Apoptosis Of MM Cells Research Articles

Doxorubicin-loaded PEG-CdTe QDs conjugated with anti-CXCR4 mAbs: a novel delivery system for extramedullary multiple myeloma treatment

Extramedullary multiple myeloma (EMM) is defined as the presence of plasma cells outside the bone marrow of multiple myeloma patients, and its prognosis is poor. High-dose chemotherapy with autologous stem cell transplantation, as a good option on early lines of therapy, has retained the survival benefit of youny EMM patients, but is intolerant for the majority of old patients because of drug cytotoxicity. To essentially address the intolerance above, we designed a CXCR4-PEG-CdTe-DOX (where CXCR4: chemokine receptor 4; PEG-CdTe: polyethylene glycol-modified cadmium telluride; DOX:doxorubicin) nanoplatform. First, CXCR4 is highly expressed in extramedullary plasma cells. Second, PEG-CdTe a drug carrier that controls drug release, can reduce adverse reactions, prolong drug (e.g, DOX) circulation time in the body, and form a targeting carrier after connecting antibodies. In vitro experiments showed CXCR4-PEG-CdTe-DOX facilitated intracellular drug accumulation through active CXCR4 targeting and released DOX into the microenvironment in a pH-controlled manner, enhancing the therapeutic efficacy and apoptosis rate of myeloma cells (U266). Therefore, targeted chemotherapy mediated by CXCR4-PEG-CdTe-DOX is a promising option for EMM treatment.Graphical abstractFigure 1 Action mechanism of drug delivery pathway: CXCR4-PEG-CdTe-DOX nanocomplex can be targeted and close to MM cell membranes, effectively transport DOX into MM cells, and promote MM cell apoptosis.

A SNAP23-Dependent SNARE Complex Mediates Free Light Chain Secretion in AL Amyloidosis and Multiple Myeloma and Its Blockade Triggers a Terminal Unfolded Protein Response and Massive Apoptosis

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder with only one FDA-approved therapy. Deposition of immunoglobulin free light chain (FLC) fibrils in target organs is the sole pathogenic mechanism in AL. However, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, the specific targets of botulinum neurotoxin (BoNT), mediate the docking and fusion of secretory vesicles. We hypothesized that targeting specific sets of SNAREs via BoNT will block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR) and apoptosis in AL cells. We postulate that SNAREs are potential novel targets in AL and other plasma cell disorders. Materials and Methods We stably expressed 7 distinct, inducible BoNT serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1 and ALMC2) and multiple myeloma (MM, KMS11) cell lines via tetracycline-inducible (Tet-On) bicistronic lentiviral vectors with a P2A-GFP which allowed us to use GFP as a biomarker of BoNT expression. We established monoclones of successfully transduced cells for each BoNT. We performed flow cytometry to assess the kinetics of GFP induction and assess for apoptosis via AnnexinV/DAPI staining. Western blot (WB) was used to assess the cleavage of SNAREs upon expression of each BoNT serotype. We used WB and ELISA to assess FLC secretion per cell upon expression of each BoNT serotype. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and used co-IP for confirmation. We used RNP-based CRISPR-Cas9 gene editing to knock out (KO) putative BoNT targets. Results By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SNAP23, and SYNTAXIN4 as the top expressed SNAREs. Their high expression was retained in a large panel of AL and MM cell lines. Expression of all BoNT serotypes, except for BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for the extent and rapidity of cytotoxicity. Dual cleavage of SNAP23 and VAMP3 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not non-toxic BoNT B, led to decreased FLC secretion and increased FLC retention and triggered the PERK pathway of the UPR with increased expression of CHOP and GADD34, preceding the onset of apoptosis. We identified SNAP23 as an interacting partner of highly cytotoxic BoNT F, but not non-toxic BoNT B. KO experiment showed single SNAP23 KO to be partially toxic for AL and MM cell lines but did not recapitulate the effects of cytotoxic BoNTs entirely. Based on our preliminary data, we hypothesize VAMP3 and SYNTAXIN4 to be synthetic lethal with SNAP23. Conclusions Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. By using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosisin AL cell lines, thus representing a novel, promising molecular target in AL amyloidosis and multiple myeloma.

Mechanisms of action of the BCL-2 inhibitor venetoclax in multiple myeloma: a literature review.

Abnormal cellular apoptosis plays a pivotal role in the pathogenesis of Multiple Myeloma (MM). Over the years, BCL-2, a crucial anti-apoptotic protein, has garnered significant attention in MM therapeutic research. Venetoclax (VTC), a small-molecule targeted agent, effectively inhibits BCL-2, promoting the programmed death of cancerous cells. While VTC has been employed to treat various hematological malignancies, its particular efficacy in MM has showcased its potential for broader clinical applications. In this review, we delve into the intricacies of how VTC modulates apoptosis in MMcells by targeting BCL-2 and the overarching influence of the BCL-2 protein family in MM apoptosis regulation. Our findings highlight the nuanced interplay between VTC, BCL-2, and MM, offering insights that may pave the way for optimizing therapeutic strategies. Through this comprehensive analysis, we aim to lay a solid groundwork for future explorations into VTC's clinical applications and the profound effects of BCL-2 on cellular apoptosis.

A phase 3, two-stage, randomized study of mezigdomide, carfilzomib, and dexamethasone (MeziKd) versus carfilzomib and dexamethasone (Kd) in relapsed/refractory multiple myeloma (RRMM): SUCCESSOR-2.

TPS8070 Background: Mezigdomide (MEZI), a novel oral cereblon E3 ligase modulator (CELMoD), induces maximal degradation of Ikaros/Aiolos leading to increased MM cell apoptosis and immune-stimulatory effects. MeziKd has shown potent synergistic antiproliferative activity in MM cell lines resistant to lenalidomide (LEN), and in a phase 1/2 study showed promising preliminary efficacy and safety in RRMM. The SUCCESSOR-2 phase 3 trial (NCT05552976) will compare the efficacy and safety of MeziKd vs Kd in patients (pts) with RRMM. Methods: This multicenter, open-label study comprises 2 stages. In Stage 1, ≥ 128 pts will be randomized 3:3:3:2 to 1 of 3 MEZI doses (1.0, 0.6, or 0.3 mg) + Kd, or to the Kd arm to select the optimal MEZI dose in combination with Kd for Stage 2. In Stage 2, ≈ 397 additional pts will be randomized 3:2 to MeziKd at the selected MEZI dose or to Kd, for efficacy and safety analyses (Stage 1 pts in the selected MeziKd dose cohort and Kd arm will also be included in these analyses). Pts will be stratified by age (≤ 70 vs > 70 y), number of prior lines of treatment (Tx; ≤ 2 vs > 2), and ISS stage at screening (I vs II vs III). MEZI dose selection will be based on Stage 1 efficacy, safety, pharmacokinetic and pharmacodynamic data, and exposure–response analyses. Primary efficacy endpoint is progression-free survival (PFS). Assuming a decrease in PFS risk by 33.3% (HR = 0.667) with MeziKd, under exponential distribution assumption of PFS (1-sided α = 0.025) and adjusted for 3 interim analyses, ≥ 273 PFS events will have ≈ 89% power to detect improvement in Tx effect. The planned interim analyses are for: MEZI dose selection at end of Stage 1, and to examine PFS futility and superiority when ≈ 82 (30%) and ≈ 205 (75%) events, respectively, have been accumulated. Secondary endpoints include determination of the recommended MEZI dose plus Kd (Stage 1 only), and assessment of overall survival, overall response rate, time to response, duration of response, time to progression, safety, and quality of life. MeziKd arm Tx consists of 28-day (D) cycles (C) with MEZI on D1–21; 20 mg/m2 intravenous (IV) carfilzomib (CFZ) on D1 of C1, then 56 mg/m2 on D8 and 15 of C1, on D1, 8, and 15 of C2–12, and on D1 and 15 of ≥ C13; and 40 mg oral/IV DEX (20 mg optional in certain pt groups) on D1, 8, 15, and 22. Tx in the Kd arm consists of 28-D cycles with 20 mg/m2 IV CFZ on D1 and 2 of C1, then 56 mg/m2 on D8, 9, 15, and 16 of C1, and on D1, 2, 8, 9, 15, and 16 of ≥ C2; and 20 mg oral/IV DEX (10 mg optional in certain pt groups) on D1, 2, 8, 9, 15, 16, 22, and 23. Tx will continue until progressive disease (PD) or unacceptable toxicity. Key eligibility criteria include age ≥ 18 y, ≥ 1 prior line of anti-MM Tx including LEN and an anti-CD38 monoclonal antibody, minimal response or better to ≥ 1 prior Tx, documented PD during or after last regimen, and no prior CFZ Tx. Enrollment began in October 2022 and is ongoing. Clinical trial information: NCT05552976 .

The STAT3 inhibitor stattic overcome bortezomib-resistance in multiple myeloma via decreasing PSMB6

Bortezomib, an FDA approved drug in 2003 for newly diagnosed and relapsed/refractory MM, had showed great efficacy in different clinical settings. However, many patients still developed resistance to Bortezomib, and the mechanism of action remains unelucidated. Here, we showed that Bortezomib resistance can be partially overcome by targeting a different subunit of 20 S complex - PSMB6. PSMB6 knock down by shRNA increased sensitivity to Bortezomib in resistant and sensitive cell line. Interestingly, a STAT3 inhibitor, Stattic, is shown to selectively inhibit PSMB6 and induce apoptosis in Bortezomib resistant and sensitive MM cells, even with IL-6 induction. Therefore, PSMB6 is a novel target for Bortezomib resistance and Stattic may offer a potential therapeutic strategy.

PB1975: THE RESEARCH ON THE MECHANISM OF RRAD GENE IN MULTIPLE MYELOMA

Background: Multiple myeloma is the second most common hematological malignancy with a high incidence. The pathogenesis of multiple myeloma is still unclear, and specific gene expression abnormalities may play a key role in the pathogenesis of MM. Aims: The research on the mechanism of RRAD gene in multiple myeloma Methods: The study collected bone marrow specimens and 9 healthy volunteers from 20 newly diagnosed MM patients admitted to the Affiliated Hospital of Zunyi Medical University from January 2019 to June 2020.The RT-qPCR method was used to detect the transcription level of RRAD in 20 newly diagnosed MM patients, 9 healthy donors and human MM cell lines; RRAD overexpression cell lines were constructed in SKO-007 and H929 by lentivirus infection, and flow cytometry Cytometry, RT-PCR and Western Blot verified the efficiency of overexpression; CCK8 method was used to study the effect of RRAD on cell proliferation; flow cytometry was used to detect cell apoptosis and cycle.Decitabine was used to treat MM cell lines, and RT-qPCR was used to detect changes in RRAD gene expression; CCK8 was used to detect the effect of cell proliferation; flow cytometry was used to detect cell apoptosis and cycle; MSP method was used to detect RRAD methylation of MM cells Level. Results: Compared with 9 healthy donors, the expression level of RRAD was lower in 20 newly diagnosed MM patients (P = 0.017).Compared with 9 healthy donors, the expression level of RRAD in the MM cell line is low.The ROC curve results show that the expression level of RRAD has a significant diagnostic value for the diagnosis of MM (P <0.001).RRAD gene overexpression can promote MM cell apoptosis and block cell cycle into S phase.DAC can inhibit the proliferation of SKO-007, H929, U266 cells in a dose-dependent manner.Promote the apoptosis of SKO-007, H929, U266 cells, block the cell cycle into S phase or G2 phase and increase the expression of RRAD.After SKO-007, H929, and U266 cells were treated by DAC, the RRAD methylation level of SKO-007 cells decreased, but the RRAD methylation level of H929 and U266 cells did not change significantly. Summary/Conclusion: 1. RRAD expression in MM is low.2. The expression level of RRAD is closely related to the presence or absence of bone disease, which affects the patient’s quality of life and prognosis assessment.3. The expression level of RRAD has significant diagnostic value for the diagnosis of MM.4. Overexpression of RRAD can block the cell cycle in S phase and promote MM cell apoptosis.5. Hypermethylation of RRAD in MM leads to epigenetic inactivation, which reduces the expression of RRAD and reduces MM Apoptosis of cells loses its anti-tumor effect, which is a new molecular mechanism for the occurrence and development of MM.

MAGE-A3 Recombinant Protein (recMAGE-A3) Immunotherapy and Autologous Peripheral Blood Lymphocyte (PBL) Infusion Is Safe and Induces Robust Humoral Immune Responses In Multiple Myeloma (MM) Patients Undergoing Autologous Stem Cell Transplantation (autoSCT)

▪ BackgroundMAGE-A3 is a cancer-testis antigen expressed in various human cancers, including MM, but not in normal non-gonadal tissues. MAGE-A3 expression inhibits apoptosis in MM cells, and has been associated with a more aggressive clinical course, making it a rational target for immunotherapy. Pre-autoSCT myeloablative conditioning provides tumor antigen release and a lymphopenic environment that may be ideal for expansion of primed PBL and generation of anti-myeloma immune responses. Patients and methodsEligibility for this pilot study required symptomatic MM within 1 year of diagnosis; MAGE-A+ MM cells by immunohistochemistry (IHC); at least VGPR following induction therapy; and meeting institutional criteria for autoSCT. The immunotherapy consists of a recombinant protein containing full-length MAGE-A3 fused to part of H. influenzae Protein D antigen, mixed with the AS15 immunostimulant (containing MPL, QS21, and CpG7909). Six weeks prior to SCT, patients received their 1st injection (300 µg recMAGE-A3 intramuscularly), followed 3 weeks later by steady-state leukopheresis to collect primed PBL. They then underwent stem cell mobilization and a melphalan 200 mg/m2-conditioned autoSCT, followed by re-infusion of unmanipulated PBL on day +3 after SCT, and 7 post-SCT injections (days +10, 31, 52, 73, 94, 180, and 270). Lenalidomide maintenance was permitted. The primary objectives were safety and tolerability. The secondary objectives were cellular and humoral immune responses and clinical outcomes. Humoral immunity was assessed by ELISA using baculovirus-generated recombinant MAGE-A3 protein. ResultsOf 93 pre-screened patients, 30 (32%) were MAGE-A+ by IHC. Of these, 13 (43%) enrolled. Reasons for non-enrollment were: did not achieve VGPR (n=6), declined/received other therapy (n=7), other (n=4). For enrolled patients, median age was 56; ISS stages I/II/III were 58%/25%/17%; 45% had high-risk cytogenetics at diagnosis (n=11 tested). Responses at enrollment were 12 VGPR and 1 CR; most (83%) had received VRD-based induction. All patients had adequate PBL collections (median 1.4 x 108 CD3+ cells/kg, range 1.0-2.6), and all collected stem cells successfully (median 11.4 x 106 CD34+ cells/kg, range 8.3 – 37.2). Autologous PBL infusions were well-tolerated, with no engraftment-like syndromes. Time to neutrophil recovery (median 11 days) and SCT-related toxicities were typical. The immunotherapy schedule was feasible with only 1 missed dose to date. Potential immunotherapy-related AE's (all grade 1 or 2) include injection site pain/reaction (n=6), myalgia/flu-like symptoms (n=5), fever/chills (n=2), fatigue (n=1), and rash (n=1). Three SAE's were observed: 1 immunotherapy-related (grade 2 myalgia requiring a 24-hour hospitalization), and 2 unrelated (grade 3 diarrhea, grade 3 atrial fibrillation with pulmonary edema). There have been no autoimmune toxicities and no deaths on study. Four patients initiated lenalidomide maintenance with no unexpected toxicities. Regarding immune responses, 12 of 12 (100%) evaluable patients have developed high-titer (>104) IgG antibodies against MAGE-A3 (Fig.). Responses were seen in all but 2 patients by day +31 post-SCT, and in all patients by day +75, persisting at 1 year post-SCT in both patients reaching this timepoint so far. All patients also developed strong IgG responses against Protein D. Clinically, at 3 months post-SCT, 50% of patients had achieved CR (33% stringent CR). With a median follow-up of 10 months post-SCT (range 1-20), 92% are progression-free, with follow-up ongoing. ConclusionsMAGE-A3 protein immunotherapy plus autologous PBL transfer is feasible, has an acceptable safety profile, and is strongly immunogenic, capable of overcoming the profound immune suppression typically seen in MM patients post-autoSCT. The presence of IgG-type anti-MAGE antibodies implies the generation of a CD4 T cell response to induce Ig class-switching, though formal assessment of T cell responses against MAGE-A3 and other antigens are needed to confirm this, and are ongoing. Importantly, the use of an “off-the-shelf” immunotherapy product and unmanipulated autologous PBL transfer allows the treatment to be given at any transplant center. Further study of this approach as a strategy to consolidate post-autoSCT responses and potentially eliminate minimal residual disease is warranted. [Display omitted] Disclosures: Cohen:Bristol Meyers Squibb: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria; Millennium: Honoraria. Bertolini:Ludwig Institute for Cancer Research: Employment. Pan:Ludwig Institute for Cancer Research: Employment. Venhaus:Ludwig Institute for Cancer Research: Employment. Fellague-Chebra:GlaxoSmithKline Vaccines: Employment. Gruselle:GlaxoSmithKline Vaccines: Employment.

Study on Tim3 Regulation of Multiple Myeloma Cell Proliferation via NF-κB Signal Pathways.

ObjectiveAs an important negative regulatory factor of immunological cells, Tim3 plays a regulating role in tumor immune microenvironment. The purpose of this study was to investigate the expression of Tim3 on MM cells and its effect on the proliferation and apoptosis of MM cells, as well as its potential mechanism.MethodsIn this study, the expression of Tim3 was detected on myeloma cells (CD38+CD138+ cells) of bone marrow by flow cytometry (FCM) from 167 patients with MM and 51 healthy donors as controls and making correlation analysis with related clinical indexes. In vitro, MM cell lines (RPMI-8226 and U266) were treated with Tim3 knock-down alone, bortezomib alone and combination of Tim3 knock-down and bortezomib, then cell proliferation, cell apoptosis and downstream signaling pathway were detected by CCK-8, FCM, RT-PCR and western blot.ResultsThe expression of Tim3 on myeloma cells in MM patients was significantly higher than normal control group and positively correlated with β2 microglobulin, creatine, and plasma cells of bone marrow, negatively correlated with hemoglobin and red blood cells. In vitro, we validated the high expression of Tim3 in RPMI-8226 and U266 cell lines. After Tim3 knock-down, the cell proliferation was inhibited and cell apoptosis was induced, the relative mRNA and protein expression of Tim3 and NF-κB signal pathway (PI3K, AKT, mTOR, NF-κB) were significantly decreased. Also, the cell proliferation was inhibited, cell apoptosis was increased, the relative mRNA and protein expression of NF-κB were decreased significantly in combination group than bortezomib or Tim3 knock-down group.ConclusionsThe high expression of Tim3 on MM cells is associated with progression of MM patients. Tim3 maybe regulate the proliferation of MM cells via NF-κB signal pathway. Down-regulation of Tim3 expression can inhibit proliferation and induce apoptosis of MM cells, also has an additive inhibitory effect of bortezomib on NF-κB signaling pathway, then inhibit proliferation and induce apoptosis. Therefore, Tim3 may be a potential target for the treatment of MM.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Disulfiram/copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways

Disulfiram (DSF) is an FDA approved anti-alcoholism drug in use for more than 60 years. Recently, antitumor activity of the DSF/copper (DSF/Cu) complex has been identified. Its anti-multiple myeloma activity, however, has barely been investigated. In the present study, our results demonstrated that the DSF/Cu complex induced apoptosis of MM cells and MM primary cells. The results indicated that DSF/Cu significantly induced cell cycle arrest at the G2/M phase in MM.1S and RPMI8226 cells. Moreover, JC-1 and Western blot results showed that DSF/Cu disrupted mitochondrial membrane integrity and cleaved caspase-8 in MM cells, respectively, suggesting that it induced activation of extrinsic and intrinsic apoptosis pathways. Interestingly, DSF/Cu induced caspase-3 activation was partly blocked by Z-VAD-FMK (zVAD), a pan-caspase inhibitor, indicating at caspase-dependent and -independent paths involved in DSF/Cu induced myeloma cell apoptosis machinery. Additionally, activation of the c-Jun N-terminal kinase (JNK) signaling pathway was observed in DSF/Cu treated MM cells. More importantly, our results demonstrated that DSF/Cu significantly reduced tumor volumes and prolonged overall survival of MM bearing mice when compared with the controls. Taken together, our novel findings showed that DSF/Cu has potent anti-myeloma activity in vitro and in vivo highlighting valuable clinical potential of DSF/Cu in MM treatment.

Therapeutic effects of oligo-single-stranded DNA mimicking of hsa-miR-15a-5p on multiple myeloma.

Despite the fact that a few novel agents improve the outcome of patients, MM remains incurable. Hence, developing a novel treatment strategy may prove to be promising for the clinical management of MM. Noncoding small RNAs, a cluster of RNAs that do not encode functional proteins, have been underlined that play a pivotal role in the pathogenesis of MM. Our previous study indicated that miR-15a acted as a tumor suppressor, which inhibited the cell proliferation and promoted the apoptosis of MM cells. The level of miR-15a was downregulated in MM cells and correlated with inferior outcome of MM patients. In the present study, we first developed an oligo-single-stranded DNA mimicking the sequence of hsa-miR-15a-5p (OMM-15a) and modified with locked nucleic acid (LNA-15a) to evaluate its anti-MM effects. Our results indicated that the LNA-15a presented an exciting anti-MM effect that showed notable cell growth suppression and apoptosis promotion in MM and other cancer cell lines through downregulating the expression level of target genes BCL-2, VEGF-A, and PHF19. Moreover, LNA-15a treatment significantly improved the anti-MM activity of bortezomib with the synergism effect in OCI-My5 MM cells. In our in vivo study, LNA-15a treatment significantly suppressed the tumor growth, and prolonged the survival of mice compared with the control group. However, our results indicated that the native form of oligo-single-stranded DNA mimic of hsa-miR-15a-5p (OMM-15a) without any modification had no effective inhibition on cell growth, even after increasing the dosage of OMM-15a in the treatment. Altogether, our finding provides the preclinical rationale to support the oligo-single-stranded DNA mimic of hsa-miR-15a with LNA modification, which is a promising tool for the therapy of both MM and other tumors with miR-15a downregulation.

C-Myc-Driven and PRMT5-Dependent Regulation of Multiple Myeloma Cell Proliferation through TNRC6B Gene

Objective: The protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetrical bimethylation of arginine residues, which plays an important regulatory role in the life process. Transcription factor c-Myc is widely proved involved in the alteration and development of tumors, while the regulatory mechanism of c-Myc-mediated gene expression has not been fully understood. In this article we try to investigate the functional role of PRMT5 and c-Myc in regulating multiple myeloma cell proliferation. Methods: The protein and mRNA expression levels of PRMT5 in myeloma cells and normal cells were detected by Western Blot and qPCR. Lentivirally transduced shRNAs targeting PRMT5 (sh-PRMT5) was constructed using lentivirus mediated RNAi technology, and was packaged to infect MM cells to select positive colonies. The effect of PRMT5 on the proliferation of MM cells was detected by cck-8 assay. Annexin V/7-AAD double staining flow cytometry was used to detect cell survival and apoptosis rate. The expression of apoptotic suppressor gene c-Myc in sh-PRMT5 cell lines was detected by Western Blot and qPCR. Finally, double-knockdown of PRMT5 and c-myc was performed and gene expression differentiation were identified by RNA sequencing (RNA-seq). Results: PRMT5 expression in MM cell lines (RPMI8226 and U266) was relatively high compared with peripheral blood mononuclear cells (PBMCs), which was correlated with progression-free survival (PFS) and overall survival (OS) in MM patients. After sh-PRMT5 infection to MM cell lines, puromycin selection was performed and efficient gene knockdown was evaluated by Western Blot and qPCR. After targeted silencing of PRMT5 expression, the proliferation of MM cells was decreased and apoptosis was significantly increased (P &lt; 0.05). C-Myc protein and mRNA levels were significantly downregulated after PRMT5 gene silencing. The results showed that PRMT5 promoted the proliferation of MM cells and inhibited the apoptosis of MM cells by regulating the expression of apoptotic suppressor gene c-Myc. Subsequently, RNA-seq confirmed SMCHD1 as the common candidate target gene of c-myc and PRMT5. Therefore, we speculated that PRMT5 and c-Myc may jointly regulate TNRC6B gene to promote the proliferation of MM cells. Conclusion: Altogether, our study not only proposed the mechanism PRMT5 and c-myc in regulating multiple myeloma related gene expression, but also provided a new strategy and theoretical basis for clinical treatment of multiple myeloma. Disclosures No relevant conflicts of interest to declare.

PF584 C‐MYC‐DRIVEN AND PRMT5‐DEPENDENT REGULATION OF MULTIPLE MYELOMA CELL PROLIFERATION THROUGH SMCHD1 GENE

Background:The protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetrical bimethylation of arginine residues, which plays an important regulatory role in the life process. Transcription factor c‐Myc is widely proved involved in the alteration and development of tumors, while the regulatory mechanism of c‐Myc‐mediated gene expression has not been fully understood.Aims:In this article we try to investigate the functional role of PRMT5 and c‐Myc in regulating multiple myeloma cell proliferationMethods:The protein and mRNA expression levels of PRMT5 in myeloma cells and normal cells were detected by Western Blot and qPCR. Lentivirally transduced shRNAs targeting PRMT5 (sh‐PRMT5) was constructed using lentivirus mediated RNAi technology, and was packaged to infect MM cells to select positive colonies. The effect of PRMT5 on the proliferation of MM cells was detected by cck‐8 assay. Annexin V/7‐AAD double staining flow cytometry was used to detect cell survival and apoptosis rate. The expression of apoptotic suppressor gene c‐Myc in sh‐PRMT5 cell lines was detected by Western Blot and qPCR. Finally, double‐knockdown of PRMT5 and c‐myc was performed and gene expression differentiation were identified by RNA sequencing (RNA‐seq).Results:PRMT5 expression in MM cell lines (RPMI8226 and U266) was relatively high compared with peripheral blood mononuclear cells (PBMCs), which was correlated with progression‐free survival (PFS) and overall survival (OS) in MM patients. After sh‐PRMT5 infection to MM cell lines, puromycin selection was performed and efficient gene knockdown was evaluated by Western Blot and qPCR. After targeted silencing of PRMT5 expression, the proliferation of MM cells was decreased and apoptosis was significantly increased (P &lt; 0.05). C‐Myc protein and mRNA levels were significantly downregulated after PRMT5 gene silencing. The results showed that PRMT5 promoted the proliferation of MM cells and inhibited the apoptosis of MM cells by regulating the expression of apoptotic suppressor gene c‐Myc. Subsequently, RNA‐seq confirmed SMCHD1 as the common candidate target gene of c‐myc and PRMT5. Therefore, we speculated that PRMT5 and c‐Myc may jointly regulate SMCHD1 gene to promote the proliferation of MM cells.Summary/Conclusion:Altogether, our study not only proposed the mechanism PRMT5 and c‐myc in regulating multiple myeloma related gene expression, but also provided a new strategy and theoretical basis for clinical treatment of multiple myeloma.

Inhibition of Autophagy with 3-Methyladenine Potentiates Bortezomib-Induced Apoptosis of Myeloma Cell Via up Regulation of Mitochondrial Reactive Oxygen Species

Backgrounds : Autophagy plays an important role in the pathogenesis of multiple myeloma. Autophagy usually acts on a pro-survival mechanism, and cooperates with the ubiquitin proteasome system in the homeostasis of myeloma cells by degrading excessive and misfolded proteins for energy recycling. Therefore, the inhibition of autophagy could effectively induce death and could synergize with proteasome inhibitors in myeloma cells. In this study, we investigated the synergistic mechanism of apoptosis with bortezomib (BTZ) and autophagy inhibitors in the MM cells.Methods : We evaluated the change of autophagy, apoptosis, mitochondrial ROS and cellular level of redox enzymes, including peroxiredoxin (Prx), thioredoxin (Trx), thioredoxin reductase (Trx-R) using human myeloma cell lines, MM.1S and MM.1R during BTZ treatment. To study the status of oxidation for redox enzyme, sulfinic acid (SO2) or multimeric form of Prx, Trx and Trx-R by western blot were measured using non-reducing or reducing gel.Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 24 hour of BTZ (2.5 nM) treatment. Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. LC3-II expression was elevated along with increment of mitochondrial ROS and apoptosis of MM cells after BTZ treatment. Oxidation of PRX4 and TRX2 was observed during BTZ-induced apoptosis of MM cells. After treatment of NAC, LC3-II and PRX4/TRX2 expression was reversed. Inhibition of autophagy with 3-Methyladenine (3-MA) not only resulted in a further increase in BTZ-induced apoptosis but also induced mitochondrial ROS in MM cells.Conclusions : Autophagy is induced during BTZ treatment in MM cells. Our experiment showed crosstalk between autophagy and mitochondrial ROS and its redox enzymes during BTZ-induced MM cell apoptosis. Inhibition of autophagy with 3-MA potentiates BTZ-induced apoptosis of MM cells via up regulation of mitochondrial ROS. Our results provide new perspective on the cellular mechanism of action of BTZ and support the synergism of BTZ with autophagy inhibitor for an improved therapy of the multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

A novel dual inhibitor of microtubule and Bruton's tyrosine kinase inhibits survival of multiple myeloma and osteoclastogenesis

Bruton's tyrosine kinase (BTK) regulates many vital signaling pathways and plays a critical role in cell proliferation, survival, migration, and resistance. Previously, we reported that a small molecule, KS99, is an inhibitor of tubulin polymerization. In the present study, we explored whether KS99 is a dual inhibitor of BTK and tubulin polymerization. Although it is known that BTK is required for clonogenic growth and resistance, and microtubules are essential for cancer cell growth, dual targeting of these two components has not been explored previously. Through docking studies, we predicted that KS99 interacts directly with the catalytic domain of BTK and inhibits phosphorylation at the Y223 residue and kinase activities. Treatment of KS99 reduces the cell viability of multiple myeloma (MM) and CD138+ cells, with an IC50 of between 0.5 and 1.0μmol/L. We found that KS99 is able to induce apoptosis in MMcells in a caspase-dependent manner. KS99 suppressed the receptor activator of NF-κB ligand (RANKL)-induced differentiation of macrophages to osteoclasts in a dose-dependent manner and, importantly, inhibited the expression of cytokines associated with bone loss. Finally, we found that KS99 inhibits the invivo tumor growth of MMcells through the inhibition of BTK and tubulin. Overall, our results show that dual inhibition of BTK and tubulin polymerization by KS99 is a viable option in MM treatment, particularly in the inhibition of refraction and relapse.

Bortezomib-Induced Apoptosis in Myeloma Cell Via Oxidation of Peroxiredoxin III and Thioredoxin 2

Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment.Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis.Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise.Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells. DisclosuresNo relevant conflicts of interest to declare.

A Novel BRD4 Inhibitor CA2 Suppresses MM Cell Proliferation in an Orthotopic Myeloma Mouse Model

We previously demonstrated that a novel BRD4 inhibitor, CA2, suppressed MM cell proliferation in vitro in the 57th ASH Annual Meeting. In the present study, we investigated the inhibitory mechanisms of CA2 on myeloma cell proliferation in vitro and in vivo. CA2 has a unique quinolinone core and inhibited the proliferation of MM cell lines, MM.1s, AMO-1, NCI-H929, OPM-2, and IM-9. As expected, CA2 suppressed the expression of c-MYC mRNA and protein in a dose- and time-dependent manner. In addition, CA2 decreased BRD4 binding to c-MYC promoters and c-MYC enhancers in a ChIP assay using IM-9 cells and an anti-BRD4 antibody (Figure 1). Flow cytometric Annexin-V/propodium iodide staining analyses demonstrated that CA2 induced apoptosis in MM cells. Taken together, CA2 may induce apoptosis in MM cells by inhibiting the binding of BRD4 to promoters and enhancers of c-MYC gene thereby suppressing c-MYC expression.We next performed a pharmacokinetic study of CA2 in mice. Half-lives (t1/2) of CA2 following oral administration (50 mg/kg) and subcutaneous administration (10 mg/kg) were approximately 61 min and 139.8 min, respectively. The values of area under the curve for oral and subcutaneous administrations were 179,000 hr·ng/mL and 31,000 hr·ng/mL, respectively. Lastly, we assessed the in vivo effects of CA2 using orthotopic MM mouse model, where IM-9Luc cells were inoculated intravenously through the tail veins of SCID mice that had received 2 Gy of irradiation. The mice in the vehicle-control arm died of MM by approximately 30 days after transplantation; IM-9Luc cells engrafted in many organs including bone marrow of spine and femurs, kidneys, ovaries, and stomach. CA2 treatment consisting of 20 cycles of 100 mg/kg/day b.i.d. oral dosing resulted in reduced luminescence intensity of IM-9Luc cells and prolonged the survival of mice (mean 33.7 days) compared to the vehicle-control arm (mean 27.1 days; p=0.029, Figure 2). In conclusion, CA2 is a BRD4 inhibitor with a novel structure and could be a promising molecular targeting-agent against MM. [Display omitted] [Display omitted] DisclosuresYoshioka:ConverGene LLC: Employment. Kado:Japan Community Health Care Organization Kyoto Kuramaguchi Medical Center: Employment. Strovel:ConverGene LLC: Employment.

Roar: A Phase Ib Trial of Oral Azacitidine (CC-486) in Combination with Lenalidomide and Dexamethasone (Rd) for Patients with Relapsed and/or Refractory (R/R) Multiple Myeloma Who Have Failed a Prior Lenalidomide-Containing Regimen

IntroductionAzacitidine (AZA) is a DNA methyltransferase inhibitor and cytotoxic agent with survival benefits in higher-risk MDS and AML. We have demonstrated that AZA rapidly induces apoptosis of MM cells, and induces synergistic killing when combined with lenalidomide (LEN). This, coupled with development of an oral AZA formulation (CC-486), provides rationale for pursuing this combination in MM. The cereblon [CBRN]-Ikaros/Aiolos-IRF4/c-MYC signalling pathway has been implicated in the mechanism of action of cytotoxicity for IMiD®compounds. This pathway, as well as immune markers (CD8, PD-1) may represent potential biomarkers for predicting outcomes/response to IMiD® compounds.Aims:In R/R myeloma patients who previously failed a LEN-containing regimen: to determine the maximum tolerated dose (MTD) of CC-486 in combination with Rd; to characterise safety/tolerability; to assess efficacy: overall response rate (ORR), progression free survival (PFS), overall survival (OS).To correlate pre-treatment MM protein expression (CRBN, Ikaros, Aiolos, IRF4, c-MYC), as well as whole marrow expression of CD8/PD-1 with response and survival (PFS/OS) in patients receiving CC-489 plus Rd.Methods:Phase Ib, single centre, 3 x 3 dose escalation study. LEN 25mg d1-21 and dexamethasone 40mg d1, 8, 15, 22 of 28 day cycle were combined with escalating doses of CC-489: initial dose 100mg for d1-14, increasing by either 7 days or 50mg/cohort, to a maximum of 200mg d1-21. Dose limiting toxicity (DLT) was assessed during cycle 1. Treatment continued until toxicity/progression.Single- and dual-IHC assays were performed on pre-treatment trephines (Single: CD8, PD-1; Dual: CD138 paired with CRBN, Aiolos, Ikaros, IRF4, c-MYC). In samples with >10% CD138, presence and sub-cellular localisation of each stain was documented, and given an H score: product of % of tumour positive and intensity of staining (0-3). Single CD8 and PD-1 IHC assays were reported as positive cells/mm2. Samples were grouped according to quartiles (Group1: lower quartile, Group2: mid-range, Group3: upper quartile) and analysis was performed using SAS statistical platform v9.4.Results:22 patients commenced therapy (F=10, M=12), median age 67yrs (50-82yrs). Median prior lines of therapy: 5 (2-8), including 16 ASCT and 2 prior allogeneic transplant. All had failed LEN (R/R=18, primary refractory (PrimR)=4), 10/22 received and failed pomalidomide (POM) (R/R=4, PrimR=6). All had received bortezomib (R/R=10, PrimR=10), 18/22 were both bortezomib and LEN refractory.CC-486 dose reached was 200mg d1-21, with no DLTs observed at time of reporting.One patient died due to unrelated causes prior to end cycle 1, therefore was not evaluable for response. ORR (≥PR) was 43% (9/21): 8 PR, 1 VGPR. Of the remaining patients, 2 achieved MR, 5 SD, and 5 PD. (clinical benefit rate (≥MR) = 52%). Median time to best response in patients with ≥MR: 2.5m (1-3.7m). Responses were seen in cohorts: [100mg d1-14 (3PR=3), 100mg d1-21 (VGPR=1, PR=3), 150mg d1-21 (PR=2, MR=2)]. Median time on study: 3m; responders (≥MR): 6.3m (2.5-15m), non-responders: 1.7m (0.9-8m). Median PFS 3m, median OS 15m. One patient remains on study.3/6 patients treated with LEN in prior 1-2 treatment lines responded (PR=2, VGPR=1), 1/6 had SD. 1/10 patients treated with POM in prior 1-2 treatment lines responded (PR), with 2 achieving MR and 3 SD.Patients with a lower expression of cMYC and IRF4 had superior PFS compared with patients with higher expression (cMYC: Group1 vs Group3 p=0.052; IRF4: Group1 vs Group2 p=0.04). Patients with lower numbers of CD8+ T-cells had better PFS than those with higher (p=0.069). There was no association between degree of expression of CBRN, Ikaros, Aiolos or PD-1 and survival (PFS/OS).As CRBN expression increased, patients were more likely to respond (≥PR) (p=0.07) and patients with low PD-1 expression were more likely to respond (≥ MR) (Group1 vs Group3 p=0.05).ConclusionCC-489 combined with Rd is well tolerated and effective with durable responses in a subset of heavily pre-treated R/R MM patients, including those who recently failed IMiD® compound therapy, suggesting that CC-489 may overcome drug resistance. IHC may have utility in identifying subsets of patients more likely to respond to CC-489 and Rd (CRBN, PD-1) and predict survival (cMYC, IRF4 and CD8). This trial has been expanded to include other sites and less heavily pre-treated patients. DisclosuresThakurta:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment. Guzman:Celgene: Employment. Cuoto:Celgene: Employment. Ren:Celgene: Employment, Equity Ownership.

Selective HDAC6 Inhibitor ACY-241, an Oral Tablet, Combined with Pomalidomide and Dexamethasone: Safety and Efficacy of Escalation and Expansion Cohorts in Patients with Relapsed or Relapsed-and-Refractory Multiple Myeloma (ACE-MM-200 Study)

Selective HDAC6 Inhibitor ACY-241, an Oral Tablet, Combined with Pomalidomide and Dexamethasone: Safety and Efficacy of Escalation and Expansion Cohorts in Patients with Relapsed or Relapsed-and-Refractory Multiple Myeloma (ACE-MM-200 Study)

Quercetin induces cell apoptosis of myeloma and displays a synergistic effect with dexamethasone in vitro and in vivo xenograft models.

Quercetin, a kind of dietary flavonoid, has shown its anticancer activity in many kinds of cancers including hematological malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and MM) in vitro and in vivo. However, its effects on MM need further investigation. In this study, MM cell lines were treated with quercetin alone or in combination with dexamethasone. In order to observe the effects in vivo, a xenograft model of human myeloma was established. Quercetin inhibited proliferation of MM cells (RPMI8226, ARP-1, and MM.1R) by inducing cell cycle arrest in the G2/M phase and apoptosis. Western blot showed that quercetin downregulated c-myc expression and upregulated p21 expression. Quercetin also activated caspase-3, caspase-9, and poly(ADP-ribose)polymerase 1. Caspase inhibitors partially blocked apoptosis induced by quercetin. Furthermore, quercetin combined with dexamethasone significantly increased MM cell apoptosis. In vivo xenograft models, quercetin obviously inhibited tumor growth. Caspase-3 was activated to a greater extent when quercetin was combined with dexamethasone. In conclusion, quercetin alone or in combination with dexamethasone may be an effective therapy for MM.