Abstract A25: In vivo evidence validating the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic cancers

A dynamic cycle of palmitoylation and depalmitoylation stably localizes N-Ras, but not K-Ras-4b, at the plasma membrane. This selective dependency suggests that targeting palmitoylation could inhibit the growth of NRAS-mutant cancers while leaving normal cells unaffected, due to their intact K-Ras-4b function. We sought to clarify the dependency of oncogenic N-Ras signaling on palmitoylation in a model relevant to human cancer. We used the lox-STOP-lox (LSL) knock-in system (Haigis et al., Nat Genet 2008) to express an NrasG12D oncogene at physiologic levels, and generated a second site mutation abrogating the palmitoylation site (C181S). We then compared Mx1-Cre; NrasLSL-G12D,C181S/LSL-G12D,C181S to Mx1-Cre; NrasWT/WT and Mx1-Cre; NrasLSL-G12D/LSL-G12D (hereafter G12D,C181S, WT, and G12D, respectively). We injected mice with poly(I:C) at weaning to excise the LSL cassette in hematopoietic cells. As reported (Wang et al., Blood 2011; Xu et al., Cancer Disc 2013), G12D mice died early (119-267 days, n=14) from an aggressive myeloproliferative disorder (MPD). In contrast, no mortality was observed in the G12D,C181S and WT cohorts until 18-19 months of age (p=3x10-6 by Kaplan-Meier analysis). Pathologic analysis of 6-month-old mice showed that key features of MPD were fully rescued in the G12D,C181S cohort. Specifically, whereas white blood cell (WBC) counts and spleen sizes were in the normal range in G12D,C181S and WT mice, G12D mice had profound leukocytosis (median WBC count: 52.19 k/μL), splenomegaly (median spleen weight: 0.884 g), and myeloid expansion (n=6). Biochemical analysis revealed that N-RasG12D,C181S accumulated in the GTP-bound conformation, but mostly localized in the cytosol, and failed to hyperactivate the MAPK pathway. We next generated NrasG12D,C181S/G12D compound heterozygous mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S mutation in vivo. Over half of these mice developed hematologic disease and died prematurely, although later than homozygous G12D mutant mice (median survivals: 429 and 225 days; n = 22 and 14, respectively). Common findings in moribund mice were splenomegaly, expansion of myeloid cells, and different degrees of leukocytosis and anemia. Importantly, genetic analysis from diseased mice revealed reduced frequency or absence of the NrasG12D,C181S allele (allelic burden 0-30%) due to secondary somatic genetic events. This observation supports the existence of high selective pressure to overcome growth-suppressive properties of unpalmitoylated N-Ras during leukemogenesis. In conclusion, these studies provide strong genetic evidence validating palmitoylation as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model for addressing this question in other cancers. Citation Format: Noemi A. Zambetti, Ari J. Firestone, Jasmine C. Wong, Amanda M. Long, Anagha Inguva, Jarrett R. Remsberg, Radu M. Suciu, Benjamin F. Cravatt, Kevin M. Haigis, Kevin Shannon. In vivo evidence validating the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A25.