A two-step in vivo CRISPR screen unveils pervasive RNA binding protein dependencies for leukemic stem cells and identifies ELAVL1 as a therapeutic target. A two-step in vivo CRISPR screen unveils pervasive RNA binding protein dependencies for leukemic stem cells and identifies ELAVL1 as a therapeutic target

Acute myeloid leukemia (AML) progression and relapse is fueled by self-renewing leukemic stem cells (LSCs) whose molecular determinants have been difficult to discern from normal hematopoietic stem cells (HSCs) or to uncover in screening approaches focused on general AML cell properties. We have identified a unique set of RNA binding proteins (RBPs) that are enriched in human AML LSCs but repressed in HSCs. Using an in vivo two step CRISPR-Cas9-mediated screening approach to specifically score for cancer stem cell functionality, we found 32 RBPs essential for LSC propagation and self- renewal in MLL-AF9 translocated AML. Using knockdown or small molecule approaches we show that targeting key hit RBP ELAVL1 impaired LSC-driven in vivo leukemic reconstitution and selectively depleted primitive AML cells vs. normal hematopoietic stem and progenitors. Importantly, knockdown of Elavl1 spared HSCs while significantly reducing LSC numbers across genetically diverse leukemias. Integrative RNA-seq and eCLIP-seq profiling revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with the mitochondrial import protein TOMM34 being a direct ELAVL1-stabilized target whose inhibition impairs AML propagation. Overall design: Human primary AML cells (AML #4) were infected in triplicate with shScramble or shELAVL1 lentivirus (pLKO.1) at an MOI of 50. At 48h, 100k 7AAD-EGFP+ transduced cells were FACS-sorted per sample and RNA was extracted using TRIzolLS.

急性髓系白血病（Acute myeloid leukemia, AML）的进展与复发由具有自我更新能力的白血病干细胞（leukemic stem cells, LSCs）驱动，但其分子决定簇难以与正常造血干细胞（normal hematopoietic stem cells, HSCs）相区分，也难以通过聚焦于普通AML细胞特性的筛选方法加以揭示。本研究鉴定出一组独特的RNA结合蛋白（RNA binding proteins, RBPs），它们在人AML LSCs中富集，却在HSCs中被抑制。我们采用体内两步CRISPR-Cas9介导的筛选方法，专门针对癌症干细胞功能进行评分，在携带MLL-AF9易位的AML中鉴定出32个对LSC增殖与自我更新至关重要的RBPs。通过敲低或小分子干预手段，我们证实靶向关键筛选命中的RBPs ELAVL1可损害LSC驱动的体内白血病重建能力，并选择性清除原始AML细胞，而非正常造血干祖细胞。值得注意的是，敲低Elavl1不会影响HSCs，却可显著降低多种遗传异质性白血病中的LSC数量。整合RNA测序（RNA-seq）与增强交联免疫沉淀测序（enhanced crosslinking and immunoprecipitation sequencing, eCLIP-seq）的分析结果显示，造血分化、RNA剪接与线粒体代谢是定义白血病ELAVL1-mRNA互作组的关键特征；其中线粒体导入蛋白TOMM34是ELAVL1直接稳定的靶标，抑制该靶标可损害AML的增殖能力。整体实验设计：将原代人AML细胞（AML #4）以多重感染复数（multiplicity of infection, MOI）为50的条件，分别用短发夹RNA对照（shScramble）或靶向ELAVL1的shRNA慢病毒（pLKO.1载体）进行三次重复感染。感染48小时后，通过流式细胞术分选（FACS）获取每例样本中10万个7-氨基放线菌素D（7-AAD）阴性、EGFP阳性的转导细胞，随后使用TRIzol LS试剂提取RNA。