Uncoupling Ral signal transduction from Exocyst functions

Ras is the most mutated oncoprotein. FDA-approved inhibitors target a small subset of Ras mutants and two of the three oncogenic Ras signaling effectors, Raf>MEK>ERK and PI3K>PDK>AKT. Yet resistance to inhibitors remains a major challenge. The consequences of oncogenic Ras signaling via its third effector, RalGEF activation of the small GTPase Ral, are

much less well understood. The inhibitory RalGAP selective for Ral is a tumor suppressor and Ral is a proto-oncoprotein. Elevated activation of Ral is most prominently observed in pancreatic ductal adenocarcinoma (PDAC), the tumor type with the highest Ras mutation rate and poor survival. Molecular details of Ral signaling have confounded interpretation of its putative role in

cancer: Ral is an essential regulator of the exocyst complex and hence exocytosis but is also thought to use the exocyst as a signaling intermediary to promote oncogenesis. This dual function of Ral presents a fundamental knowledge gap that has impeded understanding of the role of the Ras>RalGEF>Ral signal in cancer: all genetic perturbations of Ral alter both signaling and

exocytosis. We have filled this gap using C. elegans as model to study Ral signaling and exocyst function. We successfully identified a separation-of-function missense mutation that disrupts Ral signaling but not exocytosis. We hypothesize that introducing this mutation into human RALA or RALB proteins will lock them into inactive form and abrogate KRAS signaling in

KRAS-positive PDAC cell lines while, critically, retaining exocyst functionality. Aim 1 will determine the molecular mechanism underlying missense separation of Ral functions. Structures of human Ral proteins suggest our missense mutation weakens a salt bridge required for proper guanine nucleotide binding. We will analyze the GDP/GTP cycle of wild type vs. mutant human

RALA/B in vitro and with real-time NMR with PDAC cells. We will also mutate the partner residue of the salt bridge to phenocopy the original mutation in C. elegans. Aim 2 will translate our findings from C. elegans into preclinical studies using a panel of KRAS-mutant PDAC cell lines with elevated RALA/B activity. We will complement disruption of the endogenous RALA and RALB

with wild-type vs. separation-of-function mutant RALA and RALB. We will test KRAS-driven traits of PDAC cells known to be dependent on RalGEF>Ral: proliferation, survival, anchorage- independent growth, and invasion will be the key endpoints examined. We have unlocked investigation of the third oncogenic effector of Ras, RalGEF>Ral. Substantiation of our

hypothesis is expected to translate a breakthrough genetic mutation into human cancer cells.