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4049 Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Monday, December 11, 2023, 6:00 PM-8:00 PM

Eijiro Furukawa1*, Hiroki Kato1*, Sayaka Sano1*, Yan Yan2*, Daigo Michimata1*, Yuya Tanaka1*, Kazuki Sakurai1*, Koichi Onodera3*, Satoshi Ichikawa1*, Noriko Fukuhara4, Yasushi Onishi1*, Hisayuki Yokoyama1*, Daisuke Saigusa5*, Tohru Fujiwara6, Kazuhiko Igarashi7* and Hideo Harigae1

1Department of Hematology, Tohoku University Graduate School of Medicine, Sendai, Japan
2Tohoku university, SENDAI, Japan
3Tohoku University Graduate School, Sendai, Japan
4Department of Hematology and Rheumatology, Tohoku University School of Medicine, Sendai, Japan
5Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma-Science, Teikyo University, Tokyo, Japan., Tokyo, JPN
6Department of Laboratory Medicine, Iwate Medical University School of Medicine, Shiwa-gun, Iwate, Japan
7Tohoku University Scool of Medicine, Sendai, JPN

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo.

By using Mx1-Cre mediated Mat2a gene deletion in mice (Mat2afl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis (Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 (Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 (Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway (Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required.

Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

Disclosures: Onodera: Otsuka: Honoraria; Novartis: Honoraria; Nippon Shinyaku: Honoraria; Celgene: Research Funding; BMS: Research Funding; Astellas: Honoraria; Agios: Research Funding; Abbie: Research Funding. Ichikawa: AstraZeneca: Research Funding; BeiGene: Research Funding. Fukuhara: Eisai: Honoraria; Janssen: Honoraria; BMS: Honoraria; Incyte: Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; HUYA: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Research Funding; Nippon Shinyaku: Honoraria; Meiji Seika: Honoraria; Nihon kayaku: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ono Pharmaceuticals: Honoraria; SymBio Pharmaceuticals: Honoraria; Loxo Oncology: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Genmab: Honoraria, Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharma: Honoraria, Research Funding; Bayer: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Onishi: Pfizer: Honoraria; Novartis: Honoraria; Janssen: Honoraria; AsahiKASEI: Honoraria; Abbvie: Honoraria; Chugai: Honoraria; BMS: Honoraria; Amgen: Honoraria; Kyowa Kirin: Honoraria; Symbio: Honoraria; Astellas: Honoraria; MSD: Honoraria; Meiji Seika: Honoraria, Research Funding; Incyte: Research Funding; Sumitomo: Honoraria; Daiichi Sankyo: Honoraria; Nippon Shinyaku: Honoraria; IQVIA: Honoraria. Yokoyama: Astellas: Honoraria. Harigae: Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Novartis Pharma: Honoraria; Sumitomo Pharma: Research Funding; Chugai Pharma: Honoraria, Research Funding; Asahi Kasei Pharma: Research Funding; Kyowa Kirin: Research Funding.

*signifies non-member of ASH