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.

Automated summary

Acute myeloid leukemia (AML) is driven by leukemic stem cells (LSC) and identifying the determinants of LSCs is challenging. This study identified a group of RNA-binding proteins (RBPs) enriched in human AML LSCs. Through a stem cell-adapted CRISPR screen, they found 32 essential RBPs for LSCs and discovered that ELAVL1, an RBP, plays a crucial role in AML propagation by regulating mitochondrial metabolism. These findings highlight the importance of RBPs as therapeutic targets in AML.