Impaired proteolysis of non-canonical RAS proteins drives clonal hematopoietic transformation [CRISPR]

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adaptor of a Cullin-3 RING E3 ubiquitin ligase complex responsible for degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations have been identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 which escape degradation, drive hematopoietic stem cell expansion (HSC) and leukemia in vivo. LZTR1 null cells rely on multiple RAS GTPases for transformation. Consequently, RAS targeting bioPROTACs or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data thereby reveal proteolysis of non-canonical RAS proteins as novel regulators of HSC function, define the function and spectrum of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation. Overall design: Genome-scale CRISPR/Cas9 screem performed in 3x different variants of the TF-1 cell line genetically-modified to express mutant forms of RIT1 (M90I or F82C) or depleted in LZTR1 by CRISPR/Cas9, then infected with the Brunello sgRNA library (in technical duplicate) and cells collected following 7 days of selection ("day 0") or an additional 14 days in culture ("day 14") to assess drop-out of sgRNAs between days 0 and day 14 by next-generation sequencing.