UCLA

Expressing the oncogenic mutant form of Kras in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting Kras signaling plays a dominant role in normal hematopoiesis. However, the oncogenic mutant is insensitive to signal-terminating GTPase-activating proteins, resulting in unabated signal which is not encountered in normal physiology. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Kras ex3op), in which silent mutations in exon 3 render

the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that KRAS protein levels were significantly increased in bone marrow (BM) HSCs in Kras ex3op/ex3op mice, demonstrating that the translation of KRAS in HSCs is normally constrained by rare codons. Kras ex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 22 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in ERK1/2 phosphorylation and CDK4/6 activation. Furthermore, both the enhanced colony forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op were dependent on CDK4/6 activation. Finally, BM transplantation studies revealed that augmented KRAS produced expansion of HSCs, progenitor cells and B cells in a hematopoietic cell-autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence.