: Role and Mechanism of circ_0058063/miR-635 Axis in the Malignant Phenotype of Multiple Myeloma RPMI8226 Cells.

[This retracts the article DOI: 10.1155/2021/4630934.].

Proteasome Regulatory Atpases PSMC2 and PSMC6 Modulate Proteosome Inhibitor Sensitivity and Correlate with Survival in Multiple Myeloma

Background: Multiple myeloma (MM) cells synthesize and secrete massive amounts of immunoglobulins and thus are exquisitely sensitive to therapeutics that disrupt proteostasis. The 26S proteasome complex is a 2.5-MDa holoenzyme comprised of >32 individual subunits responsible for the bulk of intracellular protein degradation. 26S proteasomes are comprised of barrel-shaped proteolytic core complex, the 20S core particle (CP), capped at either or both ends by 19S regulatory particles (RPs), which confer ATPase activity and specificity for ubiquitinated proteins. Six distinct ATPases (PSMC1-6) form a heterohexameric ring-like structure that rests atop the 20S CP's α-subunits and is responsible for unfolding and shuttling target proteins for degradation within the 20S CP. The proteasome inhibitor (PI) bortezomib (BTZ) is a potent, selective, and reversible drug that dramatically improved the prognosis of multiple myeloma (MM) patients and remains the backbone of induction therapy for transplant-eligible new diagnosis MM (NDMM), but response to therapy remains highly variable. Here, we predicted that gene expression analysis of publically available datasets could identify components within the ubiquitin-proteasome system (UPS) that areprognostic in patients treated with BTZ, suggesting potential new targets in MM. Methods: We interrogated gene expression profiles (GEP) from the randomized, phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study that compared BTZ with high-dose dexamethasone (DEX) in RRMM patients who had received 1-3 prior therapies. APEX datasets served as a test cohort to identify genes that influenced survival in patients treated with BTZ. Patients consented to genomic analyses of pretreatment tumor samples. CD138+ cells were isolated from BM aspirates and analyzed by RNA microarrays to generate GEP datasets. We correlated gene expression with response to BTZ or DEX and overall survival (OS). We then validated individual genes identified from the APEX analysis using the MM Research Foundation (MMRF) CoMMpass study, a longitudinal observational analysis of the clinical and molecular data for >1000 NDMM patients receiving various standard approved treatments. Finally, we used shRNA to knockdown PSMC2 in RPMI8226 and ARH77 cells and tested their sensitivity to BTZ and carfilzomib (CFZ). Results: Analysis of the APEX dataset revealed that increased expression of genes PSMC2 and PSMC6, members of the heterohexameric ATPase ring of the 19S RP, correlated with poorer OS (PSMC2- Figure 1A, PSMC6- Median OS 320 versus 620 days for upper and lower tertile PSMC6, respectively). Using the CoMMpass database, increased PSMC2 expression also correlated with a worse OS (Figure 1B), while increased PSMC6 correlated with worse OS as well as progression free survival (PFS) (OS- HR= 2.29 p=0.0014, PFS- HR=1.63 p=0.0109). PSMC6 displayed greater expression in 50 patients above the median RNA expression (Fragments per Kb transcript per million mapped reads (FPKM)= 1 to 100) while PSMC2 expression was elevated in 400 patients above the median (FPKM= 25 to 100). shRNA-mediated PSMC2 knockdown (KD) increased PI susceptibility in ARH77 and the RPMI8226 MM cells. In ARH77 cells, the BTZ LD50 decreased from 11 nM to 2.5 nM and the CFZ LD50 decreased from 11 to 9 nM in control and KD cells, respectively. In RPMI8226 cells, the BTZ LD50 decreased from 30 nM to 20 nM and the CFZ LD50 decreased from 28 to 17 nM in control and KD cells, respectively. Conclusion: Taken together, our results indicate that increased PSMC2 and PSMC6 correlates with OS in MM in both patients treated with BTZ in the APEX and with a variety of standard therapies in the CoMMpass database. Analysis in this validation cohort extends the clinical significance of PSMC2/PSMC6 beyond the landmark APEX trial and suggests they may inform survival in patients treated with modern triplet anti-myeloma therapies. In addition to the prognostic importance across our test and validation datasets, point mutations in each ATPase subunit have been associated with PI resistance. Consistent with these findings, PSMC2 KD increased sensitivity to PI therapy. Controlled inhibition or degradation of proteasome 19S ATPase subunits using small molecules, PROTACs or RNA-based modalities now provides an opportunity to therapeutically target proteostasis in MM and other malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Effect of BECN1 Expression on Proliferation and Invasion of Human Myeloma Cell Line RPMI-8226

To investigate the effect of BECN1 expression on proliferation and invasion of human multiple myeloma (MM) cell line RPMI-8226. RPMI-8226 cells were cultured in vitro, the recombinant plasmid pcDNA3.1-BECN1 was constructed and transfected into RPMI-8226 cells, then the cells were divided into three groups: control group, negative transfection group and BECN1 transfection group. The expression of BECN1 mRNA in cells of each group was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the effect of BECN1 overexpression on cell proliferation inhibition rate was detected by cell counting kit 8 (CCK-8); the effect of BECN1 overexpression on the colony formation rate was detected by plate cloning assay; the effect of BECN1 overexpression on cell invasion was detected by Transwell assay; the effects of BECN1 overexpression on the expression of cell proliferation, invasion and autophagy-related proteins were detected by Western blot. The expression of BECN1 mRNA in BECN1 transfection group was significantly higher than that in control group and negative transfection group (P<0.05); the inhibition rate of cell proliferation and the expression of autophagy-related proteins Beclin1, Atg5 and invasion-related protein E-cadherin in BECN1 transfection group were significantly higher than those in control group and negative transfection group; the colony formation rate, invasion number and the expression of proliferation-related proteins CyclinD1, β-catenin and invasion-related protein N-cadherin were significantly lower than those in control group and negative transfection group (P<0.05). Overexpression of BECN1 can inhibit the proliferation and invasion of human MM cells RPMI-8226, which may be a potential research target of MM.

6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist.

P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

Abstract 237: Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that primarily affects the elderly. MM is the second most common hematologic cancer with approximately 25,000-30,000 new cases diagnosed annually in the United States. A challenge associated with MM therapy is unwanted drug uptake by normal healthy cells. Nanoparticles provide a promising outlook to mitigate these issues, offering more selective drug uptake, while minimizing toxicity associated with treatment. The objective of this study was to develop, optimize and evaluate physicochemical and In vitro properties of MM-CLENs in comparison to conventional liposomes and relevant cell controls. Methods: RPMI-8226 MM cell line was selected cellular model for MM disease. RPMI-8226 cells were cultured and expanded In vitro for cellular extraction purposes. Lipid extraction (LE) procedures were performed to generate the RPMI-8226 LE ingredient. Other lipid ingredients used in preparation of RPMI-8226 CLENs included DOPC, cholesterol, and DPPE-Rhodamine (fluorescence indicator). Phase I included physicochemical characterization and cellular uptake studies of CLENs preparations with varying concentration of RPMI-8226-LE (0 to 40 mol%) content. Phase II began with the optimized concentration of LE determined in Phase I, along with varying concentrations of cholesterol (0 to 40 mol %) content for the nano-targeting studies in vitro. The control cell lines represent other hematological diseases, K-562-GFP (chronic myeloid leukemia), CCRF-CEM (acute lymphoblastic leukemia), and U937 (lymphoma). Results: Phase I and II physicochemical characterization and In vitro studies, revealed that the optimized preparation of RPMI-8226-CLENs consisted of 5% LE, and 5% cholesterol content. Optimal preparation consisted of DOPC: Cholesterol: LE (90:5:5) with an average values for particle size and zeta potential of 178±38 nm (n=4), and -14±4 mV (n=4), respectively. The inclusion of LE in conventional DOPC:cholesterol liposomes resulted in significantly improved uptake of the CLENs by RPMI-8226 cells in vitro (P&amp;lt; 0.001). However, preliminary selectivity studies suggest no significant difference in the uptake of RPMI-8226-CLENs by RPMI-8226 when compared to control cells (K-562-GFP, CCRF-CEM and U937). Conclusion: Studies involving RPMI-8226 CLENs are currently underway. Such studies include target selectivity, and influence of additional components of CLENs as a function of the microenvironment. Citation Format: Christina T. Tran, Eden Park, Pedro L. Rodriguez-Flores, Robert B. Campbell. Evaluation of cell membrane lipid-extracted nanoliposomes (CLENs) as a potential drug platform for targeting multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 237.

A short peptide derived from pigment epithelial-derived factor exhibits an angioinhibitory effect

BackgroundPigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect.MethodsA series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides.ResultsThe MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice.ConclusionsThis is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.

Root Bark of Morus alba L. and Its Bioactive Ingredient, Ursolic Acid, Suppress the Proliferation of Multiple Myeloma Cells by Inhibiting Wnt/β-Catenin Pathway.

The root bark of Morus alba L. has cytotoxic activity against several types of cancer cells. However, little is known about its chemopreventive mechanisms and bioactive metabolites. In this study, we showed that M. alba L. root bark extracts (MRBE) suppressed β-catenin response transcription (CRT), which is aberrantly activated in various cancers, by promoting the degradation of β-catenin. In addition, MRBE repressed the expression of the β-catenin/T-cell factor (TCF)-dependent genes, cmyc and cyclin D1, thus inhibiting the proliferation of RPMI-8226 multiple myeloma (MM) cells. MRBE induced apoptosis in MM cells, as evidenced by the increase in the population of annexin VFITC- positive cells and caspase-3/7 activity. We identified ursolic acid in MRBE through LC/mass spectrum (MS) and observed that it also decreased intracellular β-catenin, c-myc, and cyclin D1 levels. Furthermore, it suppressed the proliferation of RPMI-8226 cells by stimulating cell cycle arrest and apoptosis. These findings suggest that MRBE and its active ingredient, ursolic acid, exert antiproliferative activity by promoting the degradation of β-catenin and may have significant chemopreventive potential against MM.

CD56 Has a Critical Role in Regulating Multiple Myeloma Cell Growth and Response to Therapies

Multiple myeloma (MM) is a disease derived from genetically abnormal clonal plasma cells. MM cells aberrantly express several surface antigens compared with normal plasma cells. Among others, CD56/NCAM1 is present at variable levels in approximately 70% of MM patients. Very little is known about its role in MM; however, CD56 positivity in MM correlates with greater osteolytic burden and lower frequency of good prognostic features, such as the presence of t(11;14).We first analyzed 569 patients with MM diagnosed between 1/1/2005 and 12/31/2014 at the Ohio State University Wexner Medical Center, stratifying them based on the percentage of CD56-expressing clonal MM cells. The mean percentage of CD56-expressing clonal MM was 26.5%, with range from 0 to 100%; the Mean Fluorescent Intensity values varied, with a quarter of patients expressing CD56 at high intensity. We then evaluated patient outcomes based on the percentage of CD56-expressing clonal MM cells. We noticed that MM patients with more than 30 or 50 percent of CD56-expressing MM clonal cells have inferior clinical outcomes than patients with less than 30 or 50 percent of CD56-expressing MM clonal cells, with median overall survival of 9.61 versus 7.64 years (log-rank p-value: 0.004) or 9.30 versus 6.47 years (log-rank p-value: 0.0009), respectively.We then demonstrated by conventional and real-time PCR analyses that the predominately expressed CD56 isoform in MM has signaling potential with a transmembrane portion and cytosolic tail. Therefore, we evaluated the functional role of CD56 in MM. By gain-of function studies in U266 and MM.1S MM cell lines, we showed that overexpression of CD56 promotes MM growth and viability; the opposite effect occurred with CD56 silencing in H929, OPM-2, and RPMI-8226 MM cell lines, which leads to reduced MM growth and increased apoptotic cell death. Overexpression of CD56 resulted in the phosphorylation and hence activation of ribosomal protein S6 kinase A3 (RSK2) and of the transcription factor, cAMP responsive element binding protein 1 (CREB1). This induced CREB1 binding to DNA consensus CRE elements, and promoted transcription of CREB1 targets, the anti-apoptotic genes BCL2 and MCL1. CD56 silencing in H929 and OPM-2 MM cell lines had opposite effects, with reduction of phospho-RSK2, phospho-CREB1, MCL1, and BCL2 levels.We then used shRNAs targeting RSK2 and CREB1 or specific inhibitors (BI-D1870 that is a RSK2 inhibitor, and 666-15 that is a CREB1 inhibitor) at 0.1-1 microM concentration. We evaluated viability by MTT assay or Zombie dye staining on CD138 positive MM cells and apoptosis by Annexin V-PI staining. We demonstrated that CD56 positive MM cell lines (H929, OPM-2, and RPMI-8226) or patients with high CD56 expression (>30% of CD56-expressing clonal MM cells) are more sensitive to RSK2/CREB1 inhibition compared with CD56 negative MM cell lines (U266, L363, and MM.1S) or patients with low CD56 expression (<30% of CD56-expressing clonal MM cells). Using similar strategies, we also proved that CREB1 is essential to CD56-protumoral phenotype, since CREB1 inhibition reduces cellular growth and viability in CD56 overexpressing U266 cells. RSK2 and CREB1 inhibition mimic CD56 silencing with decrease of BCL2 and MCL1 mRNA and protein levels. Furthermore, we observed that CD56 signaling by CREB1 activation decreases CRBN expression, reducing responses to lenalidomide. Conversely, CREB1/RSK2 blockade rescued CRBN levels in CD56 positive MM cells and increased lenalidomide response. These results support the hypothesis that targeting CREB1 is hence a so far unexplored but potentially effective synthetic lethal strategy for CD56-expressing MM patients.In conclusion, our study defines an effective threshold for therapeutic intervention in CD56-expressing MM patients. Moreover, our data pioneer the use of CREB1/RSK2 inhibition in CD56-expressing MM cells, either as single agents or in combination with lenalidomide, suggesting that CD56 can be a prognostic and predictive factor of response in MM. DisclosuresNo relevant conflicts of interest to declare.