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311 Durable Clinical Impacts and Mechanisms of Action and Resistance in EZH1/2-Targeting Epigenetic Therapy

Program: Oral and Poster Abstracts
Type: Oral
Session: 621. Lymphomas: Translational—Molecular and Genetic: Single Cell, Microenvironment, and Treatment Resistance
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Lymphomas, Translational Research, non-Hodgkin lymphoma, T Cell lymphoma, Diseases, aggressive lymphoma, Lymphoid Malignancies
Saturday, December 10, 2022: 5:00 PM

Makoto Yamagishi, PhD1*, Yuta Kuze2*, Makoto Nakashima, Ph.D.1*, Seiichiro Kobayashi, M.D., Ph.D.3, Satoko Morishima, M.D., Ph.D.4*, Toyotaka Kawamata, M.D., Ph.D.5*, Junya Makiyama, M.D., Ph.D.6*, Kazumi Abe2*, Kiyomi Imamura2*, Eri Watanabe7*, Kazumi Tsuchiya7*, Isao Yasumatsu8*, Gensuke Takayama, PhD9*, Kazumi Ito, D.V.M., Ph.D.10*, Yasuhito Nannya, M.D., Ph.D.5, Arinobu Tojo, M.D., Ph.D.11, Toshiki Watanabe, M.D., Ph.D.12*, Shinji Tsutsumi, Ph.D.10*, Yutaka Suzuki, Ph.D.13* and Kaoru Uchimaru, M.D., Ph.D.1*

1Laboratory of Tumor Cell Biology, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan
2Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
3Department of Hematology, Kanto Rosai Hospital, Kanagawa, Japan
4Division of Endocrinology, Diabetes and Metabolism, Hematology and Rheumatology, Second Department of Internal Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
5Department of Hematology/Oncology, IMSUT Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
6Department of Hematology, Sasebo City General Hospital, Nagasaki, Japan
7IMSUT Clinical Flow Cytometry Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
8Organic Synthesis Department, Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan
9Translational Science I, Daiichi Sankyo Co., Ltd., Shinagawa-Ku, Japan
10Translational Science I, Daiichi Sankyo Co., Ltd., Tokyo, Japan
11Tokyo Medical and Dental University, Tokyo, Japan
12Department of Advanced Medical Innovation, St. Marianna University Graduate School of Medicine, Kanagawa, Japan
13Laboratory of Systems Genomics, Department of Computational Biology and Medical Sciences, The University of Tokyo, Tokyo, Japan


Epigenomes allow the rectification of disordered gene expressions, thereby providing new targets for pharmacological interventions in cancer therapy. The clinical utility of targeting histone H3 lysine tri-methylation (H3K27me3) as an epigenetic hallmark has been demonstrated. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumor cells remain unclear.

Results and Discussion:

We conducted intensive molecular monitoring of three patients enrolled in the valemetostat first-in-human phase 1 clinical trial (NCT02732275) for adult T-cell leukemia/lymphoma (ATL). Valemetostat was administered orally once daily (200 mg/day) until a sign of disease progression could be observed. The number of abnormal lymphocytes (Ably) in these three patients drastically decreased after one week of treatment. All patients could stay on valemetostat single agent for more than 2 years with acceptable safety and durable response. PBMC samples (n = 55) were collected from these three patients immediately before (Pre) and during treatment, as well as during the progressive disease (PD) stage.

Flow cytometry-based histone methylation assay demonstrated that tumor H3K27me3 level was generally high in the Pre-samples, but the valemetostat treatment reduced it to a normal level in all patients. In addition, ChIP sequencing (ChIP-seq) could directly assess how valemetostat affects the tumor epigenome. The pre-treatment tumor cells showed an overall increasing trend in H3K27me3. For all H3K27me3 peaks (51,683 peaks), 85.9% of the peak signals decreased at the average fold-change of 0.742 after the valemetostat treatment. Focusing on the gene loci, an overall decrease could be observed around the TSS and across the gene body. The characteristic H3K27me3 clusters initially observed in the tumor cells were demethylated.

We also conducted integrative multilayered single-cell analyses (scATAC-seq and scRNA-seq) and showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, such as tumor suppressor genes (TSGs), in all patients. These results demonstrated that valemetostat sufficiently restored the epigenome of the tumor cells close to the healthy state, leading to clinical improvements.

Although all tested cases showed a durable response for more than 2 years, these responses were eventually interrupted due to the recurrence of ATL. We identified characteristic somatic mutations of EZH2 during clonal repopulation at PD. The identified amino acid substitutions were all located at the interface between EZH2 and valemetostat. Direct evaluation of the H3K27me3 level in the PD sample harboring the acquired EZH2 mutation showed that tumor cells in a hypermethylated state were repopulating. The scATAC-seq analysis also showed strong chromatin compaction of the same gene set occurred in the rapidly propagating resistant clone, indicating that the clonally selected mutations should be responsible for resistance.

In patients free of the EZH2 resistant mutations, we detected TET2 mutation or elevated DNMT3A expression in the propagated resistant clones. Genome-wide DNA methylation analysis revealed increased DNA methylation near the TSS in the resistant clones. The genes characterized by the H3K27me3-related chromatin condensation were restored by valemetostat treatment but were again strongly re-condensed by mCpG gain at PD. Consequently, the gene regulation of several important TSGs restored by valemetostat was repressed again. Furthermore, in the single-cell platform, we successfully identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations.


This study illustrates, for the first time, the molecular and cellular dynamics in patients in response to a molecular-targeting inhibitor designed for histone methyltransferases. Eliminating H3K27me3 leads to the reprogramming of the cancer epigenome, thereby exerting a sustained benefit on tumors. Targeting chromatin homeostasis might provide opportunities for further sustained epigenetic therapies.

Disclosures: Yamagishi: Astellas Pharma Inc.: Honoraria; Bristol Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Honoraria, Research Funding. Nakashima: Takeda Pharmaceutical Co., Ltd,: Honoraria, Research Funding. Morishima: Meiji Seika: Honoraria; Daiichi Sankyo: Honoraria; Takeda: Honoraria; Kyowa Kirin: Honoraria; Sanofi: Honoraria; Chugai Pharma: Honoraria; Bayer: Honoraria; Nippon Shinyaku: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Pfizer: Honoraria. Yasumatsu: Daiichi Sankyo RD Novare Co., Ltd: Current Employment. Takayama: Daiichi Sankyo Co., Ltd: Current Employment. Ito: Daiichi Sankyo Co., Ltd: Current Employment. Nannya: Chugai Pharmaceutical: Speakers Bureau; Pfizer: Speakers Bureau; Takeda Pharmaceutical Company: Speakers Bureau; Filgen: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceutical: Speakers Bureau; Astrazeneca: Speakers Bureau; Sumitomo Pharma: Speakers Bureau; Daiichi Sankyo Co., Ltd: Research Funding; Fuji Pharma: Honoraria; Kyowa-Kirin: Speakers Bureau; Asahi Kasei Pharma: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Daiichi Sankyo RD Novare: Research Funding; Otsuka Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Tojo: KM Biologics: Research Funding; Novartis Japan: Honoraria; Otsuka Pharmaceutical: Consultancy, Honoraria; Sysmex: Consultancy, Research Funding. Tsutsumi: Daiichi Sankyo Co., Ltd: Current Employment. Suzuki: Daiichi Sankyo Co., Ltd: Research Funding. Uchimaru: Kyowa Kirin Co., Ltd: Honoraria; Takeda Pharmaceutical Co., Ltd,: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Meiji Seika Pharma Co., Ltd.: Honoraria.

*signifies non-member of ASH