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.