Role of Distal Regulatory Elements in Cancer Progression and Therapy

Enhancer elements comprise of regions of DNA that are distal to gene promoters with a characteristic capacity to affect and regulate gene transcription. Enhancers are enriched in a highly context-specific manner allowing for intricate control of gene expression. Current studies endeavor to elucidate the mechanisms underlying enhancer activation and function to ultimately exploit their specificity in targeted therapeutics. Due to the reported addiction of cancer to aberrant gene transcription, targeting enhancer elements is a promising therapeutic target in various malignancies. In this project, we conduct a series of studies with the general aim of extending the knowledge about the molecular mechanisms by which enhancers drive aberrant transcription in cancer. We focus on epigenetic modulation to exploit enhancer elements as therapeutic or prognostic targets. In the first study included in our project, we evaluated the importance of the super enhancer subcategory of distal regulatory elements in a breast cancer cell line where estrogen plays an important role in driving gene expression through enhancers. Super enhancers are claimed to be a highly active subgroup of distal regulatory elements that is abundantly enriched with transcription factors, span long stretches of DNA, and exhibit preferential efficacy in driving major transcriptional programs in cancer. We identified super enhancers related to estrogen in this system using the standard algorithm and failed to observe a distinct high efficacy of super enhancers compared to typical enhancers. By varying the settings of this algorithm, we also uncovered biases in enhancer identification that extensively influence the results. On the other hand, we observed that major targets of estrogen activation showed a preference for association with super enhancers and concluded that they may indeed tend to regulate the transcription of master regulators. Accordingly, we concluded that the focused attention given to super enhancers should not lead to disregarding typical enhancers which also play a significant and important role in gene transcription regulation. Consequently, in the second study we reviewed the role of enhancers in pancreatic cancer, a malignancy with exceptionally low survival rates. We focused on the application of epigenetic modulators, such as bromodomain and extraterminal proteins inhibitors and histone deactylase inhibitors, in targeting enhancer elements and speculated about mechanisms underlying the reported synergy between these two inhibitors. Interestingly, we used publicly available data to further study the context-specificity of enhancers. Notably, we observed a tendency where the same oncogenic target gene is activated by different enhancers in various systems due to differential expression of transcription factors. Accordingly, we expanded our studies in pancreatic cancer and uncovered a group of subtype-specific super enhancers that drive the cells into a squamous phenotype which correlates with a particularly poor prognosis. Studying the general activation epigenetic profiles of different pancreatic cancer cell lines identified deltaNp63 as a major driver of the squamous molecular identity in cells and patient-derived xenografts. Moreover, extensive analysis of the role of deltaNp63 in driving a more aggressive phenotype uncovered the implication of super enhancers which are supported by a network of interconnected and differentially expressed transcription factors. This pattern resembles the reports of transcription factor regulatory circuitry driving the pluripotent molecular identity of embryonic stem cells. Identification of the same pattern governing differentiation into specific molecular subtypes in pancreatic cancer opens the door to precision-based medicine approaches targeting this circuitry in this particular subtype. Finally, we further investigated the role of enhancer elements in the context of chemotherapeutic resistance in pancreatic cancer. Interestingly, we observed that pro-inflammatory and migratory programs are activated in paclitaxel-resistant cells via activation of BET-dependent enhancers. Furthermore, we observed that BET inhibition sensitizes resistant and sensitive cells to paclitaxel. Notably, super enhancers that we observed to be enriched in resistant cells were associated with genes that correlate with poor prognosis. This study confirmed the patterns we uncovered in the other studies where enhancers and super enhancers drive aberrant transcription activation in cancer and present a promising target for patient treatment. Altogether, this project resulted in 2 peer-reviewed publications in the journals of Transcription and Epigenomes, one manuscript that has been peer-reviewed and is currently under revision in Proceedings of the National Academy of Sciences of the United States of America (PNAS), and another manuscript in preparation for submission. These publications/manuscripts join the growing body of literature investigating the role of enhancers in malignancy and aim to guide new approaches for precision-based medicine.