Expression of Ink4a and a p53 tumor suppression-associated gene (TSAG) signature

The CDKN2A locus, which is frequently deleted in pancreatic ductal adenocarcinoma (PDAC), encodes two tumor suppressors, ARF and INK4A, that may influence tumorigenesis through distinct mechanisms. Distinguishing their individual contributions to cancer could help improve the understanding of PDAC pathogenesis and potentially uncover targetable vulnerabilities. Moreover, whereas ARF is known to enhance p53 function, defining its p53-independent activities could elucidate new processes that drive PDAC development. In this study, we sought to understand ARF function in PDAC suppression. Analysis of gene expression and mutational patterns in human PDAC TCGA data indicated that CDKN2AARF and CDKN2AINK4A are commonly both affected by point mutations and/or deletions, suggesting that their combined inactivation contributes to PDAC development. In genetically engineered mouse models, Arf inactivation accelerated KRASG12D-driven PDAC development, both in the presence and absence of Trp53, demonstrating that ARF is a PDAC-suppressor and can act in a p53-independent manner. Transcriptomic analyses of PDACs supported a p53-independent role for ARF, with ARF deficiency promoting extracellular matrix, collagen synthesis/assembly, and epithelial-mesenchymal transition gene expression programs. Accordingly, ARF-deficient PDACs displayed extensive remodeling of the tumor microenvironment (TME), associated with collagen deposition, increased tissue stiffness, and higher fibroblast content-hallmarks of aggressive and treatment-resistant PDAC stroma. Together, this study shows how ARF deficiency associated with CDKN2A inactivation sculpts the PDAC TME in a p53-independent fashion. Given the central role of the TME in PDAC progression and therapeutic resistance, these findings may provide insight critical for improving therapeutic interventions for PDAC. SIGNIFICANCE:ARF deficiency induced by CDKN2AARF alterations promotes remodeling of the pancreatic cancer microenvironment, which could provide a genotype-specific therapeutic vulnerability to improve outcomes of pancreatic cancer patients. See related commentary by Destefanis and Mulvaney, p. 3101.

Transcriptome analysis of PDACs from KTC, KTC;Trp53fl/fl, KTC;Arffl/fl and KTC;Trp53fl/fl; Arffl/fl mice

sgRNA level data from the Lenti-U6BCsgRNASaturation/Cre library in KTC mice

GO enrichment of tumor suppressive epigenomic regulators

GO enrichment of tumor dependency epigenomic regulators

Enrichment analysis of up-regulated genes

Epigenomic dysregulation is widespread in cancer. However, the specific epigenomic regulators and the processes they control to drive cancer phenotypes are poorly understood. We used a novel high-throughput in vivo method to perform iterative functional screens of >250 epigenomic regulators within autochthonous oncogenic Kras-driven lung tumors. We identified many previously unappreciated epigenomic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and MLL1 complex are robust tumor suppressors in lung adenocarcinoma. Histone modifications generated by the HBO1 complex are frequently reduced in human lung adenocarcinomas and are associated with worse clinical features. HBO1 and MLL1 complexes co-occupy shared genomic regions, affect chromatin accessibility, and control the expression of canonical tumor suppressor genes and lineage fidelity. The HBO1 complex is epistatic with the MLL1 complex and other tumor suppressor genes in lung adenocarcinoma development. Collectively, these results provide a phenotypic roadmap of epigenomic regulators in lung tumorigenesis in vivo. SIGNIFICANCE:Using a novel functional genomics method in vivo, we investigated epigenomic regulators in lung tumorigenesis. We discovered multiple novel genes that affect tumor growth. We show that the HBO1 and MLL1 complexes interact to suppress lung adenocarcinoma. Our findings provide broad insights into the epigenomic regulatory landscape of lung cancer.

Genes included in the Lenti-U6BCsgRNAEpigenomics/Cre library

Gene level data from the Lenti-U6BCsgRNAEpigenomics/Cre library in KTC mice

Genes included in the Lenti-U6BCsgRNASaturation/Cre library

sgRNA level data from the Lenti-U6BCsgRNAEpigenomics/Cre library in KTC mice

Gene level data from the Lenti-U6BCsgRNASaturation/Cre library in KTC mice

Supplemental Figure 7. Inactivation of the HBO1 complex genes reduces H3K14ac and H4K12ac levels in lung tumors and worsens tumor grade. Supplemental Figure 8. Tumor-suppressive genes in the MLL1 complex are mutated, and genetic alterations in the HBO1JADE2-ING5 and MLL1 complexes are associated with survival in human lung adenocarcinoma. Supplemental Figure 9. H3K14ac is reduced in a subset of human lung adenocarcinomas. Supplemental Figure 10. HBO1 complex target histone modifications and H3K36me3 are disrupted in human lung adenocarcinoma. Supplemental Figure 11. Inactivation of HBO1 or MLL1 complex genes results in highly correlated chromatin accessibility landscapes. Supplemental Figure 12. Inactivating genes in the HBO1 or MLL1 complex alters chromatin accessibility at shared regions enriched for lineage transcription factor motifs and at known tumor suppressor genes. Supplemental Figure 13. Chromatin binding profiles of the HBO1 and MLL1 complexes and their target histone modifications across the genome. Supplemental Figure 14. HBO1 complex catalytic activity functions in suppressing lung cancer cell proliferation. Supplemental Figure 15. Inactivation of HBO1 or MLL1 complex genes alters transcriptional states in lung tumor cells. Supplemental Figure 16. Inactivation of HBO1 or MLL1 complex genes suppresses tumor suppressor gene expression and disrupts lineage fidelity. Supplemental Figure 17. Transcriptional impact of Kat7 deletion in normal lung AT2 cells. Supplemental Figure 18. Cancer dependency score correlation and tumor growth metrics from Lenti-U6BCsgRNAEpistasis/Cre library. Supplemental Figure 19. Kat7 is synthetic lethal with Stag2-Cohesin complex. Supplemental Figure 20. Tumor sizes at different percentiles upon perturbing the HBO1JADE2-ING5 or MLL1 complex in multiple genetic driver contexts.

Sample information for bulk ATAC-seq

Categories and percent frequency of significantly decreased chromatin accessibility regions

The CDKN2A locus is one of the most frequent alterations in pancreatic ductal adenocarcinoma (PDAC). CDKN2A locus encodes two tumor suppressors, ARF and INK4A, which are rarely studied individually, limiting our understanding of their distinct roles in tumorigenesis. In addition, although ARF is an established positive regulator of the p53 tumor suppressor, it also has proposed p53-independent activities. The p53-dependent and p53-independent roles of ARF in the context of PDAC remain unclear. Here, we sought to understand ARF function in PDAC suppression. To this end, we first characterized expression and mutational patterns of the individual transcripts encoding ARF and INK4A using human TCGA data. Because the discrete roles of these transcripts have long been obscured by the shared CDKN2A locus nomenclature, we propose that future studies should analyze them independently and adopt CDKN2A ARF and CDKN2A INK4A as nomenclature that allows for their distinction. Notably, we found that CDKN2A ARF and CDKN2A INK4A are both commonly altered in human PDACs, through point mutation and/or deletion, suggesting that their combined inactivation contributes to human PDAC development. Next, to unequivocally interrogate a role for ARF as a PDAC suppressor, we used genetically-engineered mouse models (GEMMs). We found that Arf inactivation accelerated KRASG12D-driven PDAC development in GEMMs, both in the presence and absence of Trp53, demonstrating that ARF is a PDAC suppressor, and furthermore that it can act in a p53-independent manner. Transcriptomic analyses of PDACs from mice of various genotypes provided support for a p53-independent role for ARF, with ARF deficiency promoting extracellular matrix, collagen synthesis/assembly and epithelial-mesenchymal transition gene expression programs. Accordingly, ARF-deficient PDACs exhibited extensive remodeling of the tumor microenvironment (TME), associated with collagen deposition, increased tissue stiffness, and higher fibroblast content, all of which are characteristic of aggressive and treatment-resistant PDAC stroma. Similarly, transcriptomic analyses of human PDACs supported the notion that reduced ARF expression drives changes in the TME. Taken together, our findings reveal that ARF deficiency sculpts the PDAC TME and does so in a p53-independent fashion. Given the central role of the TME in PDAC progression and therapeutic resistance, our findings offer critical insights that may inform improved therapeutic strategies for PDAC. Moreover, our observations also underscore the importance of distinguishing between the two CDKN2A-encoded tumor suppressors, ARF and INK4A, and of studying both p53-dependent and p53-independent roles for ARF as they may have distinct and complementary roles in PDAC biology. Sofia Ferreira, Brittany M. Flowers, Won-Young Choi, Maria Farina-Morillas, Alberto Gatto, Sohinee Bhattacharyya, Gábor Boross, Ghmkin Hassan, Abigail S. Mulligan, Hannes Vogel, Laura D. Wood, Valerie M. Weaver, Monte M. Winslow, Dmitri Petrov, Mara H. Sherman, Hyo Young. Choi, D. Neil. Hayes, Andrew J. Aguirre, Jose A. Seoane, Laura D. Attardi. Inactivation of CDKN2A ARF promotes p53-independent remodeling of the PDAC tumor microenvironment [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 A030.

Tumor metrics of the Lenti-U6BCsgRNAEpistasis/Cre vectors

sgRNA level data from the Lenti-U6BCsgRNAEpigenomics/Cre library in KT mice