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 (LSCs) whose determinants are challenging to discern from hematopoietic stem cells (HSCs) or uncover by approaches focused on general cell properties. We have identified a set of RNA binding proteins (RBPs) 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-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution and selectively depleted primitive malignant vs. healthy cells. Integrative multiomics revealed differentiation, splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34 being a direct ELAVL1-stabilized target 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.