Annual Report 2022

Division of Hematological Malignancy

i)Chief: Issay Kitabayashi

ii)Staff Scientists: Kazutsune Yamagata, Takuo Katsumoto, Yutaka Shima

iii)Postdoctoral Fellowship: Haruka Shinohara

iv)Graduate student: Junpei Ito, Yoko Kuroki

v)Assisting Staffs: Yukiko Aikawa, Tomoko Shoji, Kimiharu Tauchi

Introduction

 Acute myelogenous leukemia (AML) is the most common leukemia in Japan and the U.S. With current standard chemotherapy, approximately 70% of adults with AML can be expected to attain complete remission status following appropriate induction therapy. However, many of the patients suffer a relapse of AML and only 25-30% of young adults and fewer than 10% of older patients survive longer than 5 years, suggesting presence of AML stem cells that is resistant to chemotherapy. Thus, AML stem cell eradication is thought to be crucial for curing AML. AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant AML stem cells. Selective targeting of the chemo-resistant population is a promising strategy for preventing and treating AML relapse.

The Team and What We Do

 Our research purpose is to establish new therapeutic methods by identifying molecular targets that are essential for maintaining hematological malignancies, especially AML stem cells.

Research Activities

 Disease-risk stratification and development of intensified chemotherapy protocols have substantially improved the outcome of acute lymphoblastic leukemia (ALL). However, outcomes of relapsed or refractory cases remain poor. Previous studies have discussed the oncogenic role of enhancer of zeste homolog 1 and 2 (EZH1/2), and the efficacy of dual inhibition of EZH1/2 as a treatment for hematological malignancy. We therefore investigated whether an EZH1/2 dual inhibitor, DS-3201 (valemetostat), has antitumor effects on B cell ALL (B-ALL). DS-3201 inhibited growth of B-ALL cell lines more significantly and strongly than the EZH2-specific inhibitor EPZ-6438, and induced cell cycle arrest and apoptosis in vitro. RNA-seq analysis to determine the effect of DS-3201 on cell cycle arrest-related genes expressed by B-ALL cell lines showed that DS-3201 upregulated CDKN1C and TP53INP1. CRIPSR/Cas9 knockout confirmed that CDKN1C and TP53INP1 are direct targets of EZH1/2 and are responsible for the antitumor effects of DS-3201 against B-ALL. Furthermore, a patient-derived xenograft (PDX) mouse model showed that DS-3201 inhibited the growth of B-ALL harboring MLL-AF4 significantly. Thus, DS-3201 provides another option for treating B-ALL.

 Monocytic leukemia zinc finger protein (MOZ, MYST3, or KAT6A) is a MYST-type acetyltransferase involved in chromosomal translocation in acute myelogenous leukemia (AML) and myelodysplastic syndrome. MOZ is established as essential for hematopoiesis; however, the role of MOZ in AML has not been addressed. We propose that MOZ is critical for AML development induced by MLL-AF9, MLL-AF10, or MOZ-TIF2 fusions. Moz-deficient hematopoietic stem/progenitor cells (HSPCs) transduced with an MLL-AF10 fusion gene neither formed colonies in methylcellulose nor induced AML in mice. Moz-deficient HSPCs expressing MLL-AF9 were significantly reduced in terms of colony and cell numbers. Moz-deficient HSPCs expressing MOZ-TIF2 could form colonies in vitro but could not induce AML in mice. By contrast, Moz was dispensable for colony formation by HOXA9-transduced cells and AML development caused by HOXA9 and MEIS1, suggesting a specific requirement for MOZ in AML induced by MOZ/MLL fusions. Expression of the Hoxa9 and Meis1 genes was decreased in Moz-deficient MLL fusion-expressing cells, while expression of Meis1, but not Hoxa9, was reduced in Moz-deficient MOZ-TIF2 AML cells. AML development induced by MOZ-TIF2 was rescued by introducing Meis1 into Moz-deficient cells carrying MOZ-TIF2. Meis1 deletion impaired MOZ-TIF2-mediated AML development. Active histone modifications were also severely reduced at the Meis1 locus in Moz-deficient MOZ-TIF2 and MLL-AF9 AML cells. These results suggest that endogenous MOZ is critical for MOZ/MLL fusion-induced AML development and maintains active chromatin signatures at target gene loci.

 Malignant rhabdoid tumors (MRTs) are rare and highly aggressive pediatric cancers with no standard treatment. MRTs are characterized by loss of SMARCB1, which results in upregulated expression of enhancer of zeste homolog 2 (EZH2), which is responsible for the methylation of lysine 27 of histone H3 (H3K27me3), leading to the repression of gene expression. Although previous reports suggest EZH2 as an effective therapeutic target, the functions of EZH1, the other homolog of EZH, in MRT remain unknown. We showed that EZH1, as well as EZH2, contributes to MRT cell growth and H3K27 methylation. Depletion or selective inhibition of EZH2 led to a compensatory increase in EZH1 expression, and depletion of EZH1 enhanced the effect of EZH2 inhibition. EZH1/2 dual inhibitors suppressed MRT cell growth markedly, reflecting the reduction of H3K27me3 accumulation at one of the EZH1/2 targets, the CDKN2A locus. Dual inhibition of EZH1/2 in vivo suppressed tumor growth completely, with no significant adverse effects. These findings indicate that both EZH1 and EZH2 are potential targets for MRT therapy, and that EZH1/2 dual inhibitors may be promising therapeutic strategies for MRT.

Education

 One post-doctoral fellow and two graduate students were trained for research.

Future Pospects

 Based on our research, we are developing anti-cancer therapies for cancer patients.

List of papers published in 2022

Journal

1. Ito J, Yamagata K, Shinohara H, Shima Y, Katsumoto T, Aikawa Y, Kitabayashi I. Dual inhibition of EZH1/2 induces cell cycle arrest of B cell acute lymphoblastic leukemia cells through upregulation of CDKN1C and TP53INP1. International journal of hematology, 117:78-89, 2023

2. Shinohara H, Sawado R, Nakagawa M, Hattori A, Yamagata K, Tauchi K, Ito J, Kuwahara Y, Okuda T, Ogawa C, Kitabayashi I. Dual targeting of EZH1 and EZH2 for the treatment of malignant rhabdoid tumors. Molecular therapy oncolytics, 27:14-25, 2022

3. Nakagawa M, Yamaguchi M, Endo M, Machida Y, Hattori A, Tanzawa F, Tsutsumi S, Kitabayashi I, Kawai A, Nakatani F. Clinical usefulness of 2-hydroxyglutarate as a biomarker in IDH-mutant chondrosarcoma. Journal of bone oncology, 34:100430, 2022

4. Katsumoto T, Ogawara Y, Yamagata K, Aikawa Y, Goitsuka R, Nakamura T, Kitabayashi I. MOZ is critical for the development of MOZ/MLL fusion-induced leukemia through regulation of Hoxa9/Meis1 expression. Blood advances, 6:5527-5537, 2022