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HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis

Takahashi, Satoshi 京都大学 DOI:10.14989/doctor.k23803

2022.03.23

概要

MLL融合遺伝子はHOXA9やMEIS1などの様々な造血幹細胞特異的な遺伝子群を恒常的に活性化させて造血前駆細胞の異常な自己複製を引き起こし白血病の原因となる。しかし MLL 融合遺伝子蛋白が標的遺伝子を恒常的に活性化させるメカニズムはまだ不明な部分が多い。

MLL 融合遺伝子蛋白が標的クロマチンに結合する機序は解明されてきた。MLL はMENIN、LEDGF と三者複合体と結合し、H3K36m2/m3 と結合する。また、MLL は MLL内のCXXC を通して非メチル化CpG 領域と結合する。MLL はAF4 family 蛋白やENLfamily 蛋白と融合遺伝子産物を形成する。これらの MLL 融合遺伝子産物は融合遺伝子産物側でAEP(AF4 family/ENL family/P-TEFb)複合体を形成する。同AEP 複合体は ribosomal RNA 転写に寄与する SL1 を recruit し結合して MLL 融合遺伝子産物によるRNAP2 の活性化に寄与する。

今回、MLL 内に進化上、保存された領域であるtrithorax homology domain2(THD2)がMLL 融合遺伝子による白血病化に寄与することを同定した。新しくTHD2 を含むプラスミドを正しく作成し同領域の真の結合因子として質量分析によりHBO1複合体(HBO1, PHF16, MEAF6, ING4/5)を同定した。HBO1 複合体の各因子は MLL 標的遺伝子のプロモーター上でMLL やRNAP2 と共局在することをChIP-seq で確認した。また各MLL融合遺伝子により不死化した細胞群や白血病細胞株はHBO1依存的な増殖を示した。次にMLL をKO したHEK293T 細胞を用いたChIP-qPCR 法によりMLL がMYC やCDKN2C 遺伝子上でHBO1、AEP、SL1 複合体を促進させていることが示唆された。

さらにHBO1複合体とMLLが結合し協調して白血化する一例としてNUP98 融合遺伝子に注目した。NUP98-HBO1 融合遺伝子産物は不死化しMLL 依存的な増殖を示した。また HBO1 をHBO1 複合体の構成因子であるMEAF6、ING5 へ置換したNUP98 融合遺伝子産物も MLL と結合し不死化することを示した。これらの結果から MLL 複合体がHBO1複合体と結合し協調してNUP98融合遺伝子を不死化させることが示唆された。

MLL-ELL の不死化は THD2 依存性が高く、MLL-ELL の不死化の分子学的機序に関しても詳細に解明することになった。ELL には転写伸長領域と occludin homologydomain (OHD)などが存在する。ELL-OHD はAF4 family 蛋白、EAF1、TP53 などの結合が知られていた。MLL-ELL の白血化に関連する分子として重要な分子の特定をする目的で ELL-OHD 部位にアミノ酸変異を導入し各分子の結合能の変化や不死化能を評価した。変異体 ELL-S600A-K606T は AF4、TP53、EAF1 とのクロマチン上での結合能を低下させるが不死化能、白血化能は担保される変異体であった。これまでの研究から、MLL 融合遺伝子における白血化においてMLL 融合遺伝子がAEP 複合体、SL1 複合体を順次recruit し、RNAP2 の活性化を促進することを研究、主張してきた背景を鑑みて(Nature Communications 2015)、MLL-ELL 融合遺伝子産物における白血病化においても OHD を介した AEP 複合体の recruitment が重要であろうことがと示唆された。

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