Concurrent Inhibition of The RAS Pathway and Autophagy As a Treatment Approach for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of the KRAS-RAF-MEK-ERK pathway enhanced autophagic flux and dependency in several PDAC models. Furthermore, we demonstrated that concurrent treatment with the only FDA-approved autophagy inhibitors chloroquine/hydroxychloroquine (CQ/HCQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. However, CQ is limited in terms of specificity and potency, and we therefore aimed to identify more efficacious anti-autophagy strategies. To this end, we performed a CRISPR-Cas9 loss-of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a gene necessary for PDAC proliferation. PIKfyve is a lipid kinase that is essential for the production of PI(3,5)P2, and critical for the proper regulation of lysosome recycling. We evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition also impaired autophagy as demonstrated by robust accumulation of autophagy-related proteins and significantly impaired autophagic flux. This was likely due to impaired lysosome function, demonstrated by accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation and lack of normal acidity. We next showed that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC.

Learning Objectives:

• Identify the effects of PIKfyve inhibition on autophagic flux and tumor growth in pancreatic cancer.
• Analyze the efficacy of combining PIKfyve inhibitors with RAS or ERK-MAPK pathway inhibitors in KRAS-mutant PDAC.
• Describe the mechanisms through which PIKfyve inhibition enhances cell cycle arrest and apoptosis in PDAC cells.