Abstract IA20: Targeting hyperactive Ras signaling in acute leukemia
Authors: Shannon Kevin M. 
Published: May 2016

Ras proteins are molecular switches that cycle between active guanosine triphosphate (GTP) and inactive guanosine diphosphate (GDP)-bound conformations (Ras-GTP and Ras-GDP). Guanine nucleotide exchange factors (GNEFs) stimulate nucleotide exchange on Ras, resulting in increased Ras-GTP levels. Upon GTP binding, the switch I and switch II domains of Ras undergo a conformational change that allows them to interact productively with downstream effectors including Raf, phosphatidylinositol 3-kinase (PI3K), and Ral-GDS to activate kinase signaling cascades that regulate cell proliferation, differentiation, and survival. Mutational, biochemical, and cell biologic studies of human cancers and experiments in mouse models strongly implicate deregulated signaling through the PI3K/Akt/mTOR and Raf/MEK/ERK cascades in cancer initiation and maintenance. Ras-GTP is hydrolyzed to Ras-GDP through an intrinsic GTPase activity. This slow “off” reaction is greatly augmented by GTPase activating proteins (GAPs). Thus, the competing activities of GNEFs and GAPs regulate Ras-GTP levels in vivo. Neurofibromin, the protein encoded by NF1, and p120 GAP are the predominant Ras GAPs in mammalian cells. Codons 12, 13, and 61 of RAS genes are the most common targets of dominant mutations in human cancer, with NRAS codon 12 mutations predominating in AML. Substitutions in these residues result in constitutively elevated levels of Ras-GTP due to reduced intrinsic GTP hydrolysis and resistance to GAPs. RAS and NF1 mutations occur in pediatric patients with a spectrum of hematologic malignancies including juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL).

Because oncogenic Ras proteins are exceedingly challenging biochemical targets, most recent drug discovery efforts have focused on inhibiting downstream signaling molecules such as Raf, MEK, and Akt with many compounds. We have engineered and exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human acute leukemias and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable acute leuekmias characterized by hyperactive Ras signaling. This system provides a novel in vivo forward genetic strategy for introducing cooperating mutations and for generating clonal heterogeneity. Leukemia cells can be manipulated ex vivo and transplanted into congenic recipients. This experimental flexibility provides an opportunity to establish cohorts of mice that are engrafted with the same primary cancers for conducting preclinical testing and for investigating mechanisms of intrinsic and acquired drug resistance.

I will discuss biologic and preclinical therapeutic studies in which we treated primary Nras, Kras, and Nf1 mutant AMLs characterized by hyperactive Ras signaling with targeted agents in vivo, isolated drug resistant clones at relapse, and compared drug sensitive and resistant clones to discover genes that modulate resistance. This latter strategy represents a potent and unbiased strategy to address mechanisms of resistance to kinase inhibitors, which has emerged as a major clinical problem in cancer therapeutics, and for investigating therapeutic strategies that might be effective in human cancers in which oncogenic RAS is a driver mutation. Our results in these robust AML models support testing drug combinations that include Raf/MEK/ERK pathway inhibitors and other targeted and conventional agents.

Citation Format: Kevin M. Shannon. Targeting hyperactive Ras signaling in acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA20.