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Enhancer of zeste homologous (EZH) 2 and its close homolog EZH1 are catalytic subunits of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper trimethylation of H3K27 has been associated with malignant lymphoma and myeloma progression, thus several small molecules suppressing PRC2 complex activity has been developed for hematological malignancy therapy. We have developed valemetostat tosylate (DS-3201b, also known as valematostat), a potent dual inhibitor of EZH1/2, and demonstrated its superior anti-proliferative effect against DLBCL cells to tazemetostat (EPZ-6438, E7438) a selective EZH2 inhibitor currently in clinic. In addition, valemetostat synergized with wide variety of 1st and 2nd line drugs used in DLBCL therapy both in vitro and in vivo proposing its potential combination opportunities. However, it is still elusive how valemetostat modulates epigenetic landscape and represses malignant B-cell proliferation more potently than selective EZH2 inhibitors. Therefore, impact on epigenetic landscape between valemetostat and tazemetostat was analyzed by RNA/ChIP-sequencing. Though these two inhibitors significantly reduced cellular global H3K27me3 level, we observed ectopic EZH1/2 accumulation in several tumor suppressor gene loci after tazemetostat treatment resulting in partial reduction in H3K27me3 and de-repression of silenced gene expression. Meanwhile valemetostat treatment evidently triggered gene expression by depleting H3K27me3 and enhancing H3K27Ac mark without inducing ectopic enrichment of EZH1/2, suggesting that valemetostat has a distinct effect on genome wide distribution of EZH1/2 from tazemetostat. In conclusion, these results suggest that valemetostat has a capacity of averting ectopic relocation of EZH1/2 on tumor suppressor genes mainly induced by EZH2 specific inhibition and thereby exerts greater anti-B cell tumor effect than EZH2 preferential inhibitor. A phase 2 clinical study of valemetostat is now ongoing for patients with Relapse/Refractory B-cell Lymphoma [ClinicalTrials.gov Identifier: NCT04842877] Disclosures Banjo: Daiichi Sankyo Co., Ltd.: Current Employment. Hama: Daiichi Sankyo Co., Ltd.: Current Employment. Nosaka: Daiichi Sankyo Co., Ltd.: Current Employment. Takata: Daiichi Sankyo Co., Ltd.: Current Employment. Honma: Daiichi Sankyo Co., Ltd.: Current Employment. Kitagawa: Daiichi Sankyo Co., Ltd.: Current Employment. Yamamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Wada: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Yoshida: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Lim: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Okamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Katayama: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Goto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Abe: Daiichi Sankyo Co., Ltd.: Current Employment.

Introduction The World Trade Center (WTC) disaster exposed first responders to high levels of aerosolized carcinogens (Lioy et. al. Env. Health Perspect 2002). Clonal hematopoiesis is associated with exposure to smoking and genotoxic stimuli (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM 2015). We sought to determine its incidence in WTC-exposed first responders. We also assessed the effect of WTC particulate matter (WTC-PM) on genome integrity in vitro, and in murine studies. Methods Deep targeted sequencing was performed on blood collected from 481 first responders (429 WTC-exposed firefighters, 52 WTC-exposed emergency medical service workers) and 52 non-exposed first responders. Samples were analyzed for 237 genes mutated in hematologic malignancies and interpreted using reference databases. Non synonymous somatic mutations were annotated and analyzed. Results In the WTC-exposed cohort, 57 individuals with 66 somatic mutations of expected pathogenic potential were identified (overall prevalence 11.9%). In the non-exposed cohort, only one pathogenic mutation was found in the IDH2 gene (overall prevalence 1.9%). There was a strong association between increasing age and prevalence of mutations in the WTC-exposed cohort (Fig 1A). DNMT3A (16/66), TET2 (7/66), SF3B1 and SRSF2 (3/66 each) were the most common genes identified in the WTC-exposed cohort (Fig 1B). Median VAF was 12% and missense mutations were most frequent alteration. Aging, smoking, DNA repair and alkylating agent exposure related mutational signatures were observed with a cytosine to thymine (C→T) transition being most common. Next, we assessed the effect of WTC-PM on genome integrity and replication in vitro. WTC-PM that was collected in the first three days after 9/11 was used in concentrations mimicking exposure levels. Lymphocytes exposed to WTC-PM demonstrated a significant increase in phosphorylated H2AX foci accumulation, suggesting a DNA damage response (Fig 2). Since common fragile sites (CFSs) detect basal levels of stress in the cell, and activate DNA damage response (DDR), we profiled DNA replication dynamics at CFS-FRA16D at very high resolution using the single molecule analysis of replicated DNA (SMARD) assay. Treatment with WTC-PM significantly altered replication at two common fragile sites (regions 1 and 2 of FRA16D, Fig 3A) with replication pausing being observed at multiple sites (Fig 3B-I, white rectangles). Striking increase in replication initiation was seen, characterized as dormant origins activated to rescue replication pausing (Fig 3E, J). These alterations were accompanied by a corresponding increase in replication speed, conditions that lead to DNA replication errors and mutagenesis (Fig 3F, K). Next, we treated mice with WTC-PM via the oropharyngeal route to mimic first responder exposures, and then harvested and analyzed their bone marrow compartments. Significant expansion of hematopoietic stem cells (Kit+, Sca1+, Lineage-ve, KSL) was seen in WTC-PM treated mice (Fig 4A,B). Whole genome sequencing of sorted stem cells showed a significant increase in non-synonymous SNPs, deletions and indels in the WTC-PM treated samples when compared to control (Fig 4C-E). These genomic alterations were found to occur at low VAF throughout the whole genome, demonstrating widespread genotoxic effects of WTC-PM on hematopoietic stem cells in vivo (Fig 4F). Discussion We report a high burden of mutations in 11.9% (57/481) WTC-exposed first responders compared to the non-exposed cohort (1.9%, 1/52). The frequency of the somatic mutations was many fold higher than in previous studies (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM, 2015). In the 50-59 year age group, 10% of WTC-exposed individuals carried somatic mutations, compared to the frequency of 2.5% reported by Jaiswal et. al. for the same age group. Despite deeper sequencing performed in our study, the median VAF in our study was 12%, indicating that the difference in technique did not bias our study towards increased detection of small, subclinical clones when compared to previous studies. Furthermore, we demonstrate that WTC-PM can perturb DNA replication and increased genomic instability in vivo, potentially leading to higher burden of clonal hematopoiesis in WTC-exposed first responders. These results demonstrate adverse environmental exposures can be associated with a high rate of clonal hematopoiesis. Disclosures Landgren: Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Fletcher:Genoptix/Neogenomics: Employment. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Steidl:GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Research Funding. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

Enhancer of zeste homolog (EZH) 1 and its close homolog EZH2 are component of polycomb repressive complex 2 (PRC2), and play a partially redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper tri-methylation of H3K27 have been associated with lymphoma and myeloma progression, suggesting PRC2 is a therapeutic target for hematological malignancies. We have developed a novel EZH1 and EZH2 dual inhibitor valemetostat (DS-3201b), which simultaneously inhibited the enzymatic activity of EZH1 and EZH2 in nano-molar concentration. Valemetostat demonstrated anti-proliferative activities against the Activated B-cell-like (ABC) and Germinal Center B-cell-like (GCB) subtypes of Diffuse Large B-cell Lymphoma (DLBCL) cells. Furthermore, valemetostat induced apoptosis in DLBCL cell lines, regardless of subtype. We revealed that the pleiotropic effects of valemetostat on the expression levels of B-cell receptor signaling molecules by western blotting analysis. In particular, valemetostat suppressed the expression level of BCL6 protein, a key oncogene in B cell lymphoma. Transcriptome analysis of 16 DLBCL cell lines using RNA sequencing suggested that tumor suppressor genes, DNA damage response related genes and cell cycle related genes were affected by valemetostat treatment. In particular, valemetostat down regulated c-myc signaling in valemetostat-sensitive cells. Valemetostat also demonstrated synergistic anti-tumor activity with standard of care therapy against a DLBCL cell line KARPAS-422 xenografted model. In conclusion, our results suggested that valemetostat has therapeutic activity in DLBCL cells by inhibiting B-cell receptor signaling and c-myc signaling pathway. A phase 1 clinical study of valemetostat mono-therapy is now ongoing in patients with non-Hodgkin lymphoma including DLBCL (Clinical trial information: NCT02732275). Disclosures Hama: Daiichi Sankyo Co., Ltd.: Employment. Banjo:Daiichi Sankyo Co., Ltd.: Employment. Honma:Daiichi Sankyo Co., Ltd.: Employment. Takata:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Yamamoto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Hirata:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Nakano:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Inaki:Daiichi Sankyo Co., Ltd.: Employment. Goto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Totoki:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kataoka:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Lim:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Wada:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kumazawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Tsutsumi:Daiichi Sankyo Co., Ltd.: Employment.

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is universally associated with Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) infection. The prognosis of PEL is extremely poor due to its aggressiveness and resistance to conventional chemotherapy. Therefore, the understanding of the molecular pathogenesis of PEL is vital to develop novel therapeutic strategies. PU.1, one of the Ets family transcription factors, has been reported to be down-regulated in some B-cell malignancies and acute myeloid leukemia. In this study, we examined the methylation of the PU.1 promoter and enhancer regions in PEL cell lines and performed the restoration of PU.1 in PEL cells in vitro and in vivo. Methods: PU.1 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). PU.1 was conditionally expressed using tet-on system in three PEL cell lines. The bisulfite sequencing was performed to analyze the methylation of the PU.1 promoter and enhancer regions in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used as an experimental tool for demethylation. The anti-proliferative activities of restoring PU.1 or 5-Azacytidine in PEL cell lines were measured by the methylthiotetrazole method. Apoptosis was quantified using Annexin V antibody. To evaluate the association of PU.1 with TRAIL promoter, we analyzed its recruitment to the promoter by luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. BCBL-1 cells that expressed tet-inducible PU.1 were injected intraperitoneally into NOD/Rag-2/Jak3double-deficient mice to assess the in vivo efficacy of restoring PU.1. Organ invasion by BCBL-1 cells was evaluated by immunohistochemistry. Results: The expression of PU.1 mRNA in PEL cell lines was weaker than in the human monoblast leukemia cell line, U937, which expresses PU.1 mRNA. The promoter and enhancer regions of the PU.1 gene were highly methylated in PEL cells. The treatment with 5-Azacytidine up-regulated the expression of PU.1 mRNA. Restoration of PU.1 or treatment with 5-Azacytidine inhibited cell proliferation. PU.1 up-regulated TRAIL gene expression among apoptosis-related genes and caused apoptosis. PU.1 induced the transactivation of TRAIL promoter in the reporter assay. Disruption of the PU.1 binding site repressed its transactivation. PU.1 was recruited to TRAIL promoter in the ChIP assay. Although PU.1 induced the master regulator of viral replication, ORF50 expression, the expression of ORF57, which is essential for virus production as a posttranscriptional activator, decreased. PU.1 suppressed ORF 57 by the occupancy of IRF7 in the ORF57 promoter region. Restoration of PU.1 inhibited the ascites formation and organ invasion significantly in vivo compared with control mice. Conclusions : The up-regulation of PU.1 induces growth inhibition and apoptosis via the direct transactivation of TRAIL and modulates viral transactivation via the up-regulation of IRF7. PU.1 is a tumor suppressor in PEL and its down-regulation has a critical role in PEL development. The targeting of PU.1 could be a therapeutic approach in patients with PEL. Disclosures No relevant conflicts of interest to declare.

Introduction Recently, the critical roles of CD47 on the surface of resistant cancer cells and its ligand, signal regulatory protein alpha (SIRPα), have been proposed in their evasion of immunosurveillance. Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced advanced acquired immunodeficiency syndrome (AIDS). The lack of optimal therapy combined with the aggressive nature of PEL results in a short median survival of less than 6 months. There is therefore a need for the development of new therapies. In this study, we transplanted PEL cells intraperitoneally into immunodeficient mice and evaluated the effect of targeting CD47-SIRPα signal with anti-CD47 antibody (Ab) and NOD-specific SIRPα on PEL. Methods Surface CD47 expressions of six PEL cell lines and peripheral blood mononuclear cells (PBMC) from six different donors were examined by flow cytometry. The antiproliferative activities of anti-CD47Ab against PEL cell lines were measured by the methylthiotetrazole (MTT) method. Efficacy of anti-CD47 Ab-mediated phagocytosis against PEL was evaluated using mouse peritoneal macrophages and human macrophages in vitro. In a direct xenograft mouse model, primary PEL cells were injected intraperitoneally into NOD/Rag-2/Jak3 double-deficient mice to assess the in vivo efficacy of anti-CD47 Ab. To clarify the effect of NOD-specific SIRPα on PEL, we compared the phagocytic activities of peritoneal macrophages against PEL and the in vivo engraftment of PEL among Rag-2/Jak3 double-deficient mice with NOD and non-NOD (Balb/c and C57/BL6) genetic backgrounds. Organ invasion by PEL cells was evaluated by immunohistochemistry and flow cytometry. Results Surface CD47 of PEL cell lines was highly expressed compared with that of PBMC. Anti-CD47 Ab did not have a direct antitumor effect against PEL cell lines. Treatment with anti-CD47 Ab promoted phagocytic activities of mouse peritoneal macrophages and human macrophages in vitro. In addition, the expression of CD47 was significantly correlated with the fold increase of phagocytic activity. Treatment with anti-CD47 Ab inhibited the ascites formation and organ invasion completely in vivo compared with control IgG-treated mice. Anti-CD47 Ab-treated mice seemed to stay in healthy and show no apparent change. Although the numbers of peritoneal macrophages were not different among Rag-2/Jak3 double-deficient mice with NOD and non-NOD genetic backgrounds, the phagocytic activity in NOD mice was significantly decreased compared with non-NOD mice. Consistent with the data of phagocytosis assay, the development of PEL cells in NOD mice was superior to those in non-NOD mice. Conclusions CD47 and SIRPα play the pivotal role in the immune evasion of PEL. NOD-specific SIRPα polymorphism has been reported to produce signals for macrophages not to engulf human cells. Thus, NOD-specific SIRPα is considered to contribute to the attenuation of macrophage phagocytosis and enable the development of PEL. CD47-SIRPα signal could be a therapeutic target for the immunotherapy of PEL. Disclosures: No relevant conflicts of interest to declare.