KRAS mutations are drivers of oncogenesis, and historically have been considered ?undruggable.? Consequentially, therapeutic approaches have focused on downstream effectors of the mitogen-activated protein kinase (MAPK) pathway, including RAF, MEK, and ERK?though no approach has led to an effective drug for KRAS-driven disease. However, genetic studies strongly support that the MAPK signaling pathway is a critical dependency in KRAS-mutant cancers; so why do current MAPK drugs fail? Recent studies suggest that these drugs are effective inhibitors of MAPK enzyme activity, yet fail due to feedback mechanisms that rely on protein-protein interactions (PPIs) to maintain MAPK signaling even in the presence of drug. My overarching hypothesis is that current MAPK inhibitors are limited by their inability to effectively regulate critical PPIs among MAPK components. To test this hypothesis, I will alter critical PPIs by modulating a MAPK scaffold termed Kinase Suppresor of Ras (KSR). In contrast to previous MAPK targets, KSR is a pseudokinase that lacks catalytic activity, but serves as a scaffold protein to promote RAF and MEK binding. Our group showed that a lead compound termed APS-2-79 binds to KSR2 at the ATP binding site and synergizes with MEK inhibitors (MEKi) in KRAS-driven cell lines by impeding KSR?s interaction with RAF. While useful as a tool for biochemical studies, APS-2-79 has several limitations including modest affinity and selectivity for KSR. Moreover, the mechanism of KSR inhibitor (KSRi) synergy with MEKi in KRAS mutant cell lines is not known, and may be the consequence of off target kinase inhibition instead of direct KSR targeting. To investigate the mechanism of KSRi synergy with MEK inhibitors, I aim to test the dependence of KSRi synergy in KRAS mutant cells on KSR using genetic tools. I hypothesize that the synergy observed between MEKi and KSRi in KRAS mutant cells is dependent on the availability of KSR?s ATP-binding pocket. To test this hypothesis, I will use lentiviral vectors to overexpress KSR, +/- mutations in the ATP binding pocket known to prevent compound binding. To further explore the importance of KSR in mediating RAS-MAPK signaling, I also aim to induce targeted KSR degradation with small-molecule PROteolysis TArgeting Chimeras (PROTACs) These small molecule tools simultaneously bind their protein targets and E3 ubiquitin ligases, allowing for ubiquitination of the target and downstream proteolysis. I hypothesize that PROTAC mediated KSR1 degradation will mimic genetic deletion studies supporting the importance of KSR1 for oncogenic KRAS.
My aims outline genetic and pharmacological approaches to investigate KSR inactivation as a mechanism to exploit crucial PPIs within the KRAS-driven MAPK signaling pathway. Critical to this study are the development of potent and specific next-generation KSRi analogs and precise genetic tools for target validation. Ultimately this training proposal nurtures skills in synthetic chemistry, assay development, drug discovery, and target validation, which will be broadly applicable to my future goals as a physician scientist.

Public Health Relevance

A large number of cancer deaths are due to KRAS mutant cancers; our lab was the first to show that the inhibitor APS-2-79 exploits protein-protein interactions within the KRAS-driven MAPK signaling pathway, and synergizes with MEK inhibitors to decrease KRAS-mutant cell viability. APS-2-79 is purported to target Kinase Suppressor of Ras (KSR), though the mechanism of action in cancer cells remains unclear. Here we propose genetic methods to test the purported mechanism of APS-2-79, as well as a novel strategy to target KSR for degradation in KRAS mutant cell lines with chemical probes that exploit the cell?s own proteasomal machinery.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Damico, Mark W
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Icahn School of Medicine at Mount Sinai
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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