Abstract A047: Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer

Abstract To identify novel drivers of pancreatic ductal adenocarcinoma (PDAC) malignancy, we employed a systems biology approach called 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 (“master regulators”) that drive phenotypic distinctions. We applied this technique to a set of 200 laser capture microdissected human PDAC samples as well as 45 low-grade precursors for which we had matched histopathological, clinical, and epidemiological annotation. We identified the master regulators associated with 4 malignancy phenotypes: tumor initiation, tumor progression, survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top master regulator was BMAL2, a member of the PAS family. Although BMAL2 is classically associated to the circadian rhythm, gene set enrichment analysis highlighted a potential role in hypoxia response, a key phenotype in PDAC evolution. We previously demonstrated that PDAC in humans and in the genetically engineered “KPC” mouse model is hypovascularized and hypoperfused, and we have since shown that KPC tumors are profoundly hypoxic. Sequence conservation analysis of BMAL2 demonstrated close analogy to hypoxia-related genes, its activity is induced in response to it and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally inferred association with RAS activity. Strikingly, knockout of BMAL2 in human PDAC cells led to defects in viability and invasion in the setting of hypoxia. Metabolically, loss of BMAL2 impairs the ability to induce glycolysis - key metabolic pathway unregulated during hypoxia response - upon exposure to severe hypoxia. Surprisingly, knockout of BMAL2 in multiple PDAC cell lines led to a complete loss of HIF1A stabilization in response to hypoxia. By contrast, HIF2A was further upregulated under hypoxia in the setting BMAL2 loss. Additionally, metabolomic studies showed an overrepresentation of glycolysis metabolites and related metabolic pathways in hypoxic control conditions but to a lesser degree in BMAL2 knockout cells. Finally, preliminary in vivo experiments in cell line derived xenograft models displayed lower tumor growth rates in BMAL2 knock out cells. Together, our data indicates that BMAL2 loss blunts hypoxia response providing also a potential answer to the long-standing question of how these proteins are differentially regulated. Certainly, a connection between RAS signaling and glycolysis has long been apparent, but our findings provide a novel mechanistic understanding of the relationship between RAS and hypoxic metabolism. Citation Format: Alvaro Curiel Garcia, Carlo H. Maurer, Sam Holmstrom, Cristina Castillo, Pasquale Laise, Carmine F. Palermo, Steven A. Sastra, Anthony Andren, Li Zhang, Tessa Le Large, Irina Sagalovsky, Daniel R. Ross, Vilma Rosario, Kate Lu, Ethan Ferraiuolo, Nicholas Spinosa, Winston Wong, Kaitlin Shaw, John A. Chabot, Jeanine Genkinger, Hanina Hibshoosh, Gulam A. Manji, Alina C. Iuga, Roland M. Schmid Schmid, Michael A. Badgley, Kristen C. Johnson, Andrea Califano, Yatrik M. Shah, Costas A. Lyssiotis, Kenneth P. Olive. Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A047.