ALBERT

Description: Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such as Ebf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered by conditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions of these genes are downregulated in the AML cells and increased by deletion of IDH2/R140Q. Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN that hydroxylates and marks HIF1α for ubiquitin-proteasomal degradation, it is probable that mutant IDH2-produced 2HG stabilizes HIF1α through inhibition of EGLN. Furthermore, it was reported that Meis1 activates the transcription of HIF1α. In consistent with these information, we showed that IDH2/R140Q increased the protein levels of HIF1α in cultured cells. Disclosures Matsunaga: Daiichi Sankyo Co., Ltd.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Acute myeloid leukemia (AML) is a clonal malignant disorder originating from a small number of leukemic stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of LSCs is a promising strategy for the prevention and treatment of AML relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the stemeness of ES cells and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) is a catalytic component of PRC2 that trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Mutations and overexpression of EZH1/2 are associated with cancers including hematopoietic malignancies. Here, we used genetic deletion of EZH1/2 or a novel dual inhibitor of EZH1/2 activity to show that loss or inhibition of EZH1/2 eradicates dormant AML stem cells. To examine the effects of genetic deletion of EZH1/2 on AML cells, Ezh1-null, Ezh2-conditional and double knock-out mice were generated. Hematopoietic stem/progenitor cells prepared from single knock-out mice or double knock-out mice were transduced with various types of AML fusion-genes, such as MOZ-TIF2, MLL fusions, AML1-ETO, and others by retroviral infection, and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. When the cells were cultured in vitro, double deletion of Ezh1/2 induced cell differentiation and apoptosis more severely than single deletions in all subtypes of AML tested, resulting in complete loss of cells. In AML mice, deletion of Ezh1/2 induced AML cell differentiation and complete remission of AML, which was not achieved by single deletion of Ezh1 or Ezh2. Genetic deletion of both Ezh1 and Ezh2 on the LSC fraction dramatically reduced the number of LSCs (Lin-c-Kit+ CD16/32+ CD34+, L-GMP), especially quiescent LSCs, whereas deletion of either Ezh1 or Ezh2 did not have such a strong effect. The transcriptional profiles of LSCs deficient in both Ezh1 and Ezh2 were characterized by the upregulation of cell cycle-related genes such as Cyclin D1/D2, which is the main regulator of G0/G1 transition, along with differentiation-related genes. These results suggested that deletion of both Ezh1 and Ezh2 is required for eradication of LSCs. To investigate whether pharmacologic inhibition of EZH1/2 could serve as a therapeutic strategy in AML, we developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1 and EZH2. The drug induced cell differentiation and apoptosis in most subtypes of AML tested in vitro and its effects were similar to those of genetic depletion of EZH1/2. A selective EZH2 inhibitor did not affect the growth and survival of AML cells to the same extent as the dual inhibitor. Oral administration of the EZH1/2 dual inhibitor reduced the number of LSCs effectively in AML mice in a manner similar to the effect of genetic deletion of EZH1/2. Taken together, these results strongly suggest that dual inhibition of EZH1 and EZH2 is a promising therapeutic strategy to eradicate LSCs in a wide range of AMLs, which could lead to important advances in the treatment of AML. Disclosures Honma: Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Enhancer of zests homologous (EZH)1 and its close homolog EZH2 are component 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 tri-methylation of H3K27 has been associated with lymphoma and myeloma progression, suggesting that PRC2 is a therapeutic target for hematological malignancies. Selective EZH2 inhibitors induce compensatory activation of EZH1 which in turn re-activates PRC2 function. We hypothesized that dual inhibition of EZH1 and EZH2 is more effective than selective EZH2 inhibition as anti-tumor therapy. We have developed a novel EZH1 and EZH2 dual inhibitor DS-3201, which simultaneously inhibited the enzymatic activity of EZH1 and EZH2 in nano-molar concentration. DS-3201 showed anti-proliferative effect against various NHL cells, such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and peripheral T-cell lymphoma, with GI50 values less than 100 nM regardless of EZH2 gain-of-function mutations. DS-3201 also induced differentiation of undifferentiated NHL cells with increment of cell lineage specific markers and which induced cell death in vitro. DS-3201 also showed synergistic effect with the standard of care agents for NHL in vitro and in vivo. An open-label phase 1 clinical study was initiated to examine the safety and pharmacokinetics of multiple-dose monotherapy of DS-3201b which is the salt form of DS-3201 in patients with NHL (ClinicalTrials.gov Identifier: NCT02732275). Eighteen patients with relapsed or refractory NHL were enrolled. The patients received oral administration of DS-3201b once daily in a 28-day cycle at dose of 150, 200 and 300 mg. Preliminary efficacy results (D. Maruyama, et al. ASH 2017), showed that the overall response rate was 58.8% (10/17) with 1 complete response and 9 partial responses (PR). Thirteen NHL patients including five follicular lymphoma (FL) and one DLBCL were analyzed for gene mutation status by targeted gene sequencing. EZH2 mutation was detected only in one FL patient, who achieved PR. It was suggested that DS-3201b has clinical activity against NHL, regardless of the mutation status of EZH2. Disclosures Honma: Daiichi Sankyo: Employment. Nosaka:Daiichi Sankyo: Employment. Shiroishi:Daiichi Sankyo: Employment. Takata:Daiichi Sankyo: Employment. Hama:Daiichi Sankyo: Employment. Yamamoto:Daiichi Sankyo RD Novare: Employment. Adachi:Daiichi Sankyo: Employment. Maruyama:Mundipharma International: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Dai-ichi-Sankyo: Honoraria; Bristol-Myers Squibb: Honoraria; Biomedis International: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Fujifilm: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Dai-Nippon-Sumitomo: Honoraria; Asahi Kasei Pharma: Honoraria; AstraZeneca: Research Funding; Solasia Pharma: Research Funding; Pfizer: Research Funding; Nippon Boehringer Ingelheim: Research Funding; Novartis: Research Funding; Otsuka: Research Funding; Amgen Astellas BioPharma: Research Funding; Astellas Pharma: Research Funding; Abbvie: Research Funding; GlaxoSmithKline: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding. Tobinai:Chugai Pharma: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Eisai: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SERVIER: Research Funding; Abbvie: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Zenyaku Kogyo: Consultancy, Honoraria; HUYA Bioscience International: Consultancy, Honoraria. Ishida:Kyowa Hakko Kirin Co.Ltd: Honoraria, Research Funding; Celgene K.K: Honoraria, Research Funding; Bayer AG: Research Funding; Mundiparma K: Honoraria. Kusumoto:Bristol-Myers Squibb: Honoraria, Research Funding; Chugai Pharmaceutical Co. Ltd: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Tsukasaki:Daiich-Sankyo: Consultancy; Ono Pharma: Consultancy; HUYA: Consultancy, Research Funding; Chugai Pharma: Honoraria, Research Funding; Eisai: Research Funding; Celgene: Honoraria; Mundy Pharma: Honoraria; Kyowa-hakko/Kirin: Honoraria; Seattle Genetics: Research Funding. Fujioka:Daiichi Sankyo: Employment. Watanabe:Daiichi Sankyo: Employment. Kanno:Daiichi Sankyo: Employment. Kumazawa:Daiichi Sankyo RD Novare: Employment. Fujitani:Daiichi Sankyo: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo: Employment.

Description: Introduction: Enhancer of zeste homolog 2 (EZH2) and EZH1 are alternative subunits of polycomb repressive complex 2 that catalyze the tri-methylation of lysine 27 residue of histone H3. This histone modification epigenetically regulates gene expression and may play an important role in tumor progression. Valemetostat (DS-3201) is a potent and highly specific orally bioavailable dual inhibitor of EZH2 and EZH1 demonstrating anti-tumor activity against various hematological malignancies in preclinical studies (Honma D, et al. Cancer Sci 2017; Fujita S, et al. Leukemia 2018). Valemetostat demonstrated clinical activity as a novel oral therapeutic option for both B-cell and T-cell NHLs in the interim analysis of a phase I study (Maruyama D, et al. ASH 2017). Here we report the updated results of this phase I study focusing on ATL patients. ATL is a peripheral T-cell malignancy caused by human T-cell leukemia virus type I (HTLV-1), and is divided into aggressive and indolent subtypes. Aggressive ATL has an extremely poor prognosis, with a median survival time of only 8 to 10 months (Katsuya H, et al. Blood 2015). Although conventional chemotherapeutic agents, anti-CCR4 antibody mogamulizmab, and oral immunomodulator lenalidomide have been used for treatment of aggressive ATL patients, most patients eventually become resistant to treatment. In addition, allogenic hematopoietic stem cell transplantation is not available for elderly patients. Therefore, new therapeutic options are urgently needed. Methods: This ongoing open-label, single-arm phase I study consists of the dose escalation part (NHLs including ATL and peripheral T-cell lymphoma [PTCL]) and the expansion part (ATL and PTCL). The drug was administered orally once daily (QD) continuously over 28-days (1 cycle) until disease progression or intolerance. Results: Thirty-eight patients (15 females) with a median age of 69 (44-88) were enrolled in this study as of the data cut-off of 24 January 2019. Of 38 patients, 25 patients were enrolled in the dose-escalation part, and additional 13 patients (7 ATLs and 6 PTCLs) were treated in the expansion part with 200 mg of valemetostat. In all patients, adverse events (≥30%) on treatment with all grades included; platelet count decreased (73.7%), dysgeusia (52.6%), anemia (42.1%), lymphocyte count decreased (39.5%), neutrophil count decreased (39.5%), and white blood cell count decreased (39.5%). Preliminary efficacy was based on investigator's assessment with an objective response rate (ORR) of 47.2%. Fifteen patients were able to stay on valemetostat for more than 24 weeks with tumor shrinkage. Of the 9 ATL patients in the study (2 in dose escalation and 7 in dose expansion), baseline characteristics are as follows: age, median age 74 (range 61-78 yrs); sex, 7 males and 2 females; ATL subtype, 6 acute- and 3 lymphoma-subtypes; median number of prior therapies, 2 (range 1-8). Adverse events (≥30%) on treatment with all grades in the 9 ATL patients included; platelet count decreased (77.8%), dysgeusia (66.7%), neutrophil count decreased (44.4%), white blood cell count decreased (44.4%), anemia (33.3%), alopecia (33.3%), and dry skin (33.3%). No grade 4 and 5 adverse events were detected. Grade 3 adverse events included; white blood cell count decreased (33.3%), platelet count decreased (22.2%), neutrophil count decreased (22.2%), lymphocyte count decreased (11.1%), and anemia (11.1%), which are consistent with those in all population. Responses for the 9 ATL patients included; 1 unconfirmed complete remission (CRu), 3 partial remission (PR), and 3 stable disease (SD) (ORR = 44.4%; 4/9). Of the 6 prior mogamulizumab treated patients, 3 patients demonstrated a response (ORR = 50%; 3/6). Five of 9 ATL patients continued on valemetostat treatment for more than 12 weeks with tumor shrinkage (Figure). Four ATL patients continue on treatment. The latest study results will be presented. Conclusion: The updated results of this ongoing phase I study showed that the oral EZH1/2 dual inhibitor valemetostat has demonstrated acceptable safety and promising preliminary efficacy in NHL. In addition, the results of ATL patients, including mogamulizumab pretreated patients, showed promising clinical activity. A subsequent pivotal phase II study for ATL will soon be initiated. Clinical trial information: NCT02732275 Disclosures Ishitsuka: Novartis: Honoraria, Research Funding; sanofi: Honoraria; Celgene: Honoraria; Astellas Pharma: Honoraria, Research Funding; Pfizer: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria; Janssen Pharmaceutical: Honoraria; Shire: Honoraria; Eisai: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Mochida: Honoraria, Research Funding; Shire: Honoraria; Teijin Pharma: Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Genzyme: Honoraria; Genzyme: Honoraria; Eisai: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Teijin Pharma: Research Funding; MSD: Research Funding; Yakult: Research Funding; Asahi kasei: Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Eli Lilly: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; mundiharma: Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Alexion: Honoraria; Mochida: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; mundiharma: Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Ono Pharmaceutical: Honoraria, Research Funding; Yakult: Research Funding; Bristol-Myers Squibb: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Pfizer: Honoraria; Alexion: Honoraria; sanofi: Honoraria. Izutsu:Eisai, Chugai, Zenyaku: Honoraria; Chugai, Celgene, Daiichi Sankyo, Astra Zeneca, Eisai, Symbio, Ono, Bayer, Solasia, Zenyaku, Incyte, Novartis, Sanofi, HUYA Bioscience, MSD, Astellas Amgen, Abbvie, ARIAD, Takeda, Pfizer: Research Funding; Celgene: Consultancy; Eisai, Symbio, Chugai, Zenyaku: Research Funding; Kyowa Kirin, Eisai, Takeda, MSD, Chugai, Nihon Medi-physics, Janssen, Ono, Abbvie, Dainihon Sumitomo, Bayer, Astra Zeneca, HUYA Japan, Novartis, Bristol-Byers Squibb, Mundi, Otsuka, Daiichi Sankyo, Astellas, Asahi Kasei: Honoraria. Kusumoto:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding. Araki:Daiichi Sankyo: Employment. Adachi:Daiichi Sankyo: Employment. Yamashita:Daiichi Sankyo: Employment. Atsumi:Daiichi Sankyo: Employment. Tsukasaki:Eisai: Research Funding; Mundi Pharma: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Huya: Consultancy, Honoraria, Research Funding; Byer: Research Funding; Kyowa Kirin: Honoraria; Ono Pharmaceutical: Consultancy; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Consultancy. Tobinai:Eisai: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Takeda Pharmaceutical: Consultancy, Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Zenyaku Kogyo: Consultancy, Honoraria; Meiji Seika: Honoraria; Verastem: Honoraria; Solasia: Honoraria; Janssen Pharmaceutical: Honoraria, Research Funding; Yakult: Honoraria; AbbVie: Research Funding; Ono Pharmaceutical: Consultancy, Honoraria, Research Funding; Mundi Pharma: Consultancy, Honoraria, Research Funding; HUYA Bioscience: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria.

Description: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma and is refractory to currently available combination chemotherapy. The unfavorable prognosis results from an inadequate understanding of how diseases are caused and maintained in human T-cell leukemia virus type I (HTLV-1)-infected individuals. To date, direct comprehensive analyses of leukemic cells have identified the intrinsic molecular hallmarks of ATL. Among these, polycomb group (PcG)-mediated epigenetic disruption has been known to be a crucial characteristic of ATL (Yamagishi et al., Cancer Cell, 2012). However, no attempt has been made to determine the global epigenomic status explaining the deregulated gene expression pattern specific to ATL. In this study, we performed integrative analyses of epigenome (n=3) and transcriptome (n=58) of primary ATL patient cells and corresponding normal CD4+ T cells to decipher the ATL-specific 'epigenetic-code' that was critical for cell identity. We found that PcG-mediated tri-methylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at over half of genes (53.8%) in ATL cells, the pattern of which appears distinct from other cancer types and PcG-dependent cell lineages such as ES cells and peripheral T lymphocytes. Large proportion of the abnormal gene downregulation occurred at an early stage of disease progression and was explained by the H3K27me3 accumulation. The global H3K27me3 alterations were involved in determination of key genes such as miR-31, CADM1, EVC1/2, CDKN1A, and NDRG2, which are essential for ATL cell survival and other cellular characteristics. In addition, PcG generated diverse outcomes by the remote regulation of a broad spectrum of gene regulators, including various transcription factors, miRNAs, epigenetic modifiers, and developmental genes. Thus, the emerging epigenomic landscape is a fundamental characteristic of ATL. Although EZH2 mutations conferring gain-of-function were undetected in ATL (0/50; 0%), EZH2 level was significantly upregulated and inversely correlated with H3K27me3 targets, indicating that the global alteration of H3K27me3 mark depends on the abundance of EZH2 and other core components of the polycomb repressive complex 2 (PRC2). We found that EZH2 was sensitive to promiscuous signaling network including NF-kB pathway and was functionally affected by HTLV-1 Tax through both NF-kB activation and direct association. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, the majority of epigenetic silencing has occurred in leukemic cells from indolent type ATL and also in HTLV-1-infected T cells from asymptomatic HTLV-1 carriers. Collectively, our results unveiled that epigenetic reprogramming arises at an early stage of ATL development. Tracing the epigenetic marks and expression patterns in samples obtained from patients of various diagnostic categories, as well as in other biologically relevant models has supported the efficacy and relevance of targeting PRC2. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, permitting favorable treatments. According to the expression profiling, EZH2 may represent the first-choice as a druggable target. In addition, peripheral T cells highly express EZH1 that compensates for the EZH2 functions. We found that simultaneous depletion of the two H3K27me3 writers EZH1 and EZH2 significantly diminished cellular H3K27me3 level and dramatically inhibited ATL cell growth compared with single depletion, suggesting that the compensatory actions of EZH1/2 may be critical for ATL. To selectively eliminate the ATL and HTLV-1-infetced populations, we have developed a novel EZH1/2 dual-inhibitor that shows strong inhibitory effects for both of EZH1 and EZH2. Treatment with the new drugs showed significant inhibitory effects on the ATL cell survival derived from patients. Interestingly, the dual-inhibitor reversed the epigenetic disruption and selectively eliminated leukemic and immortalized cells from HTLV-1 infected individuals. Collectively, this approach will achieve potent and selective synthetic lethality by targeting the regulators of H3K27me3 in aggressive and indolent ATL cells, as well as in clonally expanded infected cells, improving medical care and the prevention of disease onset. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Epigenetic program is a molecular basis of cellular identity and functions. We have shown the transcriptome (Cancer Cell, 2012) and the underlying histone methylation patterns (Blood, 2016) of adult T-cell leukemia-lymphoma (ATL). The epigenetic landscape of ATL is defined as "genome-wide H3K27me3 accumulation". Similar accumulation has been detected in multiple cancers, particularly in hematological malignancies. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, leading to a promising treatment. Due to variation characteristics, EZH2 is recognized as a druggable target. However, extrinsic regulation of methylation pattern is still challenging without knowledge of molecule(s) and their combination that needs to be targeted to reprogram the epigenome in anticipation of synthetic lethality. To redefine the conventional concept, we first examined expression and global occupancy patterns of H3K27 methyltransferases EZH1 and EZH2. EZH2 expression was low in CD4+ T cells and significantly high in ATL. In contrast, basal EZH1 was very high in mature lymphocytes compared with undifferentiated lineages and also high in ATL. ChIP-on-chip analysis revealed significant changes in distributions of both EZH1 and EZH2 in primary ATL cells, which caused abnormal H3K27me3 accumulation. Importantly, over 80% of H3K27me3 are specifically induced by either EZH1 (28.9%) or EZH2 (14.4%), or by both (39.3%). Moreover, they have differential functions by regulating specific targets; EZH2 suppresses various transcription regulators, establishing complex gene regulatory network. EZH1 would rather directly induce H3K27me3 at genes involving functional processes such as lymphocyte activation. We detected EZH1 and EZH2 in functional polycomb repressive complex 2 (PRC2) in ATL and DLBCL cells. Unexpectedly, EZH1/2 were co-localized with H3K4me3 at a small population of the lymphoma genome. The "EZH1/2 + H3K4me3" non-canonical targets such as cell cycle activators were highly expressed in ATL. We next compared functions of EZH1/2. Single depletion of EZH1 or EZH2 suppressed T- and B-lymphoma cell proliferation, which supports their unique functions. The broad reduction of H3K27me3 was achieved by EZH2 inhibition; however, genome-wide ChIP demonstrated that EZH2 inhibition or depletion triggered strong compensatory action of EZH1, followed by rebound accumulation of H3K27me3 at many functional genes in EZH1+ lymphoma cells. We found that a transcription factor YY1 affects PRC2 recruitment and EZH1 compensation after EZH2 inhibition. Double depletion of EZH1+EZH2 or YY1+EZH2 surmounted the epigenetic homeostasis. In addition, depletion of EZH1 significantly enhanced effects of EZH2 inhibitors in ATL and DLBCL cells (5.47~205-fold). These data indicate requirements for the aberrant epigenome, namely, fluctuating EZH2 (upregulation or mutation) and underlying stable EZH1. We have succeeded in developing an unprecedented EZH1/2 dual-inhibitor (IC50 9.0 nM (EZH1); 10 nM (EZH2)). The new drug successfully inhibited genome-wide EZH1/2 occupancies without compensation, resulting in effective H3K27me3 reduction and gene reactivation compared with the single inhibitor. Of note, the dual-inhibitor suppressed expression of the non-canonical EZH1/2 + H3K4me3 target genes. Besides H3K27me3 reprogramming, EZH1/2 inhibition could modulate genes through H3K4me3 regulation and indirect influence on the secondary regulatory network and signaling pathways such as NF-kB. The EZH1/2 dual-inhibitor significantly blocked primary ATL cell survival and in vivo tumor growth of ATL and DLBCL models. Furthermore, we evaluated drug efficacy in various models of hematological malignancies including ATL, DLBCL (EZH2 WT and mutant), Burkitt lymphoma, and PTCL, which are all originated from EZH1+ differentiated lymphocytes. All of them showed higher sensitivity against the EZH1/2 inhibitor compared with the EZH2 solo inhibitor (5.2~200-fold). We did not detect strong adverse effects in normal T cells. These integrated molecular and phenotypic analyses provide a novel concept "EZH1+EZH2 dual-targeting" and demonstrate validity of the new drug in the treatment of malignant lymphomas. Taking these results, a Phase I clinical trial against T- and B-cell non-Hodgkin lymphomas including ATL is now under way in Japan. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Ohsugi:Daiichi Sankyo Co., Ltd.: Research Funding. Utsunomiya:Daiichi Sankyo Co., Ltd.: Speakers Bureau. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding. Tobinai:Zenyaku Kogyo: Honoraria; Takeda: Honoraria, Research Funding; Mundipharma KK: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Celgene: Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; SERVIER: Research Funding; Ono Pharmaceutical: Research Funding; Eisai: Honoraria, Research Funding; Chugai Pharma: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; HUYA Bioscience: Honoraria. Araki:Daiichi Sankyo Co., Ltd.: Employment.