In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose deter-minants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identifi ed a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of -func-tion approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitu-tion, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defi ning the leukemic ELAVL1- mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized tar-get whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.SIGNIFICANCE: LSC-targeted therapies remain a signifi cant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism.