RNA splicing factor mutations drive myeloid neoplasm oncogenesis through protein complex poisoning

Summary RNA splicing factor mutations (SF mut ) are founding oncogenic events which cause RNA splicing errors with unpredictable gene expression dynamics. Despite extensive transcriptomic studies, SF mut cancer-initiating mechanisms remain elusive. Among SF mut cancers, myeloid neoplasms (MN) alone offer a setting where true cancer-driving SF mut stem cells can be identified, namely the hematopoietic stem cell (HSC). Within a cohort of 62 MN patients and 20 healthy donors, we conducted long/short-read single-cell transcriptomics (10X-ONT, n = 21) and immunophenotype-resolved low-cell proteomics ( pauciproteomics, n = 78) to resolve gene expression, RNA splicing and protein expression dynamics across healthy and SF mut MN hematopoiesis. During hematopoietic differentiation, SF mut RNA mis-splicing decouples proteotranscriptomic dynamics in a mutation-specific manner. Pauciproteomics defines the functional effects of SF mut RNA mis-splicing on the protein layer, identifying early-stage protein effects which poison entire functional protein complexes and progressively disrupt the proteome-wide network during cellular maturation. Importantly, this information could neither be resolved nor predicted by transcriptomic analyses. Finally, we functionally validate the proteome dynamics of HSC through induced pluripotent stem cell culture models of early hematopoietic differentiation, identifying candidate mechanisms for SF mut oncogenesis. Overall, these data elucidate SF mut MN disease biology with unprecedented granularity, defining the molecular consequences of SF mut RNA mis-splicing and offering a generalizable framework for cancer stem cell studies.