Defining the KRAS-regulated kinome in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer. * 2D : two-dimensional 3D : three-dimensional CRISPR : clustered regularly interspaced short palindromic repeats DDR1 : discoidin domain receptor 1 ERKi : ERK inhibition FBS : fetal bovine serum GTP : guanosine triphosphate GI50 : concentration at which growth is 50% of maximum IC50 : concentration at which the target is 50% inhibited MAPK : mitogen-activated protein kinase MEKi : MEK inhibition MIB/MS : multiplex kinase inhibitor beads/mass spectroscopy NS : non-specific PDAC : pancreatic ductal adenocarcinoma RPPA : reverse phase protein array RNAi : RNA interference RTK : receptor tyrosine kinase siRNA : small interfering RNA STRING : Biological database of protein-protein interactions WT : wild-type