Abstract A111: Understanding the interplay between ULK1/2 signaling and the RAS MAPK pathway

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is dependent on mutant KRAS and elevated autophagy for growth. Previously, we and others demonstrated that pharmacological inhibition of the KRAS ERK-MAPK pathway caused increased autophagic flux. Targeting this increased autophagic flux with inhibitors that block autophagic processing led to synergistic growth suppression in several preclinical models of PDAC when combined with RAS pathway inhibition. Hence, concurrent inhibition of the RAS and autophagy pathways represents an attractive therapeutic combination. However, the only FDA-approved autophagy inhibitor, hydroxychloroquine (HCQ), is limited by low potency and specificity. This motivated us to evaluate other therapeutic strategies to block autophagy in PDAC. The Unc-51-like autophagy activating serine/threonine kinases 1 and 2 (ULK1/2) are essential for the initiation of autophagosome biogenesis, making ULK1/2 potential anti-autophagy therapeutic targets. Selective ATP-competitive ULK1/2 inhibitors have been developed and have shown pre-clinical efficacy in combination with RAS inhibition in non-small cell lung cancer (NSCLC). Using a panel of ULK1/2 inhibitors with RAS ERK-MAPK inhibitors, we showed that in contrast to previous results in NSCLC, the combination is antagonistic in most PDAC cell lines and organoids. Using a combination of reverse phase protein array (RPPA), proteomics, and phosphoproteomics, we identified robust activation of ERK and RSK signaling as a primary signaling consequence of ULK1/2 inhibition across multiple, chemically distinct inhibitors. Upregulation of ERK/RSK signaling occurs rapidly in response to ULK inhibition and upregulation persists for days. We hypothesize that upregulated ERK/RSK signaling is responsible for the antagonism observed between ULK and RAS inhibition in PDAC. Ongoing experiments are aimed at further delineating the mechanism that leads to increased ERK/RSK signaling upon ULK1/2 inhibition. Additionally, following kinase prediction analysis of our phosphoproteomics dataset, we found that ULK1/2 inbition resulted in the downregulation of novel, unexpected kinases that are associated with other endocytic processes, including, leucine rich repeat kinase 2 (LRRK2), adaptor protein 2-associated kinase 1 (AAK1), BMP-2 inducible kinase (BIKE/BMP2K), and Cyclin G-assoicated kinase (GAK). This points to ULK1/2 having a limited kinome, and instead using other kinases to affect cellular processes. We hypothesize that LRRK2, AAK1, BIKE, and/or GAK could represent novel anti-autophagy targets in the context of PDAC.