Abstract 4803: BMAL2 is a KRAS-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma

Abstract

Abstract To identify novel drivers of PDA malignancy, we employed a regulatory network analysis, which accurately infers the activity of transcription factors and other regulatory proteins based on the integrated expression of their positive and negative target genes. This highly validated approach enables the identification of the most hyper-activated and hyper-repressed regulatory proteins (i.e. the “master regulators (MRs)”) that drive phenotypic distinctions. We applied this technique to a set of 200 laser capture microdissected human PDA samples as well as 45 low-grade precursors for which we had matched histopathological, clinical, and epidemiological annotation. We identified the MRs associated with four malignancy phenotypes: precursors vs. PDA (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top MR of PDA malignancy was found to be BMAL2, a member of the PAS family of bHLH transcription factors. Although the canonical function of BMAL2 is linked to the circadian rhythm protein CLOCK, gene set enrichment analysis highlighted a potential role in hypoxia response. We previously demonstrated that PDA in humans and in the genetically engineered “KPC” mouse model is hypovascularized, hypoperfused, and profoundly hypoxic, with a partial oxygen pressure <1mmHg. Given the close homology of BMAL2 to HIF1B (ARNT) and its potential to heterodimerize with HIF1A, we investigated whether BMAL2 plays a role in the hypoxic response of PDA. Indeed, BMAL2 activity was induced in response to hypoxia and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally-inferred association with RAS activity. Strikingly, knockout or knockdown of BMAL2 in human PDAC cells led to defects in viability and invasion in the setting of hypoxia. BMAL2 knockout cells lost the ability to induce glycolysis upon exposure to severe hypoxia and this was associated with a loss of expression of the glycolysis enzyme LDHA. A large-scale CRISPR screen found that LDHA was the single most critical gene necessary for viability of PDA cells in the setting of hypoxia. Moreover, the set of inferred transcriptional targets of BMAL2 were highly enriched in hypoxia-responsive proteins, including GLUT1, the second top hit in our hypoxia survival CRISPR screen. Strikingly, knockout of BMAL2 led to a complete loss of HIF1A stabilization in response to hypoxia, consistent with the stabilizing role of HIF1A heterodimerization partners such as HIF1B. By contrast, HIF2A was further upregulated under hypoxia in the setting BMAL2 loss. We conclude that BMAL2 is a key master regulator of hypoxia responses in PDA that serves as a molecular switch between the disparate metabolic roles of HIF1A- and HIF2A-dependent hypoxia responses. This will be further validated in ongoing metabolomic studies Citation Format: Alvaro Curiel Garcia, Carlo H. Maurer, Kate Hollinshead, Pasquale Laise, Anthony Andren, Stephen A. Sastra, Carmine F. Palermo, Irina R. Sagalovskiy, Li Zhang, Sam Holmstrom, Kristen Johnson, Gulam A. Manji, Alina Luga, Costas A. Lyssiotis, Andrea Califano, Alec Kimmelman, Kenneth P. Olive. BMAL2 is a KRAS-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4803.