Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive human malignancies, with a five-year survival rate below 15%. In contrast to several other solid tumors where chemotherapy, radiotherapy, and immune checkpoint blockade have significantly improved outcomes, PDAC remains largely refractory to current therapeutic strategies. One hallmark of PDAC is its profoundly hypoxic tumor microenvironment. Extensive desmoplasia and limited vascularization restrict oxygen delivery to tumor tissues, creating regions of severe oxygen deprivation. KRAS, the primary oncogenic driver of PDAC, can activate transcriptional programs that rewire cellular metabolism and enable tumor cell survival under oxygen limitation. However, the precise molecular mechanisms connecting oncogenic KRAS signaling to hypoxia-adaptive transcriptional programs remain poorly defined. To identify the top transcriptional regulators associated with KRAS, we integrated clinical and pathological information with gene expression data derived from laser captured, microdissected human PDAC epithelium. Across 242 transcriptional profiles, including 197 PDAC tumors and 45 benign precursor lesions, regulatory network analysis identified BMAL2, a member of the PAS superfamily typically associated with the regulation of circadian rhythms, as the top KRAS-associated transcriptional regulator and one of the strongest drivers of tumor initiation, progression, and post-resection survival. We found that BMAL2 activity was consistently elevated in PDAC relative to precursor lesions and independently associated with worse clinical outcomes. Meta-analysis across multiple independent PDAC cohorts confirmed that BMAL2 activity is reproducibly increased in aggressive tumor subtypes and correlated with poor prognosis. Phylogenetic analysis showed that BMAL1 and BMAL2 are most closely related to ARNT (HIF1β) and ARNT2 (HIF2β), the obligate binding partners of HIF transcription factors. Consistent with this evolutionary relationship, BMAL2 activity correlated strongly with hypoxia transcriptional signatures compared with other PAS family members. Functional studies demonstrated that BMAL2 is a critical regulator of PDAC cell fitness. CRISPR/Cas9-mediated BMAL2 knockout across multiple PDAC cell lines significantly reduced cell viability, clonogenic capacity, and migratory behavior. In wound-healing assays, BMAL2-deficient cells displayed markedly impaired migration compared with control cells. Conversely, BMAL2 overexpression enhanced tumor cell growth and accelerated wound closure, supporting a model in which BMAL2 promotes proliferative and migratory phenotypes in PDAC cells. Seahorse metabolic profiling revealed that BMAL2 expression increases cellular respiratory capacity and overall metabolic activity, whereas BMAL2 loss reduced oxygen consumption rates and impaired metabolic responses associated with hypoxic adaptation. These findings are consistent with transcriptional and metabolomic data indicating that BMAL2 promotes glycolytic metabolism, including increased lactate production and expression of key glycolytic enzymes. Together, these observations position BMAL2 as an important mediator of metabolic plasticity in pancreatic cancer cells. Beyond metabolic regulation, BMAL2 also influenced epithelial plasticity. In PATU8902 cells, BMAL2 overexpression induced morphological changes characterized by spindle-like architecture and reduced epithelial cohesion. These changes were accompanied by increased expression of Vimentin, a well-known marker of epithelial-to-mesenchymal transition (EMT). Collectively, these findings suggest that BMAL2 promotes EMT-like phenotypic reprogramming that may enhance tumor cell adaptability and invasiveness within the hypoxic tumor microenvironment. Mechanistically, we found that BMAL2 regulated the stability and balance of hypoxia-inducible factor paralogs. Under hypoxic conditions, BMAL2 is required for stabilization of HIF1A, a central regulator of glycolytic metabolism. Loss of BMAL2 prevented HIF1A stabilization while simultaneously increasing HIF2A accumulation, suggesting that BMAL2 functions as a molecular switch between HIF1A- and HIF2A-dependent hypoxia programs. This regulatory mechanism provides a direct link between oncogenic KRAS signaling and the transcriptional machinery that enables tumor cells to survive and proliferate in oxygen-limited environments. Importantly, we found that BMAL2 activity was controlled by KRAS signaling through the RAF-MEK-ERK pathway. Pharmacologic inhibition of RAS using the pan-RAS (ON) inhibitor RMC-7977 significantly reduced BMAL2 activity both in vitro and in vivo without altering BMAL2 expression levels, indicating a post-translational regulatory mechanism. Complementarily, ERK inhibition also displayed reduction in BMAL2 activity in a panel of PDAC cell lines. This finding places BMAL2 downstream of oncogenic KRAS and identifies it as a transcriptional effector of RAS-MAPK signaling. Finally, the functional consequences of BMAL2 activity extended into living animals. In orthotopic xenograft models of PDAC, BMAL2 knockout markedly impaired tumor growth and reduced tumor engraftment. Tumors derived from BMAL2-deficient cells grew significantly more slowly than controls, and in some models tumor formation was severely compromised. Consistent with reduced tumor growth, animals bearing BMAL2-deficient tumors exhibited prolonged survival compared with those implanted with control cells. These findings demonstrate that BMAL2 is not only necessary for PDAC cell proliferation in vitro but is also essential for efficient tumor initiation and progression in vivo. Collectively, these findings identify BMAL2 as a previously unrecognized transcriptional effector downstream of oncogenic KRAS that functions as a central transcriptional node linking KRAS signaling to hypoxia adaptation, metabolic reprogramming, and phenotypic plasticity in pancreatic cancer. By coordinating transcriptional and metabolic programs required for survival under extreme hypoxic conditions, BMAL2 enables PDAC cells to adapt to and thrive within their uniquely hostile tumor microenvironment. These results provide mechanistic insight into how KRAS signaling drives pancreatic cancer biology and highlight BMAL2 as a key regulator of tumor fitness within the hypoxic tumor ecosystem. Citation Format: Alvaro Curiel Garcia, Sam R. Holmstrom, Melina Chen, Diana V. Morales, Kathryn Buscher, Carmine F. Palermo, Steven A. Sastra, Anthony Andren, Lorenzo Tomassoni, Li Zhang, Maya Stella Dixon, Tessa Y. S. Le Large, Irina Sagalovskiy, Winston Wong, Kaitlin Shaw, Jeanine Genkinger, Hanina Hibshoosh, Gulam A. Manji, Alina C. Iuga, Roland M. Schmid, Michael A. Badgley, Pasquale Laise, Iok In Christine Chio, Costas A. Lyssiotis, Yatrik M. Shah, Andrea Califano, H. Carlo Maurer, Kenneth P. Olive. BMAL2 is a KRAS-driven master regulator of hypoxic adaptation and EMT in pancreatic ductal adenocarcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr NG04.
Garcia et al. (Fri,) studied this question.
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