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造血幹・前駆細胞において変異型ASXL1はミトコンドリアの機能異常とROSによるDNA損傷を惹起する

藤野, 赳至 東京大学 DOI:10.15083/0002002459

2021.10.15

概要

ASXL1(Addition of Sex Combs Like1)遺伝子の体細胞変異は、骨髄異形成症候群(Myelodysplastic syndromes; MDS)、慢性骨髄単球性白血病(Chronic myelomonocytic leukemia; CMML)、骨髄増殖性疾患(Myeloproliferative neoplasms; MPN)、および急性骨髄性白血病(Acute myelogenous leukemia; AML)を含む骨髄系腫瘍でしばしば検出される。ASXL1変異単独では白血病の発症は誘導されないが、腫瘍発症の過程で最も早期に起こる事象の一つであることが示唆されている。さらに、近年の報告においてASXL1変異は加齢に伴うクローン性造血(Clonal hematopoiesis; CH)で認められ、造血器腫瘍の発症リスクが上昇することが明らかとなっている。しかしながら、ASXL1変異がどのように白血病の発症に寄与しているのかはあまり理解されていない。本研究では、C末端を欠失した変異型ASXL1(ASXL1-MT)を造血器特異的に発現するコンディショナルノックイン(cKI)マウスの解析を行い、ASXL1-MTが造血幹・前駆細胞(Hematopoietic stem and progenitor cells; HSPC)に及ぼす影響について検討した。

 ASXL1-MTcKIマウスでは、多能性前駆細胞(Multipotent progenitors; MPPs)や⻑期造⾎幹細胞(Long-term hematopoietic stem cells; LT-HSCs)を含む、HSPC分画の細胞が著明に減少していた。競合的移植実験より、ASXL1-MTcKIマウスではHSPCの造⾎再構築能が低下していることが⽰された。アポトーシスの検討により、ASXL1-MTは主にアポトーシスを誘導することによって、HSPCの減少を引き起こすことが⽰唆された。さらに、細胞周期の解析では静⽌期にあるHSCの減少が認められ、造⾎再構築能の低下に寄与している可能性が考えられた。次に、ASXL1-MTを発現するHSPCの機能障害の原因を、ミトコンドリアにおける代謝という観点から解析した。ASXL1-MTを発現するHSPCでは、ミトコンドリアの膜電位が上昇するとともに酸素消費量が増加していた。また、メタボローム解析によりLSK(Lin-Sca1+c-kit+)細胞の代謝産物を網羅的に解析すると、ATPやTCAサイクルを構成する代謝産物のプールが増加していた。さらに、RNAシークエンス解析では、ミトコンドリア関連遺伝⼦の発現が有意に上昇していた。以上の結果から、ASXL1-MTを発現するHSPCではミトコンドリアの⽣合成が増加し、好気的代謝によるエネルギー産⽣が亢進していることが明らかとなった。

 ミトコンドリアは活性酸素(Reactive oxygen species; ROS)の主要な産⽣源であり、その増加によってDNA損傷が引き起こされることが知られている。ミトコンドリアの活性化から予測される通り、ASXL1-MTを発現するHSPCではROSとDNA損傷が増加していることが分かった。抗酸化物質であるN-アセチルシステイン(N-acetylcystein; NAC)を投与すると、ASXL1-MT発現細胞のROSとDNA損傷は減少し、造⾎再構築能が回復することが明らかとなった。したがって、ASXL1-MTcKIマウスにおけるHSPCの機能低下は、ROSの増加とそれに伴うDNA損傷が原因であると考えられた。

 DNA損傷を引き起こすような遺伝⼦発現の変化を探索するため、RNAシークエンス解析のデータを参照したところ、ASXL1-MTを発現するLSK細胞では転写因⼦であるFoxo1とそのターゲット遺伝⼦の発現が低下していることが分かった。ASXL1-MTを有する⾻髄Lin-細胞にFoxo1を過剰発現させて移植すると、γ-H2AXの発現が抑制されるとともに、⽣着が向上することが明らかとなった。当研究室の過去の知見より、ASXL1-MTcKIマウスではH3K4me3が著明に減少することが明らかとなっている。また、ChIPシークエンス解析では、ASXL1の結合部位とH3K4me3に強い相関が認められることから、ASXL1-MTがH3K4トリメチル化を阻害することが示唆されている。Foxoの遺伝子座について調べたところ、ASXL1-MTが結合するとともに、H3K4me3の著明な減少が確認された。したがって、ASXL1-MTはH3K4トリメチル化の阻害を通じてFoxoの発現を減少させ、HSPCの機能を低下させていると考えられた。まとめると、ASXL1変異によるDNA損傷の増加と造⾎再構築能の低下は、H3K4me3の減少によるFoxo1の発現低下が⼀因となっていることが⽰唆された。

 本研究の結果は、ASXL1変異が新たな変異を促進することを⽰唆しており、ASXL1変異が⾻髄系腫瘍の発症過程で早期に起こる事象である理由を説明し得る可能性がある。また、ASXL1変異を有するCHや⾻髄系腫瘍の治療に、新たな知⾒を与えるものと考えられる。

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参考文献

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