Increased Multiple Myeloma Cell Research Articles

Ferritin Metabolism Reflects Multiple Myeloma Microenvironment and Predicts Patient Outcome.

Multiple myeloma (MM) is a hematologic malignancy with a multistep evolutionary pattern, in which the pro-inflammatory and immunosuppressive microenvironment and genomic instability drive tumor evolution. MM microenvironment is rich in iron, released by pro-inflammatory cells from ferritin macromolecules, which contributes to ROS production and cellular damage. In this study, we showed that ferritin increases from indolent to active gammopathies and that patients with low serum ferritin had longer first line PFS (42.6 vs. 20.7 months and, p = 0.047, respectively) and OS (NR vs. 75.1 months and p = 0.029, respectively). Moreover, ferritin levels correlated with systemic inflammation markers and with the presence of a specific bone marrow cell microenvironment (including increased MM cell infiltration). Finally, we verified by bioinformatic approaches in large transcriptomic and single cell datasets that a gene expression signature associated with ferritin biosynthesis correlated with worse outcome, MM cell proliferation, and specific immune cell profiles. Overall, we provide evidence of the role of ferritin as a predictive/prognostic factor in MM, setting the stage for future translational studies investigating ferritin and iron chelation as new targets for improving MM patient outcome.

FGFR1OP2 Promotes Proliferation and Survival of Myeloma Cells and Is a Potential Therapeutic Target

Background: Multiple myeloma (MM) is an incurable disease with high tendency to relapse and drug resistance. Exploring new targets and new drugs is crucial for treatment. Elevated expression of fibroblast growth factor receptor 1 oncogene partner 2 （FGFR1OP2） has been observed in relapsed and refractory MM and indicated the poor prognosis. FGFR1OP2 is expressed in diverse tissues, including bone marrow, spleen and thymus and might be a oncogene in lung cancer, acute leukemia. we hypothesize that FGFR1OP2 may exert effects in MM and will be a a potential therapeutic target. Aims: To Investigate the functional role of FGFR1OP2 in MM Methods: FGFR1OP2 knockdown and overexpression MM cell lines were generated. Molecular mechanism underlying was elucidated by qRT-PCR, Western Blot and gene expression microarray. Results: To study whether FGFR1OP2 is important for MM, we found high expression of FGFR1OP2 in different myeloma cell lines which was validated by CCLE datasets. Knockdown the FGFR1OP2 in MM cells led to a decreased proliferation, while overexpression of FGFR1OP2 increased the cell growth (Fig.1a). Next, flow cytometry was performed to evaluate the cell cycle and apoptosis in MM cells. FGFR1OP2 suppression showed cell cycle arrest and overexpression accelerateed cell cycle progression (Fig.1b). Furthermore, Bortezomib(Bor)-induced apoptosis of MM cells was significantly increased when FGFR1OP2 expression was suppressed (Fig.1c). Moreover, results of Western Blot revealed increased levels of Bor-induced caspase-3 and cleaved PARP after FGFR1OP2 knockdown. Taken together these data indicate that FGFR1OP2 can increase MM cell growth and inhibit apoptosis. To better understand the molecular mechanism of FGFR1OP2, we performed gene expression microarray profiling and compared results from FGFR1OP2-knockdown cells and control cells. Surprisingly, data showed that the expression of several members of the Hippo signaling pathway changed significantly. We performed Western Blot to validate these results, and found expression of YAP1 decreased while MST1/2 increased in FGFR1OP2-knockdown cells (Fig.1c). This suggests that the FGFR1OP2 promotes MM cell growth and anti-apoptosis through Hippo signaling pathway. Conclusion:FGFR1OP2 is critical for the proliferation and survival of MM cells through Hippo pathway and may represent a novel target for therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

Multiple myeloma (MM) cells display a reciprocal relationship with neighboring niche cells in the bone marrow (BM), particularly vascular endothelial cells (BM ECs). This relationship drives disease initiation, progression, and therapy resistance, but the mechanisms underlying this interaction remain incompletely understood. Here, we show that silencing ephrin B2 (EFNB2) blocked MM cell proliferation and survival in vitro and abrogated MM engraftment and repopulation in vivo. EFNB2 expression is also increased in MM patients with adverse-risk cytogenetics and decreased survival, suggesting an important role for EFNB2 in MM disease progression. To identify novel regulators of MM pathogenesis, we performed transcriptome analyses of ECs from multiple human tissues and analyzed their capacity to promote MM growth. The EPH receptor, EPHB1, was overexpressed in human ECs that support MM expansion in culture. Silencing EPHB1 expression in ECs using siRNA or a lentiviral shRNA abrogated MM cell expansion compared to EC co-cultures treated with non-targeting shRNA and blocked the expansion of MM cells capable of in vivo repopulation in immune deficient mice. Additionally, transgenic Vk*MYC mice, which develop indolent murine MM through activation-induced deaminase-dependent MYC activation in germinal B cells, displayed increased expression of EPHB1 and EPHB4 in BM ECs compared to wild type BM ECs. We found that expression of EFNB2, an EPHB1/4 binding partner, is upregulated in 3 human MM cell lines and in primary MM cell populations from patients, whereas other EPHB1/4 binding partners were either not detected or expressed at a low level. shRNA-mediated inhibition of EFNB2 in MM cells blocked MM cell cycling, leading to a near 70-fold reduction in MM cell growth at day 12 of culture, and blunted MM cell repopulation when transplanted into NSG mice. In order to determine the therapeutic potential of targeting EFNB2 in MM in vivo, we transplanted irradiated NSG mice with MM cells and subsequently administered a single chain variable fragment (scFv) targeting EFNB2 on days +4, +6, +10 and +12. Mice treated with the EFNB2 scFv displayed significantly decreased engraftment of MM cells in vivo compared to control mice. MM reconstitution in vivo. Silencing EFNB2 expression in MM cells downregulated several pathways including those involved in cellular movement, cell death and survival, cellular assembly, cell cycle and DNA repair. Several STAT5 target genes were also downregulated in MM cells treated with shRNA-EFNB2, supporting the notion that STAT5 signaling is activated by ephrin B proteins (Bong et al. Proc Natl Acad Sci. 2007;104(44):17305-17310). Consistent with these findings, EFNB2 overexpression via dCas9-VPR increased MM cell survival, proliferation and STAT5 phosphorylation. Conversely, silencing EFNB2 decreased STAT5 activation, increased MM apoptosis, and decreased MM proliferation. Importantly, STAT5 inhibition in MM cells overexpressing EFNB2 blocked EFNB2-mediated MM cell proliferation, suggesting that EFNB2-mediated MM growth is dependent on STAT5. Overexpressing a mutant form of human EFNB2 with GFP replacing the intracellular cytoplasmic PDZ signaling domain did not result in cell growth comparable to overexpressing the full-length EFNB2. Silencing EPHB4, the main EFNB2 binding partner expressed on MM cells, resulted in significant impairment of growth in wild-type MM cells but not those with full-length EFNB2 overexpression. Updated downstream signaling and in vivo data of these will be presented at the meeting. Interrogation of MM clinical databases, including the Total Therapy studies (Zhan F. et al. Blood. 2006;108(6):2020-2028) and the CoMMpass data (Multiple Myeloma Research Foundation, https://research.themmrf.org/, accessed on 20 October 2021, n = 773 patients), revealed that EFNB2 expression is increased in MM patients with adverse risk cytogenetics, including t(4;14) and gain of chromosome 1q, while lower EFNB2 expression is associated with the more favorable, "standard risk" disease category, hyperdiploid MM. Increased EFNB2 expression is also associated with advanced stage disease and decreased survival in MM patients. Our analyses suggest that EFNB2 signaling represents an important new mechanistic target in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myeloma cells regulate miRNA transfer from fibroblast-derived exosomes by expression of lncRNAs.

Multiple myeloma (MM) progression and drug resistance depend on the crosstalk between MM cells and bone marrow (BM) fibroblasts (FBs). During monoclonal gammopathy of undetermined significance (MGUS) to MM transition, MM cell-derived exosomes (EXOs) reprogram the miRNA (miR) profile of FBs, inducing the overexpression miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Here, we demonstrate that the miR content of MM FB-derived EXOs (FB-EXOs) overlaps the miR profile of parental FBs by overexpressing comparable levels of miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Recipient MM cells co-cultured with MM FB-EXOs selectively overexpress only miR-214-3p and miR-5100 but not miR-23b-3p, miR-27b-3p, and miR-125b-5p, suggesting a putative selective transfer. MM cells express HOTAIR, TOB1-AS1, and MALAT1 lncRNAs. Transient transfection of MM cells with lnc·siRNAs demonstrates that HOTAIR, TOB1-AS1, and MALAT1 lncRNAs are sponges for miR-23b-3p, miR-27b-3p, and miR-125b-5p. Indeed, lncRNA knockdown significantly increased miR levels in U266 MM cells co-cultured with MM FB-EXOs. Selective miR-214-3p and miR-5100 overexpression modulates MAPK, PI3K/AKT/mTOR, and p53 pathways in MM cells. Interrogation using the DIANA tools algorithm and transient overexpression using miR mimic probes confirmed the involvement of miR-214-3p and miR-5100 and their target genes, PTEN and DUSP16, respectively, in the modulation of these intracellular pathways. Finally, the uptake of EXOs as well as miR-214-3p and miR-5100 overexpression increase MM cell proliferation and resistance to bortezomib-induced apoptosis by switching the balance between pro-/anti-apoptotic proteins. Overall, these data show that MM cells are not simply a container into which EXOs empty their cargo. On the contrary, tumour cells finely neutralize exosomal miRs via lncRNA expression to ensure their survival. © 2021 The Pathological Society of Great Britain and Ireland.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 μM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

Preclinical Assessment of CDR101 - a BCMAxCD3xPD-L1 Trispecific Antibody with Superior Anti-Tumor Efficacy

BCMAxCD3 targeting therapies have demonstrated anti-myeloma activity, and high minimal residual disease negativity rates can be achieved with this approach in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM). Despite these promising clinical results, patients eventually develop resistant disease and relapse. Thus, there is a high need for novel BCMA therapies that can evade the resistance mechanisms and provide more durable responses. Recently, we reported on the promising activity of the Local Activator and T cell Engager (LocATE) technology, a trispecific molecule that targets CD3, BCMA and PD-L1, redirecting T cells to multiple myeloma (MM) cells while selectively counteracting PD-L1/PD-1 induced immunosuppression at the immune synapse (ASH, 2020). Here we present CDR101, an optimized LocATE candidate with potential for clinical development.First, we analyzed the ability of CDR101 to induce PBMC-mediated cytotoxicity in two MM cell-lines expressing BCMA (U-266 and NCI-H929) and compared it to four BCMAxCD3 bispecific formats currently in clinical development (a half-life extended BCMAxCD3 BiTE, a BCMA-TCB, and two different BCMAxCD3 bispecific monoclonal antibodies) alone or in combination with a separate PD-L1 blocking antibody. CDR101 resulted in at least 10-fold increased T cell-mediated target cell lysis compared to control BCMAxCD3 bispecifics. Strikingly, CDR101 also resulted in increased MM cell killing when compared to free, independent combinations of BCMAxCD3 bispecifics and the PD-L1 inhibitor. These results, together with the observation that MM cells upregulate the expression of PD-L1 in response to treatment with BCMAxCD3 bispecifics, suggest that the superior effect of CDR101 could be attributed to preferential and highly selective inhibition of the PD-1/PD-L1 axis at the cellular interaction within the immune synapse.Next, bone marrow aspirates from newly diagnosed and RRMM patients were treated with increasing concentrations of CDR101 or a BCMAxCD3 bispecific control. After 24h of incubation, percentage of viable CD138-positive cells and activation status of autologous T cells were analyzed by FACS. Overall, CDR101 potently induced lysis of primary MM cells independently of the E:T ratio (range of E:T ratio between 1.3:1 and 33:1). CDR101 achieved higher target cell killing in all samples compared to the bispecific control, with at least 2-fold difference in 3 out of 4 samples at the highest concentration tested. Concomitantly, CDR101 induced a dose-dependent increase of the T cell activation marker CD25, corroborating the ability of CDR101 to counteract PD-L1/PD-1 induced immunosuppression.In vivo anti-tumor activity of CDR101 was evaluated using a human MM (NCI-H929) xenograft model in NPG mice. Treatment with four different doses of CDR101 or BCMAxCD3 bispecific control demonstrated that CDR101 induced stronger and more durable responses compared to the bispecific control leading to complete tumor regression in 55 out of 60 mice at the last day of treatment (day 29) with no relapse until the end of the observation time (day 41).Collectively, CDR101 demonstrated that targeting BCMA with simultaneous blockade of PD-L1 leads to improved myeloma cell killing compared to clinically validated therapies. In contrast to high-affinity PD-L1 immune checkpoint inhibitors, CDR101 selectively inhibits PD-L1 at the immune synapse preventing on-target off-tumor effects. This is expected to translate into a decreased incidence of immune related adverse events (irAEs) and better efficacy arguing for a high clinical potential and swift translation into the clinic. DisclosuresVrohlings: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Jungmichel: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Senn: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Howald: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Schleier: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scheifele: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wendelspiess: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Richle: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Merten: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Lenherr-Frey: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Leisner: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA. Borras: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Novel BCMA Targeted ADCs, Bispecific and Trispecific Antibodies

Over the past five years, several antibody-based therapeutic approaches have been investigated in multiple myeloma (MM) beyond the naked antibodies that had targeted DKK1, CD38 and SLAMF7. The antibody-drug conjugates (ADCs) have been utilized for different solid and hematologic malignancies to deliver a toxic payload to a cancer cells with a specific surface marker. Belantamab mafodotin (Belamaf) is the first FDA approved, B-cell maturation antigen (BCMA) targeting ADC in triple-class exposed, relapsed and/or refractory MM beyond four prior lines of therapy. Belamaf is now being combined with other drug classes in earlier lines of therapy. There are other MM surface markers beyond BCMA being targeted with ADCs (CD38, CD46, CD56, CD74, CD138, etc.) but are in early clinical development. Bispecific antibodies (BsAbs) were conceived as a novel idea to direct the cytotoxic immune effector cells system towards cancer cells in the late 1960s but came to fruition clinically in the 2000s. The initial wave of BsAbs recognize CD3 on the T-cells whereas the MM specific surface markers that have been targeted include BCMA, GPRC5D, CD38, FCRH5 (Table 1). Although there are BsAbs in preclinical development exploring CD16 in place of CD3 to help direct natural killer cells towards the tumor cells, the present talk will summarize clinical data on BsAbs in MM that have been reported at recent international congresses. Lastly, the trispecific antibodies are in preclinical development with dual MM surface markers targeting in an attempt to increase MM cell specificity.

Inhibition of p62-ZZ Domain-Mediated Signaling Overcomes Bortezomib Resistance in Multiple Myeloma Cells Independent of Their p53 Status

Multiple myeloma (MM) cells depend on autophagy as well as proteasome activity for their survival. In MM cells, autophagy is essential to reduce the extraordinary ubiquitinated protein system stress derived from high levels of immunoglobulin synthesis. Thus, proteasome inhibitors (PIs) decrease tumor burden in the majority of MM patients but eventually a PI resistant MM clone(s) arises. Bortezomib (BTZ) resistance has been reported to correlate with p53 status of MM cells. Previous studies showed that MM patients harboring p53 genetic abnormalities are commonly resistant to standard therapies, increasing the need for new druggable targets.A possible mechanism of BTZ resistance involves the cargo receptor for ubiquitin-mediated autophagy SQSTM1/p62, that enhances MM cells survival by increasing their autophagic flux. We found that MM cells with wild type p53, are more sensitive to PI treatment, while MM cells harbouring mutant p53 or lacking p53 are more resistant to BTZ-mediated cytotoxicity. BTZ treatment induced a modest level of apoptosis in MM1.S cells (p53 wild type) at high doses (>5nM) by triggering the cleavage of caspase 3 and PARP-1. As expected BTZ treatment induced a significant compensatory increase in SQSTM1/p62 at the mRNA and protein levels in these cells. Conversely, BTZ treatment of p53 mutant MM cells failed to induce apoptosis, exhibiting only a mild effect on viability. This occurred in spite of a similar increase in SQSTM1/p62 expression as seen in MM.1S cells.To determine the role of p62 in the BTZ resistance of p53 mutant MM cells, we tested a novel small-molecule p62-ZZ domain inhibitor, XRK3F2 we recently identified. XRK3F2 treatment of mice with established MM, dramatically increased bone formation in MM-bearing bones, although it did not affect tumour burden in vivo . In vitro studies showed that high concentrations (10μM) of XRK3F2 were required to decrease the viability of primary and MM cell lines and induce a time-dependent activation of caspases 3, 7 and 9. XRK3F2 treatment dose-dependently increased levels of the lipidated form of LC3, LC3II, suggesting activation of pro-survival autophagy. Surprisingly, the expression levels of SQSTM1/p62 were either unchanged or increased following XRK3F2 treatment despite the increase of autophagosome formation, indicating that XRK3F2 inhibited the autophagic flux in MM cells. Defective autophagy occurs in tumor cells and leads to accumulation of p62, further promoting tumorigenesis by altering NFκB regulation and gene expression. We found that XRK3F2 treatment also significantly inhibited TNFα-induced NFκB activation, by inhibiting p65 nuclear translocation. These data suggested that XRK3F2 may sensitize MM cells to BTZ-induced cytotoxicity and possibly overcome BTZ resistance.To test this hypothesis, human MM cell lines MM1.S (p53 wild type), U266 (p53 mutant) and KMS-11 (p53 null) were exposed to different doses of a BTZ-XRK3F2 combination comprised of varying concentrations of BTZ combined with XRK3F2 at 5μM. We found treatment with the BTZ-XRK3F2 combination significantly reduced cell viability compared with either drug alone at the same doses, and increased MM cells sensitivity to BTZ, independent of their p53 status (IC50 BTZ vs BTZ-XRK3F2 for MM1.S 3 vs 1.5nM of BTZ; for U266 10 vs 1.7nM of BTZ; for KMS-11 7 vs 1nM BTZ).Mechanistic studies revealed that in p53 wild type MM cells, the combination (Bortezomib 3nM - XRK3F2 5 μM) significantly increased annexin V positive cells after 6h of treatment. This was followed by a reduction in expression of the anti-apoptotic Bcl-2 protein (mRNA and protein levels) and extensive activation of caspase 3 and cleaved PARP1 after 24h, demonstrating apoptosis-mediated cell death. Importantly, p62 expression was significantly reduced by the BTZ-XRK3F2 combination. The decrease in p62 level may result as a consequence of its caspase 3-dependent degradation. In p53 mutant cells, the combination significantly induced PARP1 cleavage, although only mildly increased caspase 3 activation. These results imply that the BTZ-XRK3F2 combination activates multiple parallel death pathways to enhance both apoptosis and necrosis.Taken together, these data suggest that p62-ZZ antagonists, alone or in combination with PIs, may have both anti-tumor and bone anabolic effects, and may overcome PI resistance in MM cells, making p62 an attractive target for the treatment of MM. DisclosuresXie:Oxis Biotech: Consultancy. Roodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

MicroRNA-1252-5p Associated with Extracellular Vesicles Enhances Bortezomib Sensitivity in Multiple Myeloma Cells by Targeting Heparanase.

IntroductionMultiple myeloma (MM) remains an incurable disease, and patient survival requires a better understanding of this malignancy’s molecular aspects. Heparanase (HPSE) is highly expressed in aggressive MM cells and related to tumor growth, metastasis, and bortezomib (BTZ) resistance. Thus, targeting HPSE seems to be a promising approach for MM treatment, and because microRNAs (miRNAs) have emerged as potential regulators of HPSE expression, the use of extracellular vesicles (EVs) can allow the efficient delivery of therapeutic miRNAs.MethodsWe used prediction algorithms to identify potential miRNAs that regulate negatively HPSE expression. RT-qPCR was performed to assess miRNAs and HPSE expression in MM lines (U266 and RPMI-8226). Synthetic miRNA mimics were electroporated in MM cells to understand the miRNA contribution in HPSE expression, glycosaminoglycans (GAGs) profile, cell proliferation, and cell death induced by BTZ. EVs derived from HEK293T cells were engineered with miRNAs to evaluate their therapeutic potential combined with BTZ.ResultsIt revealed a direct association between BTZ sensitivity, HPSE, and miR-1252-5p expressions. Moreover, overexpression of miR-1252-5p significantly reduced HPSE expression and HPSE enzymatic activity in MM cells. The higher level of miR-1252-5p was correlated with a reduction of cell viability and higher sensitivity to BTZ. Further, EVs carrying miR-1252-5p increased MM cells' sensitivity to BTZ treatment.ConclusionThese results showed that miR-1252-5p could negatively regulate HPSE in MM, indicating the use of EVs carrying miR-1252-5p as a potential novel BTZ sensitization approach in MM cells.

Ruxolitinib reverses checkpoint inhibition by reducing programmed cell death ligand-1 (PD-L1) expression and increases anti-tumour effects of T cells in multiple myeloma.

Multiple myeloma (MM) tumour cells evade host immunity through a variety of mechanisms, which may potentially include the programmed cell death ligand-1 (PD-L1):programmed cell death protein-1 (PD-1) axis. This interaction contributes to the immunosuppressive bone marrow (BM) microenvironment, ultimately leading to reduced effector cell function. PD-L1 is overexpressed in MMBM and is associated with the resistance to immune-based approaches for treating MM. Ruxolitinib (RUX), an inhibitor of the Janus kinase (JAK) family of protein tyrosine kinases, is approved for myeloproliferative diseases. We investigated the effects of RUX alone or in combination with anti-MM agents on the expression of PD-L1 and T-cell cytotoxicity in MM. We showed that the expression of the PD-L1 gene was markedly increased in BM mononuclear cells from patients with MM with progressive disease versus those in complete remission. Furthermore, RUX treatment resulted in a concentration-dependent reduction of PD-L1 gene expression in the MM tumour cells cultured alone or co-cultured with stromal cells compared with untreated cells. The results also demonstrated that RUX increased MM cell apoptosis in the presence of interleukin-2-stimulated T cells to a similar degree as the treatment with anti-PD-1 or anti-PD-L1 antibodies. In summary, these results indicate that RUX can block PD-L1 expression resulting in augmentation of anti-MM effects of T cells.

POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

Exploring POU2AF1 (BOB-1) Dependency and Transcription Addiction in Multiple Myeloma

Multiple Myeloma (MM) is a complex disease driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, copy number abnormalities and somatic mutations all contribute to myelomagenesis, yet true drivers of the disease have yet to be identified. In order to investigate the enhancer landscape and identify new dependencies and actionable therapeutic targets in MM, we have generated high-quality active enhancer landscape in MM cell lines as well as in large cohort of primary patient myeloma cells (n=70) and normal plasma cell. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining this data with gene expression and genetic dependency (CRISPR KO) enables a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified genes that are super-enhancer associated, expressed, and required specifically in MM versus other tumor types or normal tissues. Many of the specific and potent dependencies in MM are transcription factors, and especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. BOB-1 is a gene regulatory factor that regulates B-cell development, maturation and GC formation. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells, giving us the rationale to study the transcriptional program associated with BOB-1 in MM cells. To confirm CRISPR data described above, we have performed loss-of-function (LOF) studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1. Downregulation of BOB-1 in a panel of MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with G2/M cell cycle arrest and induction of MM cell apoptosis. Transcriptomic analysis by RNA-sequencing revealed a set of 72 genes (adj p-value=0.2) commonly modulated in AMO-1 and NCI-929 MM cell lines upon BOB-1 depletion, as compared with scrambled cells. Among the top 5 downregulated genes in BOB-1 knockdown samples, we found AMPD1, KCNN3, BHLHA15, HID1 and XBP1. The modulation of BOB1 target genes was confirmed at the protein level by Western blot analysis in BOB-1 LOF and gain-of-function (GOF) cell systems. Analysis of patient expression datasets revealed that these genes are positively correlated with BOB-1 expression in primary MM cells and overexpressed in MM cells compared to normal plasma cells. One of these 5 genes positively controlled by BOB-1, BHLHA15, is the gene coding for the transcription factor Mist1, which functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1. Target genes of the XBP1- BHLHA15 axis are involved in lipid synthesis, protein folding and secretion, and ER-associated protein degradation. As a result, BOB-1 knockdown increased de novo protein synthesis in MM cells compared to control cells. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, Mist1 serves as a feedback inhibitor. Moreover, we identified that LOF studies in two MM cell lines confirmed increased expression of BOB-1 and XBP1 upon KD of Mist1. However, XBP1 and/or Mist1 are dispensable for MM cell survival, as gene depletion did not affect MM cell viability. Among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 HMOX1 gene, whose expression is significantly lower in-patient MM cells compared to normal plasma cells. Moreover, its lower expression significantly correlated with poor clinical outcome; and siRNA depletion of HMOX1 increased MM cell growth in MM cell lines, suggesting important role in MM. Mechanistic studies aimed at investigating the functions of HMOX1 in MM and its relation with BOB-1 are ongoing and will be presented. In conclusion, we here report BOB1 as an important transcriptional regulator in MM and its two down-stream novel target genes (BHLHA15 and HMOX1) with potential significant biological role in MM. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy; PharmaMar: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fulciniti:NIH: Research Funding. Munshi:Janssen: Consultancy; C4: Current equity holder in private company; Karyopharm: Consultancy; Amgen: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Legend: Consultancy; Adaptive: Consultancy; AbbVie: Consultancy; Takeda: Consultancy.

Functional Genomic Characterization of Endoplasmic Reticulum-Associated Dependencies in Multiple Myeloma - Biologic and Therapeutic Implications

Background: Endoplasmic reticulum (ER)-associated degradation (ERAD) for unfolded proteins represents an important biological vulnerability for multiple myeloma (MM) cells, given the high proteostatic stress in these cells due to immunoglobulin secretion. This helps explain the efficacy of proteasome inhibitors in MM compared to most other neoplasms. Aim: To systematically investigate ER-associated genes that mediate the essential role of ERAD in MM. Methods: We integrated results from (i) genome-scale studies for CRISPR-gene knockout in 19 MM cell lines (Avana library) and CRISPR-activation (Calabrese library) in 5 MM lines, to define ER-associated dependencies and drivers of increased MM cell growth; (ii) publicly-available CRISPR-based studies for genes regulating ERAD of substrates located in the luminal (ERAD-L), membrane (ERAD-M) or cytosolic (ERAD-C) side of the ER in non-MM models; (iii) functional characterization of candidate genes in MM models in vitro and in vivo. Results: While ER function in general is globally required for all normal and malignant cells, a surprisingly large proportion of ER-related genes are not "pan-essential" genes (e.g. CERES scores <-0.4 are for <80% of human cell lines): in fact CRISPR-mediated disruption of many ER genes elicits no growth disadvantage for most non-MM tumor cell lines. Notably, though, several of these latter non-"pan- essential" genes are recurrently essential for MM cell lines, or even preferential dependencies for MM compared to other cancers. By overlaying the patterns of CRISPR essentiality in MM lines vs. the functional modules of ER function, we observe that recurrent or preferential ER-associated dependencies for MM cells include genes involved in (a) ER membrane protein complexes mediating dislocation of misfolded ER proteins to the cytosol (e.g. HERPUD1, SEL1L, AUP1 for ERAD-L substrates; and AMFR and RNF139 for ERAD-M and -C substrates,) and associated ER-specific E2/E3 enzymes (SYVN1, UBE2J1, UBE2J2, and UBE2G2); (b) upstream of the ER-to-cytosol dislocation complexes, several enzymes (e.g. DPM1, DPM3, ALG3, ALG9, ALG12, ALG6, ALG8) required for N-glycan-dependent surveillance of quality control for luminal ER glycoproteins; (c) chaperones (e.g. DNAJB11, DNAJBC3) for BiP complexes with misfolded proteins; (d) the known ER stress-sensor IRE1a (ERN1) and its downstream transcription factor XBP1. CRISPR-activation of nearly all these recurrent/MM-preferential ER-associated genes led to no significant growth advantage for MM cell lines. Several of the MM-preferential ER-associated genes (HERPUD1, SEL1L, SYVN1, UBE2J1) are expressed at significantly higher levels in MM vs. non-MM cell lines (CCLE) or patient derived samples (CoMMpass study vs. TCGA) and are proximal to binding sites of key transcription factors for plasma cell identity such as IRF4 and PRDM1. By contrast, CRISPR-mediated LOF of such genes in MM cells decreases their growth in vitro (e.g. competition assays vs. CRISPR LOF of non-essential genes not expressed in MM cells) and in vivo for MM cells growing in a bone marrow-like scaffold system with a "humanized" mesenchymal bone marrow stromal cell compartment. Knockout of MM-preferential ER-associated genes induces markers of ER stress/unfolded protein response (e.g. BiP induction by UBE2J1 or HERPUD1 LOF); and sensitizes MM cells to proteasome inhibition. Accordingly, pharmacological inhibition (LS-102) of SYVN1 (Synovolin-1) an E3 ligase which helps remove unfolded proteins from the cytosol into the ER was more effective at a lower drug concentration in MM compared to non-MM-cell lines. Conclusions: Our results provide functional insights into the role of ERAD in MM cells and nominate specific mediators which are not broadly essential across all cancers, but are recurrent or even preferentially essential for MM. Our study indicates a previously underappreciated role for the ER-to-cytosol dislocon complex that includes HERPUD1, SEL1L, SYVN1 and UBE2J1; distinct enzymes of the N-glycan-dependent ER glycoproteins quality control system; and ER chaperones in MM. These findings provide a rationale for development of agents selectively targeting these key regulators of ER protein folding processes in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Arch Oncology: Research Funding.

Identification of a Novel c-Myc Inhibitor 7594-0037 by Structure-Based Virtual Screening and Investigation of Its Anti-Cancer Effect on Multiple Myeloma.

IntroductionMultiple myeloma (MM) is an extremely malignant and incurable hematological cancer. Increased expression of the c-Myc oncoprotein is closely associated with shorter overall survival of MM patients, implying that c-Myc is a potential therapeutic target.Main MethodsWe identified a potential c-Myc inhibitor 7594–0037 by structure-based virtual screening from the ChemDiv database. CCK8 assay and flow cytometry were used to detect MM cell viability, cell cycle and apoptosis. Q-PCR and Western blot were used to measure corresponding mRNA and protein expression levels. Protein stability assay measured the stability of c-Myc.ResultsCompound 7594–0037 exhibited stronger anti-proliferative activity against MM cells, and induced MM cell cycle G2 phase arrest and apoptosis. More importantly, compound 7594–0037 overcame myeloma resistance to bortezomib and exhibited a synergistic effect with bortezomib, resulting in increased MM cell death. The mechanism consists of compound 7594–0037 facilitating c-Myc protein degradation via decreasing the c-Myc S62 phosphorylation levels mediated by PIM1 kinase. Molecular dynamics simulation with the c-Myc/7594-0037 complex showed that compound 7594–0037 bound tightly to the N-terminus of c-Myc, and blocked the binding interaction of the two termini of c-Myc, which resulted in c-Myc entering into an unstable state.ConclusionOverall, our study provides preliminary data for compound 7594–0037, which can be used as a novel c-Myc inhibitor and is a potential candidate therapeutic drug for multiple myeloma.

Pivotal Role of OCL-Derived IGF1 in Drug Resistance and Bone Destruction in MM

Multiple myeloma (MM) is largely incurable, and is characterized by devastating bone destruction caused by increased osteoclast (OCL) differentiation and bone resorption in more than 85% of MM patients. OCLs in MM not only promote bone resorption but also increase MM cell growth and drug resistance. Despite recent advances in anti-myeloma treatment, development of anti-MM drug resistance is a major limitation of MM therapy. Therefore, new treatment modalities are urgently needed to overcome drug resistance and decrease bone resorption. IGF1 is a crucial factor for tumor cell growth and survival of malignant cells, especially in MM. IGFI also contributes to development of drug resistance of MM cells to anti-MM agents, including proteasome inhibitors and immunomodulatory agents, but how OCLs contribute to drug resistance is still not clearly delineated. We found that IGF1 was highly expressed in OCLs attached to bone and bone marrow myeloid cells in vivo, and the expression levels of IGF1 in OCLs from MM bearing mice is higher than in normal OCLs. Intriguingly, OCLs produced more IGF1 (0.8 ng/ml/protein) than MM cells (not detected) and bone marrow stromal cells (BMSCs) (0.4 ng/ml/protein) in vitro. In addition, IGF1 protein expression in OCLs was upregulated (1.8 fold) by treatment with conditioned media (CM) from 5TGM1 murine MM cells, TNF-α or IL-6, major paracrine factors that are increased in the bone marrow microenvironment in MM. These results suggest that OCLs are a major source of local IGF1 in the MM bone marrow microenvironment. To further characterize the role of OCL-derived IGF1, we generated a novel mouse with targeted deletion of Igf1 in OCLs (IGF1-/--OCL), and assessed the role of OCL-derived IGF1 in drug resistance of MM cells and bone destruction. Treatment of 5TGM1 cells with bortezomib (BTZ) (3 nM, 48 hours) decreased the viability of 5TGM1 cells by 50%. Importantly, the cytotoxic effects of BTZ on MM cells were decreased (by 5%) when MM cells were cocultured with OCLs from wild type (WT) mice. In contrast, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1 neutralizing antibody (IGF1-ab) did not block BTZ's effects on MM cell death. Consistent with these results, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1-ab resulted in BTZ-induced caspase-dependent apoptosis in MM cells. We next examined the effects of OCLs on the signaling pathways responsible for MM cell survival. WT-OCL-CM promptly induced the phosphorylation of Akt and activation of p38, ERK and NF-κB in MM cells. However, these pathways were not activated by MM cells treated with IGF1-/--OCL-CM or IGF1-ab-treated WT-OCL-CM. Since adhesion of MM cells to BMSCs via interaction of VLA-4 and VCAM-1 plays a critical role in cell adhesion-mediated drug resistance (CAMDR) in MM, we tested if treatment of human BMSCs with human OCL-CM upregulated VCAM-1 expression. We found that OCL-CM upregulated VCAM-1 expression on BMSCs (x fold). In contrast, treatment of BMSCs with OCLs treated with IGF1-ab blocked VCAM-1 induction. These data suggest that OCL-derived IGF1 can contribute to MM cell drug resistance in the bone marrow microenvironment. We then examined the role of IGF1 inhibition on osteoclastogenesis and the bone resorption capacity of OCLs. RANK ligand induced the expression of cathepsin K and NFATc1 in CD11b+ bone marrow cells from WT mice, differentiation markers of OCLs, and the formation of TRAP-positive multinucleated OCLs. However, OCLs formed by RANK ligand treatment of CD11b+ bone marrow cells from IGF1-/- mice had markedly decreased cathepsin K and NFATc1 expression and OCL formation. Next, we tested the bone resorption capacity of OCLs formed by CD11b+ bone marrow cells from IGF1-/- mice vs. WT mice. Similar numbers of OCLs were cultured with RANK ligand on bone slices for 72 hours. The bone resorption activity of Igf1-/--OCLs was significantly decreased (70%) compared with WT-OCLs. These results suggest that OCL-derived IGF1 plays a critical role in MM drug resistance and bone destruction, and that inhibition of the effect of IGF1 in OCLs should decrease MM drug resistance and bone destruction. Disclosures Roodman: Amgen trial of Denosumab versus Zoledronate: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Ruxolitinib Reverses Checkpoint Inhibition By Downregulating PD-L1 and PD-L2 Expression on Both Tumor and Stromal Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of PD-L1 and PD-L2 to PD-1 on T-cells. This creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The immune inhibitory proteins PD-L1 and PD-L2 are highly expressed in MM BM. Moreover, increased expression of these proteins are associated with resistance to treatment in MM. Ruxolitinib (RUX) is a JAK1/2 inhibitor that is effective for the treatment of myeloproliferative diseases. In this study, we examined PD-L1 and PD-L2 gene and protein expression in the BM of MM patients with progressive disease (PD) or in complete remission (CR). We further investigated the effects of RUX on expression of PD-L1 and PD-L2 in MMBM, and the effect of RUX in combination with anti-MM agents in vitro and in vivo. Material and Methods: BM mononuclear cells (MCs) and serum were collected from MM patients and healthy subjects after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the mice. The cells were cultured and treated with or without RUX and then were determined by qPCR, flow cytometric analysis, ELISA, and western blot. Results and Discussion: The results from qPCR and flow cytometric assays showed that PD-L1 and PD-L2 gene expression was markedly increased in BMMCs from MM patients with PD compared with patients in CR or with healthy controls. We further investigated the effects of RUX on PD-L1 and PD-L2 expression in primary and stromal cells from MM patients' BM samples in vitro. RUX treatment markedly reduced PD-L1and PD-L2 gene and protein expression in the MM tumor cells cultured alone or co-cultured with stromal cells in a concentration dependent pattern. We then determined whether RUX can augment the anti-MM effects of T-cells in vitro. RUX (0, 0.1, 0.5, 1, and 5 µM) increased MM cell apoptosis in the presence of IL-2 stimulated T-cells in a concentration dependent fashion, to a similar degree to anti-PD-1 (0, 0.5, 1, 5, and 10 µg/ml) or anti-PD-L1 (0, 0.5, 1, 5, and 10 µg/ml) antibody treatment. Moreover, the combination of RUX with anti-PD-1 or anti-PD-L1 antibody increased T-cell-induced MM cell apoptosis more than the agents alone. To evaluate the efficacy of drugs in vivo, severe combined immune deficient mice implanted with the human MM xenograft LAGκ-2 were treated with RUX (30mg/kg). The results showed PD-L1 expression in the xenograft was significantly decreased in RUX-treated mice compared with the untreated control group. In contrast, RUX had no effect on PD-1 expression on T-cells. Conclusion: The PD-L1/PD-1 pathway delivers inhibitory signals that regulate both peripheral and central tolerance, and inhibit anti-tumor immune-mediated responses. This study demonstrated that the JAK inhibitor RUX downregulated PD-L1 and PD-L2 expression in both MM tumor and stromal cells. We also demonstrated that RUX alone increased T-cell-induced apoptosis of MM cells; and, moreover, the combination of RUX with anti-PD-1 and anti-PD-L1 further increased apoptosis. The results suggest that JAK inhibitors may be effective for treating MM patients through their ability to reduce expression of checkpoint proteins involved in the development of immune resistance. Thus, JAK inhibitors should help overcome the immune resistance generated by these proteins for patients with this B-cell malignancy. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Incyte Corporation.: Consultancy, Research Funding; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; Incyte Corporation.: Consultancy, Research Funding; Janssen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Amag: Consultancy, Speakers Bureau.

Toll-Like Receptor 4 Activation Promotes Multiple Myeloma Cell Growth and Survival Via Suppression of The Endoplasmic Reticulum Stress Factor Chop

Despite recent biomedical improvements in treating Multiple Myeloma (MM), the disease still remains incurable. Toll like receptors (TLRs) provide a link between innate and adaptive immune responses and hence potentially correlate inflammation to cancer. Although the regulatory role of TLRs in MM has been under investigation the underlying mechanisms remain unclear. In this study we assayed the function of TLR4 in MM cell lines and in MM patients’ samples. We found that lipopolysaccharide-mediated TLR4 activation increased MM cells proliferation and decreased endoplasmic reticulum (ER) stress-induced apoptosis. Furthermore, we observed that either the endogenous CHOP expression or the ER stress-mediated CHOP induction, were suppressed by TLR4 activation or its overexpression in MM cell lines; TLR4 induction also suppressed ER stress-induced apoptotic signals. In support, TLR4 gene expression silencing in MM cell lines significantly decreased cell proliferation and promoted CHOP and ATF4 upregulation. TLR4 activation was also able to partially abrogate the effect of bortezomib in MM cell lines by suppressing PERK, ATF4 and phospho-eIF2A. We suggest that TLR4-mediated disruption of ER stress responses contributes to MM cells proliferation and suppresses ER-dependent death signals.

IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies

AbstractProteasome inhibitors (PI) are extensively used for the therapy of multiple myeloma (MM) and mantle cell lymphoma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PI, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of US Food and Drug Administration–approved PI with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, mantle cell lymphoma, and Burkitt lymphoma cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone marrow–derived stromal cells. Mechanistically, the CFZ/AGI-6780 combination significantly decreased tricarboxylic acid cycle activity and adenosine triphosphate levels as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PI, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PI to other malignancies.

The Lysine-Specific Demethylase KDM4A/JMJD2A Acts As a Tumor Suppressor in Multiple Myeloma

Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown.Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response.Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse.Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM.This work was supported by NNSFC (81471165, 81670189, and 81670190). DisclosuresKumar:AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract LB-269: IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies

Abstract The introduction of proteasome inhibitors (PIs) into the clinic has transformed the treatment of patients affected by multiple myeloma (MM) and mantle-cell lymphoma (MCL) establishing new standards of care. Despite these improvements, patients continuously relapse or are intrinsically resistant to PIs. Here, to identify druggable targets that synergize with PIs, we carried out a functional screening in MM cell lines using a short hairpin RNA library targeting 152 cancer driver genes, highly representative of all signaling pathways. The Isocitrate Dehydrogenase 2 (IDH2) gene was identified as a top candidate, showing a synthetic lethal activity with the PI Carfilzomib (CFZ). IDH2 is a NADP(+) dependent mitochondrial enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate in the tricarboxylic acid (TCA) cycle. We demonstrated that combinations of the pharmacological IDH2 inhibitor AGI-6780 with FDA approved PIs significantly increased apoptotic cell death in ten MM cell lines, both sensitive and resistant to PIs. Combined treatments triggered synergistic cytotoxicity also in four MCL and in two Burkitt's lymphoma cell lines. Importantly, CFZ/AGI-6780 treatment increased death of primary CD138-positive cells from nine MM patients and exhibited a favorable cytotoxicity profile towards peripheral blood mononucleated cells and bone marrow-derived stromal cells. Mechanistically, CFZ/AGI-6780 combination significantly decreased TCA cycle activity and ATP levels, as a consequence of enhanced IDH2 enzymatic inhibition. In contrast, only a slight increase of mitochondrial reactive oxygen species (ROS) was observed. CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme required for IDH2 activation through the NAD(+)-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT (FK866) or SIRT3 (AGK7) inhibitors impaired IDH2 activity and increased MM cell death, thus phenocopying CFZ/AGI-6780 effects and putting the proteasome in a direct link with IDH2 inhibition. Finally, inducible IDH2 knock-down enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. In conclusion, our data demonstrate that IDH2 inhibition increases the therapeutic efficacy of PIs, thus providing compelling evidence for treatments with lower and less toxic doses, and broadening the application of PIs to other malignancies. Citation Format: Elisa Bergaggio, Chiara Riganti, Giulia Garaffo, Elisabetta Mereu, Nicoletta Vitale, Cecilia Bandini, Elisa Pellegrino, Paola Omedè, Katia Todoerti, Valentina Audrito, Antonio Rossi, Francesco Bertoni, Silvia Deaglio, Antonino Neri, Antonio Palumbo, Roberto Piva. IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-269.