Recent studies have thoroughly described genome-wide expression patterns defining molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), with different prognostic and predictive implications. Although the reversible nature of key regulatory transcription circuits defining the two extreme PDAC subtype lineages “classical” and “basal-like” suggests that subtype states are not permanently encoded but underlie a certain degree of plasticity, pharmacologically actionable drivers of PDAC subtype identity remain elusive. Here, we characterized the mechanistic and functional implications of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in controlling PDAC plasticity, dedifferentiation, and molecular subtype identity. Utilization of transgenic PDAC models and human PDAC samples linked EZH2 activity to PDAC dedifferentiation and tumor progression. Combined RNA- and chromatin immunoprecipitation sequencing studies identified EZH2 as a pivotal suppressor of differentiation programs in PDAC and revealed EZH2-dependent transcriptional repression of the classical subtype defining transcription factor Gata6 as a mechanistic basis for EZH2-dependent PDAC progression. Importantly, genetic or pharmacologic depletion of EZH2 sufficiently increased GATA6 expression, thus inducing a gene signature shift in favor of a less aggressive and more therapy-susceptible, classical PDAC subtype state. Consistently, abrogation of GATA6 expression in EZH2-deficient PDAC cells counteracted the acquisition of classical gene signatures and rescued their invasive capacities, suggesting that GATA6 derepression is critical to overcome PDAC progression in the context of EZH2 inhibition. Together, our findings link the EZH2-GATA6 axis to PDAC subtype identity and uncover EZH2 inhibition as an appealing strategy to induce subtype-switching in favor of a less aggressive PDAC phenotype. Significance: This study highlights the role of EZH2 in PDAC progression and molecular subtype identity and suggests EZH2 inhibition as a strategy to recalibrate GATA6 expression in favor of a less aggressive disease.

ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity. To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms. Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes. We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.

Osteosarcoma (OS) is a common type of bone tumor for which there has been limited therapeutic progress over the past three decades. The prevalence of transcriptional addiction in cancer cells emphasizes the biological significance and clinical relevance of super-enhancers. In this study, we found that Max-like protein X (MLX), a member of the Myc-MLX network, is driven by super-enhancers. Upregulation of MLX predicts a poor prognosis in osteosarcoma. Knockdown of MLX impairs growth and metastasis of osteosarcoma in vivo and in vitro. Transcriptomic sequencing has revealed that MLX is involved in various metabolic pathways (e.g., lipid metabolism) and can induce metabolic reprogramming. Furthermore, knockdown of MLX results in disturbed transport and storage of ferrous iron, leading to an increase in the level of cellular ferrous iron and subsequent induction of ferroptosis. Mechanistically, MLX regulates the glutamate/cystine antiporter SLC7A11 to promote extracellular cysteine uptake required for the biosynthesis of the essential antioxidant GSH, thereby detoxifying reactive oxygen species (ROS) and maintaining the redox balance of osteosarcoma cells. Importantly, sulfasalazine, an FDA-approved anti-inflammatory drug, can inhibit SLC7A11, disrupt redox balance, and induce massive ferroptosis, leading to impaired tumor growth in vivo. Taken together, this study reveals a novel mechanism in which super-enhancer-driven MLX positively regulates SLC7A11 to meet the alleviated demand for cystine and maintain the redox balance, highlighting the feasibility and clinical promise of targeting SLC7A11 in osteosarcoma.