MYC Overexpressing Multiple Myeloma Are Dependent on GLS1

Introduction. MYC alterations trigger transition from monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to multiple myeloma (MM). They also represent secondary genomic events inducing tumor progression. MYC localization to the nucleus and the short life of the protein are key factors that limit its direct targeting. To overcome these issues, we sought to determine the top genomic dependencies in MYC overexpressing MM by analyzing large-scale knockdown screening, followed by functional validations. Methods. We performed in silico analyses from the Dependency Map (Achilles 2.4.3) together with CCLE (Affymetrix U133+2 expression array), CLUE (Connectivity Map) and MM patient datasets (Chng et al. 2007, Gutiérrez et al. 2010, MMRF RG Dataset), to look for gene dependencies and differentially expressed pathways in MYC OE cancer cell lines and MM patient samples. We generated an isogenic model of MYC OE in U266 MM cell line by using EF1A-C-MYC lentiviral vector, and performed RNA sequencing, a quantitative proteomic analysis by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. To further investigate dependency on glutamine metabolism in MYC OE cell lines, we treated them with GLS1 inhibitor CB-839 and siRNA targeting GLS1 in several cell lines with various MYC expressions and in our isogenic model. Results. By analyzing correlations between MYC expression and gene ATARiS scores corresponding to the effect of over 9000 knockdowns in 236 cell lines, we identified specific vulnerabilities of MYC overexpressing cells for the genes involved in glutamine metabolism and cell cycle pathways. Top dependencies were observed with MYC binding protein MAX (r = -0.51, p < .001), representing an internal control as it is a co-activator of MYC, followed by GLS1 (r = -0.48, p < .001) and SLC1A1 (r = -0.42, p < .001), both involved in glutamine metabolism, together with E2F6 (r = -0.41, p < .001), involved in cell cycle. To further validate dependencies obtained from Achilles data, we generated an isogenic model of MYC OE in U266 (a low c-myc expressing MM cell line). GSEA analysis of RNA seq data showed strong enrichments of translation and cell cycle pathways, with similar results observed in CCLE and MM patient data. Quantitative proteomics analysis of U266 isogenic model showed overexpression of genes involved in glutamine transport (SLC1A5; FC = 1.28, p < .05), glucose metabolism (HK2; FC = 3.68, p < .001) and cell cycle progression (CDK6; FC = 2.85, p < .001). To explore the therapeutic potential of these dependencies, we performed a primary screen of 1902 small-molecules and identified 47 compounds with potent activity on U266/MYC model. Validation screen of these hits identified three leading compounds to which U266/MYC cells showed highest sensitivity at 10 µM concentration - Torin-2 (U266/C 40.28 ± 6.74% vs. U266/MYC 16.05 ± 3.21%), LY2835219 (U266/C 52.70 ± 9.63% vs. U266/MYC 5.52 ± 0.89%) and AT7519 (U266/C 43.03 ± 4.02% vs. U266/MYC 30.13 ± 4.90%), targeting proteins involved in translation and cell cycle pathways. For the functional validation of GLS1 dependency in MYC overexpressing cells, MYC OE cell lines were treated with GLS1 inhibitors CB-839 and 968. MYC high MM cell lines showed higher sensitivity to CB-839 inhibitor compared to MYC low cell lines at 1 µM concentration, after 48 (KMS-12-BM 14.19 ± 0.07%, KMS-18 31.56 ± 2.84%, MM.1S 23.21 ± 1.21% vs. NCI-H1650 46.49 ± 3.48%, U266 52.72 ± 4.99%, LOUCY 37.14 ± 1.14%, OVCAR-3 64.14 ± 5.19%) and 72 h (KMS-18 19.69 ± 3.15%, MM.1S 15.09 ± 1.28% vs. NCI-H1650 34.82 ± 0.95%, U266 61.73 ± 1.70%, LOUCY 46.27 ± 6.27%, OVCAR-3 65.34 ± 1.23%). This suggests that GLS1 dependency in MYC OE cells offers a therapeutic window for the use of GLS1 inhibitors in MM. Conclusion. By using a combination of different datasets and models, we characterized the main dependencies in MYC overexpressing MM. Glutamine metabolism and cell cycle emerged as strong dependencies by using therapeutic inhibitors. Altogether, our results demonstrate that MYC OE MM cells are dependent on glutamine metabolism and cell cycle, and these findings can potentially lead to development of new therapeutic approaches in MM patients. Disclosures Leleu: Oncopeptide: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Carsgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Karyopharm: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Merck: Honoraria. Facon:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Manier:Amgen: Research Funding; Celgene: Research Funding; Janssen: Research Funding.

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CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽

10.1182/blood.v114.22.1729.1729 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1729-1729

Author(s):

Melissa G Ooi ◽

Robert O'Connor ◽

Jana Jakubikova ◽

Justine Meiller ◽

Steffen Klippel ◽

...

Keyword(s):

Lung Cancer ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Significant Activity ◽

Cancer Resistance ◽

Advisory Committees ◽

Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: 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, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

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Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.1741.1741 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1741-1741

Author(s):

Steffen Klippel ◽

Jana Jakubikova ◽

Jake Delmore ◽

Melissa G. Ooi ◽

Douglas McMillin ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cancer Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Energy Production ◽

Advisory Committees ◽

Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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Indirubins: A Potential Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.3259.3259 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3259-3259

Author(s):

Tina Bagratuni ◽

Nicolas Gaboriad-Kolar ◽

Roubini Zakopoulou ◽

Vassilios Myrianthopoulos ◽

Efstathios Kastritis ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Virtual Screening ◽

Cell Line ◽

Board Of Directors ◽

Signaling Pathway ◽

Cell Lines ◽

Inhibitory Effects ◽

Advisory Committees

Abstract Current drugs in the treatment of Multiple Myeloma (MM) result in cell death via a number of mechanisms including a direct effect on plasma cells as well as alteration in the BM microenvironment. Although effective to some extent, none of the drug mechanisms of action are fully targeting a biological process essential and necessary for PC survival. Among the FDA approved kinase inhibitors, few are based on natural scaffolds. 6-bromoindirubin-3'-oxime (6BIO) is a potent kinase inhibitor based on the natural 6-bromoindirubin scaffold. Indirubin and 6-bromoindirubin are two natural products that have found a particular interest in dye chemistry as the main constituent of indigo and Tyrian purple dyes. Recent findings discovered that 6BIO was a promising anti-cancer agent acting on the JAK/STAT signaling pathway mediating cell proliferation. After enhancement of the chemical structure of 6BIO, further reports exposed that MLS-2438 and MLS-2384 were Akt signaling pathway inhibitor (MLS-2438) and potent c-Src kinase direct inhibitors. A library containing 2000 natural molecules was constructed using several data platforms. Each molecule was processed through different filters such as tautomeric studies, protonation and steroisomerism status in order to be used for calculations of virtual evaluation. Two approaches were followed: the structure-based virtual screening and the ligand-based virtual screening. To achieve structural based virtual screening, binding and evaluation of the chemical relation of each molecule in the crystallographic structure of the proteasome β5 subunit was performed. In the ligand-based virtual screening, calculations were made to identify the structural similarities of each molecule with the known proteasome inhibitor, bortezomib. The results of both approaches were combined, the molecules ranked, and 100 out of 2000 were identified as strong potential bioactive hits for the β5 subunit. Out of these 100 molecules, the chemical structures of high interest were the following: indole alkaloids derivatives (indirubins), flavonoids, secoiridoids, simple phenolic acids and acetophenone. A rational selection of indirubins derivatives was conducted in order to study their cytotoxic effects on MM. Fifty indirubins derivatives were selected based on different criteria: structure, known/unknown targets, chemodiversity in substitution patterns. To explore the inhibitory effects of indirubins in MM, we performed the WST1 proliferation assay in three MM cell lines (H929, JJN3, L363). Initially, all the selected indirubins (~50 indirubins) were tested at 7.5μM in L363 cell line and proliferation results from the WST1 assay extracted after 24 hours of treatment. More than half of the indirubins tested displayed more than 50% reduction of the proliferation at 7.5μM. Interestingly, 10 out of the 50 indirubins tested reduced more than 80% proliferation after 24 hours. The most active indirubins were tested in H929 and JJN3 cell lines, where similar effects were seen after 24 hours of treatment. All tested indirubins acted in a dose-dependent manner. Based on our first set of data, we suggest that indirubins have significant anti-proliferative effects on MM cell lines. Among the most active indirubins, two molecules namely 805 and 673 emerged as attractive for further development. Compound 805 is an analog of MLS-2384 while compound 673 is an analog of MLS-2438. The latter derivative represents a promising candidate displaying an IC50below the micromolar range on H929 and JJN3 cells. To determine the kind of cell death caused by one of the most active indirubins, 673, cell cycle analysis was performed before and after treatment in H929 cell line. In particular changes in RNA expression of 84 genes key to cell cycle regulation were analyzed in H929 cell line. Our results show that among other genes, the ones which have a dramatic increase in their expression (>5 fold) are mainly involved in cell cycle arrest such as GADD45A, CDC34, TP53, CHEK1 and CHEK2. A more detailed analysis of the profiler array will be presented at the meeting. In conclusion, this is the first study to show the inhibitory effects of indirubins in MM. Further investigation of these compounds may offer a therapeutic advantage that would affect MM pathogenesis and treatment. Disclosures Kastritis: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood-2018-191 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 191-191

Author(s):

Fengyan Jin ◽

Shaji K. Kumar ◽

Yun Dai

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Tumor Suppressor ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Committees ◽

Apoptotic Genes

Abstract 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). Disclosures Kumar: 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.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial: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.

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Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽

10.1182/blood.v122.21.273.273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 273-273

Author(s):

Salomon Manier ◽

John T Powers ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Michele Moschetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Loss Of Function ◽

Advisory Committees ◽

Expression Levels ◽

Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽

10.1182/blood.v122.21.2520.2520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2520-2520

Author(s):

Hua Wang ◽

Veerabhadran Baladandayuthapani ◽

Zhiqiang Wang ◽

Jiexin Zhang ◽

Heather Yan Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Advisory Committees ◽

Over Expression ◽

Resistant Disease ◽

Drug Naïve ◽

Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

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Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽

10.1182/blood-2021-153451 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4356-4356

Author(s):

John S Manavalan ◽

Ipsita Pal ◽

Aidan Pursley ◽

George A. Ward ◽

Tomoko Smyth ◽

...

Keyword(s):

T Cell ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Tnf Alpha ◽

Sensitive Cell ◽

Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

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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

Blood ◽

10.1182/blood-2019-123399 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3160-3160

Author(s):

Wilson I Gonsalves ◽

Srinivas Devarakonda ◽

Rachid Baz ◽

Natalia Neparidze ◽

Alex A Adjei ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Dose Escalation ◽

Mitochondrial Respiration ◽

Research Funding ◽

Phase 1 ◽

Tca Cycle ◽

Glutamine Metabolism ◽

Cellular Respiration ◽

Advisory Committees

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.

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