Our proposed studies address three FY20 PCARP Focus Areas, including "New drug development targeted toward cancer sensitivity and resistance mechanisms including immune mechanisms of resistance"; "Understanding the relationship between oncogenic signaling and the tumor microenvironment that drives drug resistance and therapeutic response"; and "Development of pharmacological, immunological, or genetic interception approaches". The rationale and directions for our proposed research are based on our recent identification and validation of a novel therapeutic approach for the treatment of KRAS oncogene-mutated pancreatic cancers. Since KRAS mutations are found in 95% of all pancreatic cancers, the potential clinical impact on treatment is enormous. Our studies identified a novel combination, concurrent treatment with an inhibitor of KRAS effector signaling (MEKi/ERKi) and the metabolic process autophagy (hydroxychloroquine/HCQ), that caused tumor regression in preclinical models of pancreatic cancer. We then leveraged these findings to initiate two ongoing phase 1/2 clinical trials for KRAS-mutant pancreatic cancer patients who have failed standard-of-care chemotherapy. Early reports of compassionate use treatment of pancreatic cancer patients identified some exceptional responders to this treatment. However, recognizing that improvements of this drug combination will be needed to achieve a long-term patient response, we now propose to develop second-generation combinations to achieve more potent inhibition of KRAS-driven metabolic processes.

Herein we propose an innovative experimental plan aimed at identifying new combination treatment approaches for future clinical trial evaluation. To identify novel targets, we propose the use of a genetic screen that utilizes a technology called CRISPR/Cas9, which was recognized with the 2020 Nobel Prize in Chemistry, and is a method of genome editing. We will construct a novel metabolism-focused screen that targets a set of ~2,500 metabolic genes, with a particular focus on genes that encode for proteins involved in nutrient scavenging pathways, such as autophagy. We will then perform inhibitor sensitization screens to determine genes that modulate ERK and autophagy inhibitor-induced antitumor activity. Importantly, rather than performing our screens in tissue culture dishes, we will perform them in vivo, as intact tumors, in an immune-competent mouse. Therefore, we will be able to model the true microenvironment and immune landscape of a pancreatic tumor. The validation of top hits from our in vivo CRISPR/Cas9 genetic loss-of-function metabolism screen will be performed using both patient-derived pancreatic cancer organoids, as well as patient-derived, orthotopic tumors and syngeneic, orthotopic mouse models of PDAC. As autophagy has been implicated in immune evasion, we will utilize specialized techniques to assess T cell activation and the intratumoral immune landscape. The utilization of both immune-competent mouse models, as well as human patient-derived tumor samples grown as organoids and as tumors in an immunosuppressed mouse will allow us to assess our top combinations as thoroughly as possible preclinically.

There is a dire need for new therapies for PDAC patients. Our recent studies identified a novel combination that caused tumor regression in preclinical models of PDAC, that we then leveraged to initiate two ongoing phase 1/2 clinical trials for KRAS-mutant PDAC patients. Thus, we have a track record of performing translational research. We hypothesize that combination inhibition of the cellular/biochemical mechanisms that drive elevated autophagy in PDAC will overcome acquired resistance mechanisms that will limit single-agent inhibition of autophagy. We now propose to develop second-generation combinations to achieve more potent inhibition of KRAS-driven metabolic processes. We anticipate that our genetic screens will identify multiple potential targets. We will prioritize those that already have pharmacological inhibitors developed, in order to expedite the potential positive impacts of this study on patients with pancreatic cancer. We are also aware that the field is in need of new novel directions to pursue. We hypothesize that the use of more advanced screening techniques will elucidate the most clinically important directions and therefore we will also validate mechanistically any novel combinations that we identify.