Splicing regulatory dynamics for precision analysis and treatment of heterogeneous leukemias.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations remains poorly understood. Prompted by the finding that splicing uniquely resolved genetic subtypes of cancer, we developed an unsupervised computational workflow called OncoSplice to comprehensively define tumor molecular landscapes. In adult and pediatric acute myeloid leukemia (AML), OncoSplice identified the spectrum of driver genetics from splicing profiles alone, defined more than a dozen previously unreported molecular subtypes recurrent across AML cohorts, and discovered a dominant splicing subtype that partially phenocopies U2AF1-mutant splicing. Although pediatric leukemias lack splicing factor mutations, this U2AF1-like subtype similarly spanned pediatric and adult AML genetics and consistently predicted poor prognosis. Using long-read single-cell RNA sequencing, we confirmed that discovered U2AF1-like splicing was shared across cell states, co-opted a healthy circadian gene program, was stable through relapse, and induced a leukemic stem cell program. Pharmacological inhibition of an implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia missplicing and inhibited leukemic cell growth. Finally, genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treatment, blocked leukemia development in xenograft models and induced differentiation. This work suggests that broad splicing dysregulation, in the absence of select mutations, is a therapeutic target in heterogeneous leukemias.