Multiple Myeloma Cell Viability Research Articles

Anti-cancer activity of dinitrosyl iron complex (no donor) on the multiple myeloma cells

The results of a study of the effect of a mononuclear dinitrosyl iron complex (DNIC7) with functional sulfur-containing ligands (NO donor) on the cell viability of multiple myeloma cells are presented. It has been shown that DNIC7 decreased cell viability and inhibited the proliferation of cells of multiple myeloma, i.e. possesses cytotoxic properties. Fluorescent analysis revealed that the DNIC7 compound lowers the level of intracellular glutathione and increases the level of reactive oxygen species in cells of multiple myeloma. It is assumed that DNIC7 has the therapeutic potential for the treatment of cancer.

HDAC6‑selective inhibitor synergistically enhances the anticancer activity of immunomodulatory drugs in multiple myeloma.

Nonselective histone deacetylase (HDAC) inhibitors have therapeutic effects, but exhibit dose‑limiting toxicities in patients with multiple myeloma(MM). The present study investigated the interaction between the HDAC6 inhibitor, A452, and immunomodulatory drugs (IMiDs) on dexamethasone (Dex)‑sensitive and ‑resistant MM cells compared with the current clinically tested HDAC6 inhibitor, ACY‑1215. It was shown that the combination of the HDAC6‑selective inhibitor, A452, with either of the IMiDs tested (lenalidomide or pomalidomide) led to the synergistic inhibition of cell growth, a decrease in the viability of MM cells and in an increase in the levels of apoptosis. Furthermore, enhanced cell death was associated with the inactivation of AKT and extracellular signal‑regulated kinase(ERK)1/2. Of note, A452 in combination with IMiDs induced synergistic MM cytotoxicity without altering the expression of cereblon and thereby, the synergistic downregulation of IKAROS family zinc finger(IKZF)1/3, c‑Myc and interferon regulatory factor4(IRF4). Furthermore, combined treatment with A452 and IMiDs induced the synergistic upregulation of PD‑L1. More importantly, this combination treatment was effective in the Dex‑resistant MM cells. Overall, the findings of this study indicate that A452 is more effective as an anticancer agent than ACY‑1215. Taken together, these findings suggest that a combination of the HDAC6‑selective inhibitor, A452, and IMiDs may prove to be beneficial in the treatment of patients with MM.

MiR-520g and miR-520h overcome bortezomib resistance in multiple myeloma via suppressing APE1

ABSTRACT Background: Nowadays, microRNAs (miRNAs) attract much attention in regulating anticancer drug resistance in cancers including multiple myeloma (MM). Bortezomib is the first-line choice in MM treatment, and bortezomib resistance caused by aberrant DNA repair leads to the recurrence and therapeutic failure of MM. Objective: Our study aims to identify a miRNA that overcomes bortezomib resistance in MM. Methods: We established bortezomib-resistant MM cell lines, and screened several miRNAs that have aberrant expressions in MM cell lines. The expression of DNA-repair-related proteins were assessed by western blot, and cell viability was determined by the MTT assay in bortezomib-resistant cell lines. The binding between miRNAs and 3ʹ-UTR of APE1 mRNA was confirmed by luciferase reporter assay. The mouse bortezomib-resistant xenograft was established to verify the therapeutic effect of miRNA overexpression. Results: miR-520g and miR-520h were significantly downregulated in bortezomib-resistant MM cell lines, and overexpression of miR-520g and miR-520h together inhibited expression of homologous recombination-related protein Rad51 and cell viability of bortezomib-resistant MM cells in vitro by binding with 3ʹ-UTR of APE1 mRNA. Combined overexpression of miR-520g and miR-520h inhibited bortezomib-resistant MM tumor growth in vivo. Conclusion: Our findings demonstrated that combined overexpression of miR-520g and miR-520h overcomes bortezomib resistance in MM through inhibition of DNA repair, offering a promising therapeutic target for MM treatment.

IL-17A Increases Multiple Myeloma Cell Viability by Positively Regulating Syk Expression

PURPOSE: Elevated IL-17 produced by Th17 cells was reported to promote myeloma cell growth and inhibit immune function in multiple myeloma (MM). IL-17A was also reported to promote MM growth through IL-17 receptors and enhance adhesion to bone marrow stromal cells (BMSCs). Spleen tyrosine kinase (Syk) influences MM cell survival and migration. Herein we aimed to investigate whether Syk was involved in the regulative role of IL-17A in the viability of MM cells. METHODS: Cell viability was determined using CCK8 assay. The production of cytokine including IL-17A was evaluated with ELISA. Western blotting assay was used to determine protein expression levels of Syk and nuclear factor κB (NF-κB) related molecules. mRNA expression level of RORγt was detected with reverse transcription quantitative polymerase chain reaction. RESULTS: IL-17Awas highly expressed in MM patients and was able to induce MM cell viability. Following analysis indicated that the effects of IL-17A were mediated by Syk/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Immunoprecipitation also indicated that Syk is involved in IL-17A–induced Act1-TRAF6 complex formation and TRAF6 polyubiquitination in MM cells. CONCLUSIONS: Taken together, our study indicated that IL-17A increases MM cell viability through activating NF-κB signal pathway via positively regulating Syk expression. Syk also participates in the formation of IL-17R-proximal signaling complex (IL-17R-Act1-TRAF6), which is essential for IL-17A–mediated NF-kB activation. These investigations highlight that inhibition of Syk may be a potential therapeutic option for neoplastic diseases such as MM.

Relationship between vitamin D nutritional status and metabolic syndrome in childhood acute lymphoblastic leukemia survivors: A petale study

PURPOSE: Elevated IL-17 produced by Th17 cells was reported to promote myeloma cell growth and inhibit immune function in multiple myeloma (MM). IL-17A was also reported to promote MM growth through IL-17 receptors and enhance adhesion to bone marrow stromal cells (BMSCs). Spleen tyrosine kinase (Syk) influences MM cell survival and migration. Herein we aimed to investigate whether Syk was involved in the regulative role of IL-17A in the viability of MM cells. METHODS: Cell viability was determined using CCK8 assay. The production of cytokine including IL-17A was evaluated with ELISA. Western blotting assay was used to determine protein expression levels of Syk and nuclear factor κB (NF-κB) related molecules. mRNA expression level of RORγt was detected with reverse transcription quantitative polymerase chain reaction. RESULTS: IL-17Awas highly expressed in MM patients and was able to induce MM cell viability. Following analysis indicated that the effects of IL-17A were mediated by Syk/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Immunoprecipitation also indicated that Syk is involved in IL-17A–induced Act1-TRAF6 complex formation and TRAF6 polyubiquitination in MM cells. CONCLUSIONS: Taken together, our study indicated that IL-17A increases MM cell viability through activating NF-κB signal pathway via positively regulating Syk expression. Syk also participates in the formation of IL-17R-proximal signaling complex (IL-17R-Act1-TRAF6), which is essential for IL-17A–mediated NF-kB activation. These investigations highlight that inhibition of Syk may be a potential therapeutic option for neoplastic diseases such as MM.

Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase‑dependent manner

Chidamide, a novel histone deacetylase (HDAC) inhibitor, induces antitumor effects in various types of cancer. The present study aimed to evaluate the cytotoxic effect of chidamide on multiple myeloma and the underlying mechanisms involved. Viability of multiple myeloma cells upon chidamide treatment was determined by the Cell Counting Kit-8 assay. Apoptosis induction and cell cycle alteration were detected by flow cytometry. Specific apoptosis-associated proteins and cell cycle proteins were evaluated by western blot analysis. Chidamide suppressed cell viability in a time- and dose-dependent manner. Chidamide treatment markedly suppressed the expression of type I HDACs and further induced the acetylation of histones H3 and H4. In addition, it promoted G0/G1 arrest by decreasing cyclin D1 and c-myc expression, and increasing phosphorylated-cellular tumor antigen p53 and cyclin-dependent kinase inhibitor 1 (p21) expression in a dose-dependent manner. Treatment with chidamide induced cell apoptosis by upregulating the apoptosis regulator Bax/B-cell lymphoma 2 ratio in a caspase-dependent manner. In addition, the combination of chidamide with bortezomib, a proteasome inhibitor widely used as a therapeutic agent for multiple myeloma, resulted in enhanced inhibition of cell viability. In conclusion, chidamide induces a marked antimyeloma effect by inducing G0/G1 arrest and apoptosis via a caspase-dependent pathway. The present study provides evidence for the clinical application of chidamide in multiple myeloma.

Anticancer Activity of Dinitrosyl Iron Complex (NO Donor) on the Multiple Myeloma Cells.

The results of the study of the effect of a mononuclear dinitrosyl iron complex (DNIC7) with functional sulfur-containing ligands (NO donors) on the viability of multiple myeloma cells are presented. It was shown that DNIC7 decreased cell viability and inhibited the proliferation of multiple myeloma cells, i.e., exhibits cytotoxic properties. Fluorescent analysis showed that the DNIC7 compound decreases the level of intracellular glutathione and increases the level of reactive oxygen species in multiple myeloma cells. It is assumed that DNIC7 has a therapeutic potential for the treatment of cancer.

Sanguinarine Induces Apoptosis Pathway in Multiple Myeloma Cell Lines via Inhibition of the JaK2/STAT3 Signaling.

Sanguinarine (SNG), a benzophenanthridine alkaloid, has displayed various anticancer abilities in several vivo and in vitro studies. However, the anticancer potential of SNG is yet to be established in multiple myeloma (MM), a mostly incurable malignancy of plasma cells. In this study, we aimed to investigate the potential anti-proliferative and pro-apoptotic activities of SNG in a panel of MM cell lines (U266, IM9, MM1S, and RPMI-8226). SNG treatment of MM cells resulted in a dose-dependent decrease in cell viability through mitochondrial membrane potential loss and activation of caspase 3, 9, and cleavage of PARP. Pre-treatment of MM cells with a universal caspase inhibitor, Z-VAD-FMK, prevented SNG mediated loss of cell viability, apoptosis, and caspase activation, confirming that SNG-mediated apoptosis is caspase-dependent. The SNG-mediated apoptosis appears to be resulted from suppression of the constitutively active STAT3 with a concomitant increase in expression of protein tyrosine phosphatase (SHP-1). SNG treatment of MM cells leads to down-regulation of the anti-apoptotic proteins including cyclin D, Bcl-2, Bclxl, and XIAP. In addition, it also upregulates pro-apoptotic protein, Bax. SNG mediated cellular DNA damage in MM cell lines by induction of oxidative stress through the generation of reactive oxygen species and depletion of glutathione. Finally, the subtoxic concentration of SNG enhanced the cytotoxic effects of anticancer drugs bortezomib (BTZ) by suppressing the viability of MM cells via induction of caspase-mediated apoptosis. Altogether our findings demonstrate that SNG induces mitochondrial and caspase-dependent apoptosis, generates oxidative stress, and suppresses MM cell lines proliferation. In addition, co-treatment of MM cell lines with sub-toxic doses of SNG and BTZ potentiated the cytotoxic activity. These results would suggest that SNG could be developed into therapeutic agent either alone or in combination with other anticancer drugs in MM.

The anti-myeloma effects of the selective JAK1 inhibitor (INCB052793) alone and in combination in vitro and in vivo.

The Janus kinase (JAK) pathway has been shown to play key roles in the growth and resistance to drugs that develop in multiple myeloma (MM) patients. The anti-MM effects of the selective JAK1 inhibitor INCB052793 (INCB) alone and in combination with anti-MM agents were evaluated in vitro and in vivo. Significant inhibition of cell viability of primary MM cells obtained fresh from MM patients, and the MM cell lines RPMI8226 and U266, was observed with single agent INCB and was enhanced in combination with other anti-MM agents including proteasome inhibitors and glucocorticosteroids. Single-agent INCB resulted in decrease in tumor growth of the MM xenograft LAGκ-1A growing in severe combined immunodeficient mice. Mice dosed with INCB (30mg/kg) showed significant reductions in tumor volume on days 28, 35, 42, 49, 56, and 63. Similarly, INCB at 10mg/kg showed anti-tumor effects on days 56 and 63. Tumor-bearing mice receiving combinations of INCB with carfilzomib, bortezomib, dexamethasone, or lenalidomide showed significantly smaller tumors when compared to vehicle control and mice treated with single agents. These results provide further support for the clinical evaluation of INCB052793 alone and in combination treatment for MM patients.

The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown.Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio).We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1.Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. DisclosuresRoodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Aryl Hydrocarbon Receptor (AHR) Antagonism As a Transformative, Dual-Mechanism Novel Therapy for Multiple Myeloma

BACKGROUNDThe aryl hydrocarbon receptor (AHR) is a transcription factor that regulates xenobiotic-metabolizing enzymes in response to exogenous polycyclic aromatic hydrocarbons. However, we have described a critical role for AHR in normal lymphocyte development and identified the functional expression of AHR in multiple myeloma (MM). Interest is growing in AHR as a novel anti-cancer therapeutic target in solid tumors, but very little comparatively is known about AHR in hematologic malignancies. Epidemiologic data suggest that exposure to the AHR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzene and dioxin may be linked to increased rates of MGUS and MM in Vietnam-era veterans as well as firefighters at the World Trade Center attacks in September, 2001. Whereas we have shown that AHR antagonism facilitates normal, healthy natural killer (NK) cell lymphocyte development and acquisition of cytolytic potential, herein, we show that AHR antagonism impairs MM cell viability. In addition, we characterize the direct effects of AHR antagonism on MM cells as well as complementary mechanisms by which the NK cell versus MM effect is augmented, further strengthening ongoing interest in targeting AHR as a novel anti-MM therapy. METHODSThe MM cell lines MM.1S, MM.1R, OPM-2, and U266 were obtained from ATCC. Primary cells from healthy donors and patients with MM were procured under IRB-approved protocols. AHR expression was studied by Western blot, flow cytometry and real time PCR. AHR function was assessed by expression of the downstream target CYP1A1 by real time PCR. Clinical MM outcomes were determined with data obtained from a publically available database. Cell proliferation and viability were studied by light microscopy (trypan blue exclusion) and Sytox. Immunophenotyping was conducted by flow cytometry. Cytotoxicity was measured by 51Cr release assay. The AHR antagonists CH233191 and CB7993113 and the high affinity AHR agonist 5,11-dihydroindolol[3,2-b]carbazole-6-carboxaldehyde (FICZ) were utilized to study the in vitro effects of AHR modulation. RESULTSAHR transcript and protein are expressed in human NK cells, plasma cells and B cells, as well as MM cell lines and primary MM samples. AHR agonism increases transcription of CYP1A1 in MM cells whereas AHR antagonism suppresses CYP1A1; these observations confirm that AHR is functionally active in MM. AHR is abundantly expressed from the CD38brightCD27(+) MM stem cell compartment to primary plasma cell leukemia samples. MM cases with high AHR expression are associated with inferior survival as compared to MM cases with low AHR expression (median OS = 50 months versus not reached at 70 months, p = 0.00002). AHR stimulation reduces expression of CD38, CD56, and CD138 and upregulates CD10, CD11a, CD13, CD19, CD20, CD27, CD40, CD45, and CD117 consistent with MM clonogenic progenitor cells previously described as “stem-like” and drug resistant. AHR antagonism suppresses MM cell line and primary MM cell viability, including cytogenetically high-risk, plasma cell leukemia primary samples. These effects are preserved in 7- and 14-day washout experiments. AHR antagonism increases expression of CD38 (2.1-fold) and SLAMF7 (CS1, 1.54-fold) among other antigens. AHR antagonism enhances NK cell cytolytic potential by promoting expression of CD94, TRAIL, perforin and granzyme B, whereas in MM cells, AHR antagonism upregulates surface expression of NK cell activating ligands: CD112, CD54, ULBP1, ULBP2, ULBP3, ULBP4, CD155, MICA/B, and DR4 and DR5. Pretreatment of MM cell targets with an AHR antagonist enhances susceptibility to NK-cell cytotoxicity (p < 0.005) and augments NK-cell mediated ADCC in combination with daratumumab (p = 0.001) and elotuzumab (p = 0.001) versus controls. CONCLUSIONSThese data confirm functional expression of AHR in MM and may provide insight to growing epidemiologic data indicting a link between environmental exposure to aromatic hydrocarbons and subsequent risk of MM. In addition, as opposed to the direct, deleterious effects of AHR antagonism on MM cell viability, AHR antagonism promotes NK cell development, surveillance of MM, and direct as well as ADCC-mediated cytotoxicity against MM targets. Taken together, these data strongly support the concept of targeting AHR as a novel direct anti-MM therapy and anti-MM immunotherapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

Erwinia Chrysantemi-Derived L-Asparaginase Strongly Enhances Proteasome Inhibitors Activity By Deregulating Cell Metabolic Programs

Background: Human tumours rely on Glutamine (Gln) metabolism to sustain wide range of metabolic processes and macromolecules synthesis. Such dependence defined as “Gln-addiction” has been recently observed also for Multiple Myeloma (MM) cells, where Gln synthetase deficiency make them more sensitive to Gln depletion. L-Asparaginase (L-ASP), the enzyme that catalyzes asparagine (Asn) and Gln hydrolysis, represents a key drug for B-cells derived malignancies treatment such as acute lymphoblastic leukemia.Aims: The aim of the present study was to screen MM cells sensitivity to pharmacological Gln depletion achieved by Erwinia chrysantemi-derived L-ASP treatment as single agent as well as in combination with proteasome inhibitors (PIs). Based on its high glutaminase activity, E. chrysantemi L-ASP was selected for this screening.Materials and methods: The IC50 value of L-ASP was determined by MTS assay in a panel of human MM cell lines (HMCLs) harboring different genetic background as well as on primary CD138+ cells derived from newly diagnosed (NDMM) and and relapsed/refractory (RRMM) MM patients. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. L-ASP treatment effect on cell viability and mitochondrial membrane potential was evaluated by FACS analysis following PI and TMRE staining, respectively. Intracellular ATP levels was also measured by HPLC-based method. Drugs combination screening with L-ASP plus PIs including bortezomib (BZ) or carfilzomib (KAR) was investigated by using isobologram analysis (CalcuSyn software program). Finally, western blot analysis was employed to fully elucidate biological mechanisms of our findings.Results: L-ASP treatment showed potent anti-MM activity in all HMCLs tested with an IC50 values ranging from 0,03 U/mL to 0.2 U/mL. Importantly, such drug preserved its effect also on primary tumor cells obtained from NDMM and RRMM patients (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible “metabolic shutdown” in MM cells treated with L-ASP which led to late mitochondrial stress (48-72h). To further confirm Gln specific role in the observed finding, MM cells viability was measured in presence of different culture media conditions. Asn depletion alone did not modify significantly MM cells viability, instead it strongly contributed to L-ASP induced toxicity of Gln-starved cells. Co-treatment experiments showed massive increase of its anti-MM activity when L-ASP was used in presence of low doses of BZ and KAR, with a combination index below than 1 in all tested drugs-concentrations. Accordingly, caspase-3 and PARP cleavage showed apoptotic cell death features of this co-treatment. Importantly, IL6 or IGF-1 addition did not reduce anti-MM activity of this combination suggesting that MM microenvironment does not reduce efficacy of this strategy. Finally, mechanistic studies revealed higher ATP shortage in co-treated cells together with c-MYC down-regulation, genomic instability and misfolded proteins accumulation, suggesting a massive proteasome activity impairment triggered by these drugs-combination. Ongoing experiment will help to further elucidate this hypothesis.Conclusions: Our preliminary data show that E. chrysantemi L-ASP exerts a potent anti-MM activity mainly due to its glutaminase activity, which results in a massive metabolic program deregulation. The combination with low-doses of PIs further increases anti-MM activity of this small molecule likely due to huge proteasome activity injury. DisclosuresGobbi:Amgen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Janssen: Consultancy; Pfister: Membership on an entity's Board of Directors or advisory committees.

Blockade of Ubiquitin Receptor PSMD4/Rpn10 Triggers Cytotoxicity and Overcomes Bortezomib-Resistance in Multiple Myeloma

Background and Rationale Proteasome inhibitors (PIs) are standard of care therapy for patients diagnosed with multiple myeloma (MM). However, prolonged treatment can be associated with toxicity and development of drug resistance. Our research efforts have therefore focused on designing therapeutic strategies that can overcome PI-resistance. In this context, our RNA-interference-based loss-of-function studies have identified 19S proteasome-associated Ubiquitin Receptor (UbRs) Rpn10 and Rpn13 as potential therapeutic targets. Both Rpn10 and Rpn13 UbRs are associated with the 19S regulatory lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. We have previously reported the role of Rpn13 in MM (Song et., al Luekemia 2016 Sep;30(9):1877-86). Here we functionally characterized the role of Rpn10 in MM.Materials and Methods Cytotoxicity assays were performed using a panel of MM cell lines, primary patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST-1, MTT, and Cell Titer-Glo assay.Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Statistical significance of data was determined using a Student's t test.Results Functional characterization of Rpn10 in MM:Gene expression (GEP) analysis showed inverse correlation between Rpn10 and overall patient survival (n=175) (p= 0.00064). Immunoblot analysis showed high Rpn10 protein levels in primary patient cells and MM cell lines versus normal plasma cells or PBMCs. Rpn10 knockdown by siRNA significantly decreased MM cell viability in both bortezomib-sensitive and -resistant MM cell lines. To assess the Rpn10 function, we generated a doxycycline-inducible Rpn10-shRNA knockout stable MM cell line. Rpn10-shRNA knockdown decreased MM cell proliferation. Mechanistic studies show that Rpn10 knockdown-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; 3) arrest of cell cycle; 4) induction of ER stress; and 5) activation of caspases mediating apoptotic pathways. In order to determine if blockade of Rpn10 affects cellular proteasome function, we next utilized a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Interestingly, Rpn10-siRNA increased accumulation of the Ub-GFP reporter, reflecting impaired proteasome-mediated protein degradation. Finally, treatment of MM cells with peptides targeting UIM2 domain of Rpn10 significantly decreased MM cell viability, suggesting a potential therapeutic approach in MM.ConclusionOur preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of the proteasome in the ubiquitin proteasomal cascade, and provides the framework for clinical evaluation of Rpn10 inhibitors to overcome PI resistance and improve patient outcome in MM. DisclosuresAnderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.

A Rapid Functional Screen for Small Molecule and Monoclonal Antibody Drug Sensitivity in Multiple Myeloma Patients

Background: The oncogenic drivers and progression factors in multiple myeloma (MM) are heterogeneous and difficult to target therapeutically. As a result, personalized medicine approaches have not yet been realized. However, clinical availability of numerous anti-myeloma drugs and readily obtainable bone marrow (BM) aspirates raises the possibility to benefit patients by profiling the drug sensitivity of their MM. Despite newly available drugs, resistance inevitably develops and there is no defined rationale for sequencing therapy. Treatments now consist of >15 agents, including IMiDs, protease inhibitors (PIs), alkylator chemotherapies, steroids, monoclonal antibodies, and options are still expanding. To profile sensitivity to the wide array of options, we developed a rapid functional assay of primary MM cells, distinct from prior assays by forgoing CD138+ cell selection to optimize MM cell viability and allow measurement of monoclonal antibody activity.Methods: Myeloma drug sensitivity testing (My-DST) was performed on patient BM aspirates to measure the MM sensitivity in heterogeneous mononuclear cell (MNC) mixtures. My-DST is performed by culturing samples with a 7-drug panel consisting of bortezomib (Bor), carfilzomib (Car), lenalidomide (Len), pomalidomide (Pom), dexamethasone (Dex), cyclophosphamide (Cy, metabolite 4HC), and daratumumab (Dara) for 48h, followed by multiparameter, high-throughput flow cytometry to measure the surviving MM population. All conditions are performed in triplicate and in parallel, clonality verified by kappa/lambda staining.Results: MM cell viability was substantially better in unselected MNC cultures than CD138-selected cultures (data not shown). Dose-response was evaluated for each agent in MNC cultures to select single active concentrations for high throughput screening (data not shown). Single concentration profiling was completed on 55 patient samples from diagnosis (n = 24), first relapse (n = 11) and after multiple relapses (n = 20) with 2.5 nM PIs, 10 µM IMiDs, 1 µM Dex, 3.75 µM Cy and 20 nM Dara. Each was scored as sensitive or resistant using a cutoff of 80% surviving MM cells normalized to untreated controls (Fig 1A). Mild inherent resistance to varied tested agents was evident at diagnosis and progressively increased with lines of therapy (LOT) until multidrug resistance predominated (Fig 1B). My-DST displayed significant correlation for LOT with increasing resistance to Bor (r = 0.27, P = 0.046), Car (r = 0.27, P = 0.044), Len (r = 0.44, P = 0.0006) and Pom (r = 0.43, P = 0.0009), but not Dara (r = 0.27, P = 0.10), Cy (r = 0.17, P = 0.24) and Dex (r = 0.032, P = 0.82) (Fig 1C). The ex vivo sensitivity for Bor and Car were highly correlated (r = 0.67, P < 0.0001) from sensitive to both to resistant to both, but still differential results favoring Bor or Car were observed (Fig 1D). The ex vivo results for the tested IMiDs were even more highly correlated (r = 0.79, P < 0.0001), with fewer samples showing greater sensitivity differentially to Len or Pom (data not shown). In patients at diagnosis, the ex vivo sensitivity to all drugs given during treatment led to significantly deeper clinical responses than those with resistance to ≥1 drugs received (Fig 1E). As an example of potential clinical utility of My-DST, the test for #847 at diagnosis identified sensitivity to PIs and resistance to IMiDs, which correlated with inadequate response to Len/Dex, followed by complete response (CR) after change to Bor/Dex (Fig 1F). Dara had ex vivo activity in most naïve patients, but patients already exposed showed significant acquired resistance (Fig 1G). We also found that CD38 expression was significantly lower in patients that showed ex vivo Dara resistance (Fig 1H). The ex vivo Dara activity was partially blocked by inhibiting macrophages with clodronate-liposomes (CL) (Fig 1I).Conclusion: My-DST on unselected MM BM aspirates with minimal perturbations ex vivo was clinically predictive of resistance to IMiDs and PIs. For the first time, this approach is able to measure the antibody-mediated cytotoxicity of daratumumab in clinical MM samples. Our data indicate that personalized regimens based on rapidly obtained ex vivo sensitivity may lead to optimized depth of clinical response. We next plan to translate this approach into clinical use in an effort to improve patient outcomes and avoid wasted time, money and complications that may arise from the use of ineffective drugs. [Display omitted] DisclosuresMark:Janssen, Takeda, Celgene, Amgen: Consultancy; BMS, Celgene: Research Funding; Celgene: Honoraria.

Preclinical Validation Studies Support Causal Machine Learning Based Identification of Novel Drug Targets for High-Risk Multiple Myeloma

IntroductionRegardless of significant advances in the therapy of multiple myeloma (MM) there is still a lack of effective treatment options for patients with high-risk disease. In this context, we recently developed a network of high-risk disease based on more than 30 000 genomic and clinical variables from 645 patients of the CoMMpass dataset (Gruber et al., ASH 2016). Validation of these findings has been performed in the IFM/DFCI 2009 trial dataset (Furchtgott et al., ASH 2017). This comprehensive computational approach revealed a network of 17 genes driving high-risk (defined as progression or myeloma-related death within 18 months). Here, we performed preclinical validation of potential novel drug targets to confirm the utility of in silico guided target discovery in high-risk MM.MethodsTTK (CFI402257, BAY-1217389), PLK4 (CFI400945, Centrinone), MELK (OTSSP167) and CDK1 (CPG71514) inhibitors were studied in a panel of human MM cell lines (n=11) for their activity in cell viability, cell growth, cell cycle, apoptosis, colony formation, drug combination and co-culture experiments. PKMYT1, TTK and PLK4 were targeted with doxycycline-inducible shRNAs. Analysis of gene expression (GEP) data (GSE24080) was used to link candidate genes to certain MM subgroups.ResultsThe network of 17 genes driving high-risk disease contained eight kinases that serve as attractive drug targets (AURKA, NEK2, CDK1, BUB1B, MELK, TTK, PKMYT1, and PLK4), all of them involved in cell cycle regulation. Accordingly, expression levels of all kinases (except PKMYT1) were enriched in the GEP-defined proliferation associated subgroup of MM and thus linked to poor outcome. To study the interconnectedness of the individual network genes we first investigated the impact of previously reported CDK1 and MELK inhibitors on other network members. This demonstrated rapid loss of CDK1, NEK2, MELK, PKMYT1 and FOXM1 protein levels.We then selected TTK and PLK4 as putative novel MM targets with available inhibitors undergoing clinical testing in solid tumors. Protein and mRNA expression of both genes was confirmed in all MM cell lines. Two selective compounds per gene were used for preclinical studies. All four inhibitors significantly reduced MM cell viability and single dose IC70 treatment impaired cell growth up to 10 days (60-98% reduction, P<0.01). This growth inhibitory effect was confirmed with inducible shRNAs. Mechanistically, growth impairment was linked to G2M cell cycle arrest followed by the accumulation of polyploid cells (15-90% of cells 72h post treatment) which is in line with the role of both genes in chromosome segregation. The formation of aberrant mitoses led to the induction of apoptosis 3-5 days post treatment (≤20% viable cells at day 5 post treatment with 3/4 inhibitors) and was accompanied by the presence of active caspase 3 and cleaved PARP. Importantly, the activity of these drugs persisted in the presence of BMSCs and showed potent activity in colony formation assays (DMSO: 168±15 and 131±57, BAY-1217389: 39±29 and 41±3, CFI-400945: 19±26 and 30±6 colonies in KMS12BM and OPM2 cells at day 14, P<0.05). Drug combination studies pointed to favorable activity in combination with dexamethasone and lenalidomide. Furthermore, confirmatory TTK knockdown with two independent shRNAs sensitized MM cells to dexamethasone.Finally, PKMYT1 was chosen as putative target based on its role as major driver of high-risk in our model. We transduced MM cells with three doxycycline-inducible PKMYT1-targeting shRNAs and observed an impressive impact on myeloma cell growth upon doxycycline induction compared to non-targeting control shRNA (up to 85% reduction at day 10, P<0.01). Furthermore, PKMYT1 knockdown led to the induction of apoptosis in all MM cell lines tested. Based on these encouraging results we currently perform in-depth in silico and in vitro analyses of the underlying PKMYT1 signaling network. Detailed results of these sub-studies will be presented at the meeting.ConclusionsOur results confirm the utility of computational based modelling of high-risk disease. This strategy not only revealed a genetic network closely associated to adverse prognosis, but also enabled the identification of so far unnoticed drug targets. Importantly, inhibitors of TTK and PLK4 are already in clinical testing and thus enable rapid clinical translation of our findings to MM patients in need of alternative therapeutic options. DisclosuresGruber:GNS Healthcare: Employment. Furchtgott:GNS Healthcare: Employment. Raut:GNS Healthcare: Employment. Wuest:GNS Healthcare: Employment. Runge:GNS Healthcare: Employment. Khalil:GNS Healthcare: Employment. Munshi:OncoPep: Other: Board of director. Hayete:GNS Healthcare: Employment. Ludwig:Amgen: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Cilag-Janssen: Speakers Bureau; Celgene: Speakers Bureau.

Preclinical Studies and a Phase I Trial of the TGF-β Receptor Inhibitor, Vactosertib (TEW-7197), in Combination with Pomalidomide in Patients with Multiple Myeloma Refractory to Bortezomib or Lenalidomide

Multiple myeloma (MM) is characterized by the loss of critical mediators of immune surveillance and the activation of osteoclast. The transforming growth factor-beta (TGF-β) signaling pathway has been implicated in MM progression through promotion of catabolic bone remodeling, IL-6 secretion and Th17 T cell development, promoting osteolytic bone disease, immune suppression and MM progression. Here, we conducted preclinical studies in the syngeneic 5T3MM immunocompetent mouse model of MM to assess the single agent activity of vactosertib (Vacto, TEW-7197), a TGF-β type I receptor kinase inhibitor, and the potential to act synergistically with the third-generation immunomodulatory drug, pomalidomide (Pom). These data support the first clinic trial to evaluate this combination regimen in patients (pts) with MM (NCT03143985, open and recruiting).MethodsPreclinical: Mice bearing 5T33MM cells expressing luciferase were treated with Vacto, Pom and the combination for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Cellular and molecular assays were performed in human RPMI8226 and U266 as well as murine 5T33MM cells via apoptosis, real-time PCR and western blotting. Peripheral blood monoclonal protein concentration, M-spike, was measured by ELISA. Distal femur trabecular bone structure was assessed by 3D micro-CT. Phase I clinical trial: In this open-label study, pts with relapsed MM with at least two lines of therapies received escalating dosages of Vacto in combination with a standard dose of Pom (4 mg) without corticosteroids. The study was conducted as a modified Fibonacci 3 + 3 dose escalation design to assess safety, maximum tolerated dose, recommended phase 2 dose, and efficacy of Vacto/Pom combination in compared to historical control of Pom without corticosteroids (PFS-6: 20% in randomized Phase II study by Richardson et al. Blood. 2014). Vacto tablets are administered orally in 28-day cycles, taken once daily for 5 days followed by 2 days without treatment, repeated either for 6 cycles or until progression of disease or intolerable toxicity. Dosing was initiated at 60 mg and increased to 120 and 240 mg.ResultsPreclinical: Vacto attenuated the growth and viability of human and murine MM cells by inducing apoptosis and inhibited TGF-β-induced activation of Smad2/3 in MM cells in vitro. In the 5T33MM preclinical model, single agent Vacto inhibited MM progression as measured by peripheral blood monoclonal protein concentration and BLI. Vacto also prolonged survival (Figure-1), prevented weight loss and increased trabecular bone thickness (Figure-2) in mice bearing MM. Vacto either alone or in combination with Pom also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b⁺Gr-1⁺ myeloid derived suppressor cells (MDSCs) in the bone marrow and diminished the population of Foxp3⁺ regulatory T cells in the spleen (Figure-1). Vacto showed anti-myeloma activity in cells from three MM pts (Figure-3) at 700-750 nM in vitro. Phase I: As of 01 July 2018, 5 pts were enrolled; 3 pts received 60 mg daily and 2 pts received 120 mg daily. Patient demographics include: median age of 70 years (range: 64-77); 3-5 lines of prior therapy; ISS stage II or III; two pts had high risk cytogenetics including 17p deletion; three pts were refractory to bortezomib and four pts were refractory to lenalidomide. None of five enrolled pts developed any DLT or needed Vacto dose reduction .The most non-hematologic common adverse event (AE) was grade II fatigue and pain in one pt with no grade III or IV non-hematologic AEs. No pt experienced any progression of disease (PFS-6: 100%). Figure-4 illustrates fluctuations in disease markers at each cycle interval. Two pts had grade III hematologic AE, no grad IV hematologic AE.Conclusions: Vacto has activity against myeloma in the syngeneic 5T33MM murine MM mouse model and in both human MM cell lines and primary patient MM samples. Remarkably, the effect of Vacto on the tumor microenvironment is augmented in combination with Pom in the Cereblon-negative immunocompetent 5T33MM model. The phase I data show safety of this agent in combination with Pom. The preliminary efficacy assessment is PFS-6: 100% which is higher than historical controls (Richardson et al. Blood. 2014) with Pom only (PFS-6: 20%) or Pom with corticosteroids (PFS-6:40%). Advancement of Vacto in clinical trial pipelines for MM and other malignant hematological disorders is planned. [Display omitted] DisclosuresMalek:Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau. Caimi:Kite Pharma: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees. Kim:MedPacto: Other: Founder/CEO.

Vactosertib, a TGF-ß Receptor I Kinase/ALK5 Inhibitor, Diminishes Tumor Progression and Bone Disease in a Mouse Model of Multiple Myeloma and Overcomes Resistance to Proteasome Inhibitors

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by osteolytic bone disease and immunosuppression. Transforming growth factor-beta (TGF-β) supports MM progression through its role in stimulation of IL-6, Th17/regulatory T cell development, hematopoietic suppression, and its promotion of catabolic bone remodeling. Inhibition of the proteasome has emerged as a powerful strategy for treatment of MM. Proteasome inhibitors (PIs) have improved overall survival in patients with MM, but the majority develop drug resistance. Therefore, a novel therapeutic strategy is needed for better treatment of MM. Vactosertib (VACTOSERTIB) is an orally bioavailable small molecule inhibitor of the TGF-β type I receptor kinase (TβRI) currently in a phase I clinical trial (NCT03143985) in combination with the immunomodulatory drug, pomalidomide. In this study, we first investigate if Vactosertib is capable of diminishing myeloma progression either as a single agent or in combination with the PI, ixazomib.Methods: Expression levels of TGF-β1 and phospho-Smad2/3 was examined in human and mouse MM cells and bone marrow by Western blot and immunohistochemistry (IHC). Cellular and molecular assays were performed in human RPMI8226, ARH-77 and PIs-resistant RPMI8226 as well as murine 5T33MM cells via MTT, apoptosis, real-time PCR and Western blot. For preclinical evaluation, C57BL/KaLwRij mice bearing 5T33MM cells expressing luciferase were treated with Vactosertib, ixazomib, or the combination of Vactosertib plus ixazomib for 3 weeks, and evaluated for MM growth by bioluminescence imaging (BLI). Peripheral blood IL-6 and monoclonal protein concentration, M-spike, were measured by ELISA. Fluorescence-activated cell sorting (FACS) analyses were used for immunological evaluation, such as myeloid derived suppressor cells (MDSCs), Foxp3+ regulatory T cells (Tregs) and NK cells. Femur trabecular bone density and microarchitecture were assessed by 3D micro-CT system.Results: We first evaluated the expression of the TGF-β1 ligand and its receptor activated signaling intermediates, Smad2/3 in bone marrow of patients with MM. There is an accumulation of the type 1 isoform of the TGF-β ligand within the tumor microenvironment (TME) and of phospho-Smad2/3 in myeloma cells within the bone marrow of patients (Figure 1). In vitro studies utilizing the TbRI inhibitor Vactosertib (TEW-7197) with murine 5T33MM, human RPMI8226 (Figure 2A), U266, ARH-77 cell lines, and tumor cells from patients with MM (Figure 2B) cultured demonstrate a dose-dependent reduction in cell viability in cultures of these MM cells continuously exposed to Vactosertib. Synergistic inhibition of Vactosertib in combination with PIs was also observed in parent (Figure 2C), bortezomib- (Figure 2D), ixazomib- (Figure 2E), and carfilzomib-resistant RPMI8226 cells (Figure 2F). We also found that there is an accumulation of the TGF-β1 within the TME and phospho-Smad2/3 within the MM at advanced stages in the bone marrow of C57BL/KaLwRij mice harboring the 5T33MM myeloma cells. Oral administration of Vactosertib as a single agent inhibited MM progression as measured by mortality (Figure 3A), peripheral blood monoclonal protein concentration and BLI (Figure 3B). While the expansion of CD11b+Gr-1+ MDSCs (Fig. 3C) and the population of Tregs (Fig. 3D) in the spleen were reduced, NK cells were increased by Vactosertib alone (Fig. 3E). Combination therapy of Vactosertib plus ixazomib exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by a decrease in mortality (Figure 4A) and significant reduction in both the BLI and M-spike before and after treatment (Figure 4B, C). Furthermore, Vactosertib single therapy enhanced the bone thickness and reduced trabecular separation in comparison to control mice.Conclusions: Taken together, our data demonstrate that Vactosertib (a small molecule inhibitor of the TGF-β type I receptor kinase) suppresses viability of human MM cells, and is associated with a potent anti-myeloma effect, suppressing bone resorption and modulating the MM TME in a immunocompetent preclinical model of MM. These data provide a rationale for clinical evaluation of the combination therapy of Vactosertib and ixazomib as a potential therapeutic strategy to improve outcomes in patients with MM. [Display omitted] DisclosuresKim:MedPacto: Other: Founder/CEO. Malek:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau.

Functional Role of Linc-RNAs in Multiple Myeloma: Linc-MIR17HG Affects Fatty Acid Biosynthesis Via transcriptional Regulation of ACC1 with Potential Therapeutic Implications

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. DisclosuresAnderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Targeting Distinct Promoter- and Enhancer-Driven Dependencies in Multiple Myeloma

Uncontrolled proliferation is a hallmark of tumorigenesis and is associated with perturbed transcriptional profiles. The proliferative program in Multiple myeloma (MM), a complex disease with heterogeneous genetic changes, is controlled by transcription factors (TFs) and chromatin-associated factors. The dependency on these transcriptional regulators, leading to the dysregulated proliferation, is not predicted by genetic changes, making the tumor cells more sensitive to inhibition of these regulators than normal cells.The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs is not well-defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. Our genome-wide epigenomic profile in myeloma has identified the existence of two non-overlapping regulatory axes controlled by promoter and enhancer-driven processes, governing distinct biological functions. We have utilized E2F1 as promoter proximal transcription factor, and evaluated its transcriptional and functional interrelationship with enhancer-associated factors, such as BET bromodomain transcriptional co-activators. We identified that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in MM cells. Global chromatin analysis revealed two distinct regulatory axes, with E2F and MYC predominantly localized to active gene promoters of growth/proliferation genes and CDK9 and BETs disproportionately at enhancer-regulated tissue-specific genes. This divergent BRD4 enhancer and E2F promoter axes is also observed in diffuse large B-cell lymphoma, suggesting a more broader transcriptome control process.Dual inhibition of E2F and BETs displays a superior activity against MM cell growth and viability, both in vitro and in vivo, compared to single perturbation alone providing an important molecular mechanism for combination therapy. Moreover, at low doses of BRD4 inhibitor JQ1, the addition of E2F1 depletion down-regulates the promoter controlled proliferation gene expression axis.As for many TFs, direct pharmacologic inhibition of E2F remains a difficult challenge in drug discovery. However, E2F is not entirely “undruggable” as inhibitors of upstream regulators of the pRB-E2F axis are available. For example, a number of Cyclin dependent kinases (CDK) 4/6 inhibitors, including Palbociclib are now being investigated in clinical trials in in fact approved by the FDA in select malignancies. CDKs are serine threonine kinases that modulate cell cycle progression. CDK4 and CDK6 together with D-type cyclins and cyclin E/CDK2 complexes control the commitment to cell cycle entry from quiescence and the G1 phase. These kinase complexes can phosphorylate RB, releasing E2F to modulate the expression of E2F target genes that are required for S phase entry. We investigated combination of low doses of JQ1 and Palbociclib and observed a profound effect on E2F promoter driven transcriptional activity, and was highly synergistic with JQ1 in a large panel of MM cell lines and primary MM cells from newly diagnosed and relapsed patients. Cell cycle analysis revealed complete G1 arrest after treatment. Importantly, the combination regimen was not effective in healthy donor PBMCs activated with PHA, suggesting a favorable therapeutic index. Transcriptomic changes to assess the impact on promoter and SE-driven processes are ongoing and will be presented.In conclusion, these data implicates the existence of a sequestered cellular functional control that may be perturbed in cancer to maintain the tumor cell state. Simultaneous targeting of non-overlapping promoter and enhancer vulnerabilities impairs the myeloma proliferative program, with potential for development of a promising therapeutic strategy in MM and other malignancies. DisclosuresYoung:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder. Munshi:OncoPep: Other: Board of director.

Discovery platform for inhibitors of IgH gene enhancer activity

ABSTRACTImmunoglobulin heavy chain (IgH) translocations are common and early oncogenic events in B cell and plasma cell malignancies including B cell non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM). IgH translocations bring oncogenes into close proximity with potent enhancer elements within the IgH locus, leading to oncogene up-regulation. As IgH enhancer activity is tightly controlled by B cell lineage-specific signaling and transcriptional networks, we hypothesized that IgH enhancers are potentially druggable targets/elements. To test this, we developed a molecular imaging-based high-throughput screening platform for discovering inhibitors of IgH enhancer-driven transcriptional activity. As proof of concept, we identified a low micromolar potency molecule (compound 30666) that inhibited immunoglobulin production by MM cells and blocked expression of an array of IgH translocation-induced oncogenes (CCND1, FGFR3/MMSET, and MYC) in MM and NHL cell lines. Prolonged exposure to 30666 significantly reduced the viability of IgH translocation-positive NHL and MM cells, but was less effective against cells lacking IgH translocations. Compound 30666 exhibited suitable pharmacological properties, including metabolic stability in liver microsomes and oral bioavailability in mice, and demonstrated preclinical anti-MM activity in a plasmacytoma mouse model. Our work suggests that IgH enhancers are attractive and potentially druggable targets for IgH translocation driven malignancies.