Kung, Andrew L.

Abstract Abstract 1409 MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement. Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures: Richardson: Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

Abstract Abstract 1534 + equal contributions Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate and infrequent somatic mutations of TP53 and genes encoding cell cycle pathway components. Despite recent advances in the molecular understanding of DLBCL pathogenesis, clinical models such as the International Prognostic Index (IPI) are still used to identify high-risk patients. By integrating high-resolution high-density SNP array data with transcriptional profiles and performing pathway analysis in 180 primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary DLBCLs with single copy loss of 17p13.1 (TP53/RPL26/KDM6B) often had CNAs of an additional p53 modifier – 9p21.3 (CDKN2A/ARF), 19q13.42 (BCL2L12), 12q15 (MDM2) or 1q23.3 (MDM4/RFWD2). CNAs of the respective p53 modifiers, CDKN2A (ARF, 9p21.3), MDM2 (12q15) and MDM4/RFWD2 (1q23.3), occurred in largely separate groups of tumors. DLBCLs with CNAs of p53 pathway members frequently exhibited concurrent alterations of additional cell cycle components such as CCND3 (6p21.32), CDK6 (7q22.1), CDK2/CDK4 (12q15) and/or RB1 (13q14.2) or RBL2 (16q12.2). When the primary DLBCLs were clustered in the space of CNAs that perturb p53 pathway and cell cycle components, 66% of tumors had multiple alterations (“complex” pattern) whereas the remaining 34% of tumors lacked these lesions (“clean” signature). Tumors with “complex” alterations of p53 pathway and cell cycle components also had more total CNAs and more frequent TP53 mutations, highlighting the association between p53 deficiency, cell cycle deregulation and increased genomic instability in DLBCL. The patterns of “complex” vs. “clean” CNAs of p53 pathway and cell cycle components and the association between the “complex” signature and total CNAs were confirmed in an independent series of primary DLBCLs. To further characterize DLBCLs with “complex” vs. “clean” CNA patterns, we performed gene set enrichment analyses with publicly available series of p53 target genes and a RB-deficiency gene set which included multiple E2F targets. The p53 target transcripts were significantly less abundant in “complex” DLBCLs, directly linking their genetic signature of p53 deficiency with decreased p53 activity. Furthermore, the RB-deficiency gene set was significantly enriched in “complex” DLBCLs suggesting that these tumors had increased E2F-mediated cell cycle progression. Consistent with these observations, DLBCLs with “complex” CNA patterns also had significantly higher proliferation indices as determined by Ki67 immunostaining. We next assessed the prognostic significance of the “complex” CNA pattern in the subset of patients who were treated with R-CHOP (rituxan, cyclophosphamide, adriamycin, oncovin, prednisone) and had long-term follow-up. R-CHOP treated patients with “complex” CNA patterns had a 5-year overall survival of only 62%; in contrast, those with “clean” CNA signatures had an 100% overall survival (p =.001). The association between CN complexity and outcome was independent of transcriptionally defined categories and additive to the clinical IPI risk model. Taken together, these data provide a structural basis for deregulated cell cycle, increased cellular proliferation and unfavorable outcome in DLBCL and suggest targeted treatment strategies. Disclosures: No relevant conflicts of interest to declare.

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.