PTBP1 controls oncogenic transcript processing and maintenance of acute myeloid leukemia.

RNA-binding proteins are critical regulators of gene expression in both normal physiology and cancer. Here we set out to systematically annotate the functions of RNA-associated proteins across multiple cancer types via domain-focused CRISPR screens targeting RNA-modifying enzymes and RNA-binding proteins. We utilized 3182 sgRNAs targeting 527 RNA-enzymatic and binding domains in 341 RNA-associated proteins and identified multiple RNA-binding proteins as dependencies in acute myeloid leukemia (AML), including the RNA splicing factor PTBP1 and the N6-methyladenosine reader RBM15, with a bias toward the aggressive KMT2A-rearranged (KMT2A-r) subtype of AML. Genetic and cellular validation confirmed all four RNA-binding domains of PTBP1 as required for KMT2A-r AML proliferation. In contrast to the lack of requirement for PTBP1 in myelopoiesis, PTBP1 suppression in AML caused cell cycle arrest, apoptosis, and induction of myeloid differentiation programs. Transcriptomic analysis revealed that PTBP1 loss disrupted the KMT2A-r-essential transcriptional program and caused widespread dysregulation of splicing. CLIP-seq analysis further identified that PTBP1 preferentially binds to a subset of transcripts critical for KMT2A-r AML proliferation, including IKZF1, MEF2C, EZH2, SIK3, and PBX3. Collectively, these findings demonstrate that PTBP1 supports AML proliferation by fine-tuning the expression and splicing of AML-essential genes, providing a workflow for systematically annotating RNA-associated protein dependencies in cancer.