Primary Patient Myeloma Cells Research Articles

B Cell Transcriptional Coactivator POU2AF1 (BOB-1) Is an Early Transcription Factor Modulating the Protein Synthesis and Ribosomal Biogenesis in Multiple Myeloma: With Therapeutic Implication

Multiple Myeloma (MM) is a malignancy driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, somatic mutations and copy number abnormalities all contribute to myelomagenesis, however true drivers of the disease have not been well defined.To identify new targetable dependencies in MM, we generated high-quality active enhancer landscape using large cohort of primary patient myeloma cells (n=70), MM cell lines, and normal plasma cells. 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 these data with gene expression and genetic dependency (CRISPR KO) enabled a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among these, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. 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 as symptomatic MM compared to normal plasma cells.To functionally validate the role of BOB-1 in MM, we performed loss-of-function studies using shRNA, siRNAs and antisense GapMers specific for BOB-1 and observed a significant impact on MM cell viability and cell cycle arrest. Transcriptomic analysis upon BOB-1 depletion by RNA-sequencing revealed a small set of genes commonly modulated in all 3 MM cell lines tested including the plasma cell differentiation related transcription factor XBP1 and heme oxygenase (HMOX1). Importantly, we observed ribosome biogenesis, RNA polymerase 1A transcription and mRNA translation and elongation processes to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Bob1 KD resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). In addition, ChiP assay revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region in BOB-1 depleted cells compared to control. These data indicate that BOB1 downregulation results in the suppression of RNA-polymerase I activity in MM cells.RNA Pol I-dependent transcription governs abundance of rRNA and directly regulates cellular translational and proliferative capacity. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. We observed that in MM cells compared to control cells, BOB-1 KD decreased, while its overexpression significantly enhanced de novo protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular light chains (kappa or lambda) production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result of decreased protein production, BOB-1 depletion was associated with induction of resistance to proteasome inhibition suggesting that high expression of BOB-1 may be one the factors driving the exquisite sensitivity of MM cells to proteasome inhibitor. Interestingly, mass spectrometry analysis revealed BOB1 in a protein complex with mTOR, Raptor and mLST8 proteins which are members of mTORC1 complex which is also involved in ribosomal function and may suggest the mechanism of action of Bob-1 at molecular level.In conclusion, here we report BOB1 as a specific proximal dependency in MM cells with potential role in modulating the protein load/capacity balance and ribosomal biogenesis essential for MM cell protein production function and therefore their sensitivity to proteasome inhibition. DisclosuresShirasaki: FIMECS: Consultancy. Mitsiades: BMS: Research Funding; Nurix: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Karyopharm: Research Funding; Sanofi: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria. Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria. Munshi: Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy.

Integrative Oncogenomic Analysis Combining Whole Genome, Transcriptome and Epigenome Identifies Altered Chromatin Accessibility Landscape in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Genomic investigations have identified few recurrently mutated protein-coding genes and various aberrations at the transcriptomic level. However, these changes do not accurately reflect the disease behavior. With accumulation of data in large patient population, it is becoming clear that analysis integrating various genomic correlates will be required to understand chromatin architecture that drives the disease behavior.To gain further insight into MM biology, we integrated whole-genome sequencing, transcriptome sequencing, and epigenome profile using various assays (including the assay for transposase-accessible chromatin sequencing [ATAC-seq] to detect open chromatin regions, multiplexed, ChIP-seq and indexed T7 ChIP-seq [Mint-ChIP] to identify active enhancers and super-enhancers) to characterize the chromatin state of MM. We have analyzed 33 samples including primary MM cells from 18 newly diagnosed and 5 relapsed patients, 5 normal plasma cells from healthy donors and 5 Myeloma cell lines.We first investigated the open chromatin landscape in myeloma cells, identifying over one million open chromatin regions (OCR) in our cohort, with an average of over 60,000 regions per sample. Despite a high heterogeneity between myeloma samples, unsupervised cluster analysis clearly separates primary myeloma samples, normal plasma cells and MM cell lines suggesting pathogenetic relevance. Although there is clear link between transcribed genes and OCR, we observe a significant number of OCR in the intergenic region with an as yet unclear biological significance. Furthermore, ATAC-seq data predicted several copy number abnormalities including the distinction of hyperdiploid from hypodiploid samples. We next characterized enhancers and super-enhancers in myeloma cells and their assigned genes using H3K27ac ChIP-Seq to identify important genes in MM biology. Despite heterogeneity between myeloma samples and myeloma cell lines, we could identify common SE-assigned genes as compared with normal plasma cells. These genes correspond to both known myeloma specific dependencies such as MYC, IRF4, CCND1, and TNSFR17 (BCMA) as well as new therapeutic targets that are currently under investigation.Next, we used WGS data from 10 samples matching with ATAC and RNA sequencing to evaluate the mutational landscape of open chromatin regions and the consequences of the mutational processes targeting myeloma epigenome. Of 53,798 single nucleotide substitutions (SNV) (average of 5380 per sample), 2.7% of SNVs involve OCR (total of 1470 SNV within ATAC peaks, median - 104 mutations involving OCR per sample). We evaluated the presence of mutations within OCR targeting known binding motifs for transcription factors (TFs). Out of 1470 mutations affecting OCR, we identified 547 (37%) SNV targeting a known binding motif for transcription factors such as Sp1, NFkB1 or YY1. Using RNA seq data, we filtered the mutations affecting expressed TFs and assigned genes and observed that the presence of these mutations could significantly impact the assigned gene expression level. Indeed, these mutations generate either a de novo binding motif or alter the binding motif of a given TFs and are potentially responsible for TF hi-jacking to modulate the expression of number of genes important for MM biology. Finally, using non-negative matrix factorization (NMF), we evaluated the mutational signatures affecting the OCR and observed that a single predominant mutational signature affects open chromatin regions, as opposed to other processes such as APOBEC, which targets other regions of the genome.In conclusion, global analysis of primary patient myeloma cells combining whole genome, transcriptome as well as epigenomic characterization reveals dysregulated chromatin landscape in myeloma that may be significantly influenced by specific mutational processes. Such integrated oncogenomic analysis provides an early view on perturbed genomic landscape with a view to understand the biology and specific targetable dependencies. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

T Cell Engagement without Cytokine Storm: A Novel Bcma x CD3 Antibody Killing Myeloma Cells with Minimal Cytokine Secretion

IntroductionMultiple myeloma (MM) is a plasma cell malignancy with an annual incidence of ~25,000. Improvements in standard of care have extended median survival to greater than 5 years; however, disease refractory to bortezomib and lenalidomide is still associated with a median life expectancy of less than 9 months. Novel therapies are critical to the treatment of these patients. Chimeric antigen receptor T cells (CAR-Ts) targeting B-cell maturation antigen (BCMA) -a protein found exclusively on the surface of plasma cells- have shown efficacy against relapsed/refractory MM in early phase clinical trials. However, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to BCMA circumvent the latter problem but to date have shown similar T-cell over-activation. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline using in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αBCMA HCA and a library of αCD3 Flic antibodies that show a >2 log range of EC50s for T cell activation in vitro . Of note, the library contained a low-activating αCD3 that induced T-cell dependent lysis of MM cells (when paired with an αBCMA HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we combined both a high- (TNB-384B) and the low-activating (TNB-383B) αCD3 moiety with a high-affinity bivalent αBCMA arm (αBCMA x αBCMA x CD3) and compared these constructs in vitro, in vivo, and ex vivo for T-cell activation, efficacy in killing MM cells, and toxicity.MethodsT-cell activation was measured via flow cytometry (CD69 expression) and cytokine ELISA (IL-2, INF-ɣ, TNFα, among others) in vitro. Lysis of MM cell lines (RPMI-8226, U266, and H929) was measured via flow cytometry in vitro . Ex vivo, survival of patient MM cells was measured via flow cytometry after incubation with TNB-383B, TNB-384B, or negative control antibody (NCA) with or without supplemental exogenous activated T cells. In vivo, NSG mice were engrafted with RPMI-8226 MM cells and human peripheral blood mononuclear cells (huPBMC), and the mice treated with TNB-383B, TNB-384B, or NCA. Tumor burden was evaluated via Bioluminescent Imaging (BLI). Pharmacokinetic (PK) studies were performed in mice and Cynomolgus monkeys using ELISA.ResultsBoth TNB-383B and TNB-384B bound to cell surface BCMA with nanomolar affinity. EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-383B and single-digit picomolar for TNB-384B against MM cell lines in vitro . TNB-383B showed markedly reduced (ie IFN-ɣ) or absent (ie IL-2) cytokine release compared to TNB-384B even at saturating concentrations (Figure 1); these findings were consistent across 10 healthy huPBMC donors. Ex vivo, both TNB-383B and TNB-384B efficiently lysed primary MM cells in the presence and absence of supplementary T-cells. In vivo, both TNB-383B and TNB-384B mediated clearance of established MM tumor from NSG mice at doses as low as 10ng q1w x3 in the presence of huPBMC. PK profiles of both molecules were consistent with those of an IgG in mice (T1/2 ~5.5 days) and Cyno (T1/2 ~8.5 days). No activation of T-cells was observed in vitro (Figure 1) or in vivo in the absence of BCMA expressing target cells.ConclusionsBoth low-activating TNB-383B and high-activating TNB-384B αBCMA x αBCMA x αCD3 bispecific antibodies kill multiple myeloma cells in vitro and in vivo in a BCMA-dependent manner, and kill primary patient myeloma cells ex vivo . While TNB-384B showed T-cell activation comparable to other CD3-engaging bispecifics, TNB-383B induced significantly reduced cytokine secretion by T-cells without appreciable reduction of efficacy in vivo or ex vivo. In summary, both TNB-383B and TNB-384B show promise as MM therapeutics differentiated from current T-cell targeted therapies in clinical trials. [Display omitted] DisclosuresBuelow:TeneoBio: Employment. Pham:TeneoBio, Inc.: Employment. Dang:TeneoBio, Inc.: Employment. Pratap:TeneoBio, Inc.: Employment. Clarke:TeneoBio, Inc.: Employment. Harris:TeneoBio, Inc.: Employment. Jorgensen:TeneoBio, Inc.: Employment. Force Aldred:TeneoBio, Inc.: Employment. Schellenberger:TeneoBio, Inc.: Employment. Shah:Indapta Therapeutics: Other: Stock Ownership; Celgene: Research Funding; Celgene, Indapta Therapeutics, Takeda: Consultancy; TeneoBio, Inc.: Honoraria. Trinklein:TeneoBio: Employment. Wiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding. Iyer:TeneoBio, Inc.: Employment. Van Schooten:TeneoBio, Inc.: Employment.

Phosphatase of regenerating liver-3 regulates cancer cell metabolism in multiple myeloma.

Cancer cells often depend on microenvironment signals from molecules such as cytokines for proliferation and metabolic adaptations. PRL-3, a cytokine-induced oncogenic phosphatase, is highly expressed in multiple myeloma cells and associated with poor outcome in this cancer. We studied whether PRL-3 influences metabolism. Cells transduced to express PRL-3 had higher aerobic glycolytic rate, oxidative phosphorylation, and ATP production than the control cells. PRL-3 promoted glucose uptake and lactate excretion, enhanced the levels of proteins regulating glycolysis and enzymes in the serine/glycine synthesis pathway, a side branch of glycolysis. Moreover, mRNAs for these proteins correlated with PRL-3 expression in primary patient myeloma cells. Glycine decarboxylase (GLDC) was the most significantly induced metabolism gene. Forced GLDC downregulation partly counteracted PRL-3-induced aerobic glycolysis, indicating GLDC involvement in a PRL-3-driven Warburg effect. AMPK, HIF-1α, and c-Myc, important metabolic regulators in cancer cells, were not mediators of PRL-3's metabolic effects. A phosphatase-dead PRL-3 mutant, C104S, promoted many of the metabolic changes induced by wild-type PRL-3, arguing that important metabolic effects of PRL-3 are independent of its phosphatase activity. Through this study, PRL-3 emerges as one of the key mediators of metabolic adaptations in multiple myeloma.

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.

Trial in Progress: Phase I Dose-Escalation and Dose-Expansion Trial of a Novel Glutaminase Inhibitor (CB-839 HCl) in Combination with Carfilzomib and Dexamethasone in Relapsed and/or Refractory Multiple Myeloma

Background: c-MYC activation is an early event of myeloma pathogenesis. It upregulates the expression of the glutaminase 1 (GLS1) enzyme which converts glutamine to glutamate in the mitochondria. Glutamate is required for the biosynthesis of various molecules in the tricarboxylic acid (TCA) cycle (i.e., glutamine anaplerosis). CB-839 HCl is a first-in-class, orally available, selective, noncompetitive inhibitor of GLS1. This inactivation of GLS1 results in an increase of glutamine and a decrease of glutamate and several TCA cycle intermediates within cancer cells, leading to a decrease in their proliferation and/or an increase in cell death. In the phase 1 study, CX-839-002, the safety and tolerability of CB-839 HCl was evaluated in patients with hematological tumors (multiple myeloma (MM) and non-Hodgkin's lymphoma), either as monotherapy or in combination with pomalidomide and dexamethasone or with dexamethasone alone. It was determined to be well tolerated and the maximal tolerated dose (MTD) was not reached. Proteasome inhibitors (PI) are the cornerstone agents in the treatment of myeloma. They disrupt normal protein homeostasis causing an induction of cellular proteotoxic stress, thus, making it an effective strategy against myeloma plasma cells, which naturally mass-produce large quantities of immunoglobulin proteins. PI-resistant MM cells are associated with changes in cellular bioenergetics that favor the increased use of mitochondrial respiration for energy production. Given the increased reliance of PI-resistant MM cells on mitochondrial respiration, and the critical role of glutamine for cellular respiration, inhibition of glutamine metabolism is a rational molecular strategy for the treatment of PI-resistant MM. Furthermore, pre-clinical studies demonstrate the in vitro and ex vivo synergism of CB-839 HCl with carfilzomib (CFZ) in terms of its cytotoxicity and anti-proliferation capacity in various primary human myeloma cell lines and primary patient myeloma cells respectively. As a result, this novel combination of glutaminase inhibition with proteasome inhibition appears promising as a therapeutic combination in MM and warrants further clinical investigation. Methods: This study is a phase 1, multicenter clinical trial of CB-839 HCl in combination with carfilzomib and dexamethasone for patients with relapsed and/or refractory myeloma. Part A of this trial is a 3+3 dose escalation design and Part B is a dose expansion cohort at the RP2D determined in Part A. Up to a maximum of 42 patients will be enrolled at participating ETCTN sites. CFZ will be administered in its usual weekly dosing schedule of days 1, 8 and 15 of a 28 day schedule along with dexamethasone on days 1, 8, 15 and 22. CB-839 will be started at a dose level of 400 mg twice daily and will be investigated to a maximum dose of 800 mg twice daily. Prior to day 1 of Cycle 1, we will administer a 7 day lead in of CB-839 monotherapy before combining it with CFZ. Key inclusion criteria are having relapsed/refractory myeloma with at least 2 prior lines of therapy and prior exposure to PIs, immunomodulators and Anti-CD38 monoclonal antibodies, having measurable disease, adequate hematologic reserve, kidney function and liver function. Key exclusion criteria are being refractory or intolerant to CFZ, adverse cardiac history, central nervous system disease and AL amyloidosis. The primary objective of this trial is to determine the MTD or recommended phase II dosing (RP2D) of CB-839 HCl in combination with carfilzomib and dexamethasone. The secondary objective is to evaluate the safety and tolerability as well as the overall response rate (ORR) associated of CB-839 HCl in combination with carfilzomib and dexamethasone. Correlative objectives will evaluate plasma pharmacokinetic profiles of CB-839 HCl and carfilzomib when used in combination. They will also evaluate potential predictive and prognostic biomarkers as well as resistance mechanisms using genomic DNA, RNA, flow cytometry, immunohistochemistry and metabolomics-based assessment platforms. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT03798678. Disclosures Baz: Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Karyopharm: 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; AbbVie: Research Funding; Merck: Research Funding. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding.

NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib

Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM.Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells.Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated.Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Targeting Aberrant Non-Homologous End Joining in Multiple Myeloma: Role of the Classical and Alternative Pathways in Genomic Instability

Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM.To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes.In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. DisclosuresNo relevant conflicts of interest to declare.

Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

AbstractHM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Inhibition of HDACs and Aminopeptidases Is Highly Synergistic in Myeloma Cells Resulting in Cell Death Via the Upregulation of BIRC3, a Key Mediator of NF-KappaB Signalling.

Abstract 607Myeloma cells rely on a number of mechanisms to maintain their survival, including the silencing of genes that would normally check uncontrolled proliferation and lead to apoptosis. Epigenetic alterations such as histone deacetylation contribute significantly to the pathogenesis of both solid and haematological malignancies and are associated with the silencing of tumour suppressor genes. Pan-histone deacetylase inhibitors (HDACs), such as SAHA, panobinostat, depsipeptide, and numerous others have been published to have anti-myeloma activity. Here we report the effects of a novel pan HDAC inhibitor, CHR-3996, on multiple myeloma cells. Results: CHR-3996 was potently cytotoxic against a panel of myeloma cell lines (H929, KMS11, LP-1, MM1s, RPMI-8226, and U266) with a GI50 ranging from 9 to 65 nM and was equally as potent in primary patient myeloma cells. The loss of cell viability was associated with an increase in apoptotic cells; EndonucleaseG and Noxa were both up-regulated and caspase 9 was cleaved. Furthermore a decrease in apoptosis was demonstrated in the presence of a pan-caspase inhibitor indicating cell death is largely dependent on caspase-mediated apoptosis. There was no effect on bone marrow stromal (BMS) cell viability but there was an observed decrease in the IL-6 and VEGF the BMS cells secrete, both of which promote the growth and survival of myeloma cells in the bone marrow microenvironment. CHR-3996 caused an increase in acetylated H3K9 but there was minimal change in the levels of ubiquitinated proteins in the cell or acetylated alpha-tubulin, indicating low activity against HDAC6 or the proteasome (also demonstrated by an assay to specifically measure proteasome function). In addition to induction of apoptosis, cell cycle analysis showed an increased proportion of cells in G0/G1 indicating cell cycle arrest. In-keeping with data from treating myeloma cells with SAHA and panobinostat, an increase in cell cycle inhibitor p21 was observed. Gene expression profiling (GEP) identified changes in cell cycle regulators, indications of increased cell stress (elevated CHOP, ATF3, and TAO kinase 3), repression of Wnt (up-regulation of NLK, GSK3beta) and mTOR (decreased 4E-BP1) signalling, and changes in key pro- and anti-apoptotic proteins (for example SMAD3, BCL2, BIM, BID, and BIRC5). When used in combination studies CHR-3996 was highly synergistic in vitro with tosedostat, an aminopeptidase inhibitor, which we have previously shown to have anti-myeloma activity via the induction of the amino acid deprivation response and autophagy. One of the largest changes the GEP analysis identified was BIRC3 (CIAP2), an inhibitor of NF-kappaB signalling, which increased 23.5 fold with CHR-3996 as a single agent and over 100-fold when added to H929 cells in combination with tosedostat. Additionally both CHR-3996 and todestat independently up-regulated expression of members of the IkappaB family. Altered expression and nuclear localisation of canonical and non-canonical NF-kappaB family members were observed by immuno-fluorescence and immunoblotting, suggesting targeting of NF-kappaB signalling as a reason for the high degree of synergy between these two compounds. Early data suggest oral CHR-3996 is effective in a subcutaneous in vivo myeloma model and, reflecting the in vitro data, there is a degree of synergy when administered with tosedostat. Conclusions: The novel compound CHR-3996 is a potent HDAC inhibitor that leads to increased H3K9 acetylation but has no detectable activity against HDAC6 or proteasome activity in myeloma. It induces apoptosis of myeloma cells without affecting BMS cell viability. CHR-3996 shows a very high degree of synergy with an aminopeptidase inhibitor, tosedostat, potentially through targeting the NF-kappaB pathway. It has exciting therapeutic potential either as a mono-therapy or in combination with other agents. Disclosures:Krige:Chroma Therapeutics Ltd: Employment, Equity Ownership. Hooftman:Chroma Therapeutic Ltd: Employment, Equity Ownership. Drummond:Chroma therapuetics: Employment, Equity Ownership.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Abstract 3830Poster Board III-766Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures:Ecsedy:Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Mi-63, a Small Molecule Inhibitor of MDM2-p53 Interaction, Has Significant In Vitro Activity in Multiple Myeloma.

Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma.Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells.Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

AT-101, a Small Molecule Inhibitor of Bcl-2 Family Proteins, Has Significant In Vitro Activity in Multiple Myeloma.

Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. Studies have shown elevated expression of anti-apoptotic proteins of the Bcl-2 family in MM cells, which appear to correlate with resistance to therapy with certain drugs. Hence, accelerating the apoptotic process by targeting the Bcl-2 family of proteins appears to be an attractive strategy for the treatment of MM. AT-101 is an orally bioavailable derivative of gossypol in cancer clinical trials, and is being developed by Ascenta Therapeutics. AT-101 behaves as a small molecule inhibitor of Bcl-2 and Bcl-XL, binding to the BH3-binding pocket of these proteins and inhibiting their ability to suppress the activity of pro-apoptotic proteins, resulting in apoptosis.Methods and Results: AT-101 was cytotoxic to several different myeloma cell lines with a median effect observed at around 5μM concentration using an MTT cell proliferation assay. Additionally, at similar doses AT-101 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. AT-101 was cytotoxic to myeloma cells under these conditions as well with a median effect at concentrations of 5–10μM. AT-101 was able to induce apoptosis in myeloma cells in a dose- and time dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. AT-101 also resulted in cytotoxicity of freshly isolated primary patient myeloma cells, inducing apoptosis in a dose dependent manner. We also studied the effect of AT-101 on levels of different pro- and anti-apoptotic proteins using flow cytometry on permeabilized cells. A time-dependent increase in the level of BAX was observed following treatment with AT-101 without any associated change in levels of Bcl-xL or Bcl-2. Further studies evaluating the combination of AT101 with other active myeloma agents as well as a detailed evaluation of its mechanisms in myeloma are ongoing.Conclusion: AT-101 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. More importantly, it has activity against cell lines resistant to conventional anti-myeloma agents. In addition, Phase I studies with this agent are currently ongoing in patients with solid tumors. The results from these studies form the rationale for early phase clinical trials in MM, either alone or in combination with other active therapies.