Biology, Therapy, and Resistance of Myeloid Malignancies Initiated by Hyperactive Ras.

Ras proteins regulate cell fates by cycling between active GTP-bound and inactive GDP-bound states (Ras·GTP and Ras·GDP). Ras·GTP modulates cell fates by activating effector pathways that include the Raf/MEK/ERK, phosphoinositol 3’-kinase (PI3K)/Akt, and Ral·GDS cascades. Signaling terminates when Ras·GTP is hydrolyzed to Ras·GDP. GTPase activating proteins (GAPs) are negative regulators of Ras output that increase the rate of GTP hydrolysis. Mammalian cells express two major GAPs – p120GAP and neurofibromin. The latter is encoded by the NF1 tumor suppressor gene, which is mutated in persons with neurofibromatosis type 1 (NF1). Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and other cancers. Somatic RAS mutations are also common in myeloid malignancies, and other leukemia-associated mutations, such as FLT3 internal tandem duplications, PTPN11 point mutations, and the BCR-ABL fusion protein deregulate Ras signaling. Together, the prevalence of oncogenic RAS mutations and the existence of these alternative genetic mechanisms establish hyperactive Ras as a major therapeutic target. However, developing inhibitors of oncogenic Ras proteins is extremely challenging due to structural considerations and because an effective drug must restore normal biochemical activity (i.e., repair a broken enzyme). Strains of mice carrying conditional mutant alleles of Nf1, oncogenic Kras, and oncogenic Nras are novel reagents for understanding how cells remodel signaling networks in response to hyperactive Ras and for performing pre-clinical trials. Use of the Mx1-Cre transgene to ablate Nf1 or to activate oncogenic KrasG12D or NrasG12D expression in hematopoietic cells causes myeloproliferative disorders (MPDs) that model JMML and chronic myelomonocytic leukemia (CMML). We are using retroviral insertional mutagenesis (RIM) to identify cooperating mutations that might induce progression from MPD to acute myeloid leukemia (AML). CI-1040, a potent inhibitor of MEK, unexpectedly had no beneficial effects in Nf1 mutant mice with MPD. By contrast, MEK inhibition induced regression of Nf1-deficient AMLs. These AMLs uniformly developed resistance in vivo, despite equivalent biochemical inhibition of the target in paired sensitive and resistant clones. Analysis of retroviral insertions in resistant AMLs revealed outgrowth of a pre-existing clone during CI-1040 administration, and we have implicated RasGRP1 and p38α as modulating resistance in vivo. These data emphasize the importance of cell context in the response to targeted agents and establish a tractable in vivo system for identifying genes that modulate therapeutic efficacy and for probing mechanisms of acquired resistance.