Ekstrom, Thomas L.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with a 5-year survival rate of 13%. Despite recent molecular stratification of tumors into distinct classical and basal-like cell states, most tumors are heterogeneous and contain both subtypes. Therefore, therapeutic approaches targeting only one subtype are unlikely to be effective as standalone PDAC treatments. Here, we integrated chromatin accessibility [assay for transposase-accessible chromatin with sequencing (ATAC-seq)], genome-wide occupancy [chromatin immunoprecipitation sequencing (ChIP-seq)] for epigenetic status (H3K27ac), and H3K4me3-anchored chromatin topology (HiChIP) to uncover subtype-independent highly interactive enhancers that interact with essential genes in PDAC. Motif analysis revealed that these common enhancers were bound by KLF5 with subsequent depletion leading to decreased cell viability via induction of apoptosis. To elucidate the transcriptional and epigenetic mechanisms by which KLF5 functions in PDAC, we used rapid depletion of KLF5 with dTAG technology and profiled the effects on the open and active chromatin landscape and transcription with nascent RNA and messenger RNA sequencing over time. Enhancer inactivation via KRAB domain Zim3-dCas9 fusion protein confirmed KLF5-bound enhancers regulate target genes, including the anti-apoptotic gene BCL2L1 . Multiplex immunofluorescence confirmed costaining of KLF5 and Bcl-xL in patient samples and overexpression of Bcl-xL rescued the induction of apoptosis after KLF5 depletion. Together, this study provides insights into common mechanisms to target highly heterogeneous PDAC tumors.

Abstract This short review highlights recent findings related to transcription factor-mediated RNA polymerase II (Pol II) pause release at enhancers and its role in regulating cell identity. We discuss how the transcriptional state of Pol II, either paused or released, shapes enhancer–promoter interactions, thereby influencing gene expression programs that define cell identity. We further summarize current knowledge of DNA-binding transcription factors that regulate Pol II pause release at enhancers and explore how this process influences cell identity. We propose a novel concept of therapy-induced molecular amnesia, where temporary inhibition of enhancer-proximal Pol II pause release and enhancer RNA transcription affects enhancer–promoter interactions differently in distinct cellular contexts. In some contexts, preservation of enhancer–promoter interactions allows rapid transcriptional reactivation and maintenance of cell identity upon recovery. In other contexts, repeated inhibition of enhancer RNA transcription leads to the permanent dissociation of enhancer–promoter interactions and loss of cell identity. Our model suggests that manipulating Pol II pause release at enhancers could selectively reverse certain aggressive tumor cell identities, limit tumor cell plasticity, and improve therapy responsiveness.

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. Objective This study sought to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. Design We integrated primary tumour single-cell RNA-seq, patient-derived xenograft RNA-seq and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analysed chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), H3K4me3-anchored chromatin topology (HiChIP) and nascent RNA capture sequencing (PRO-seq). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Results Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional programme by interacting with GATA6. GATA6 regulates classical-specific transcription through promoter-proximal pause release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilising enhancer–promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, in basal PDAC ΔNp63 promotes Pol II recruitment and stabilises enhancer–promoter interactions. Conclusion This study provides new insights into the transcriptional control and role of GR agonists in controlling PDAC molecular subtype identity.

Abstract Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN amplification. The subset of patients with tumors expressing high levels of USP44 had significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that are regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to the pathophysiology of this disease.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and highly aggressive type of pancreatic neoplasm. Unfortunately, most patients will present with unresectable, locally advanced, or metastatic disease at the time of diagnosis. Current treatment methods (gemcitabine and nab-paclitaxel, FOLFIRINOX) are not sufficient for patients with late-stage disease and only modestly improve survival. Difficulties in treating PDAC lie in the characteristics of PDAC tumors. They are highly heterogeneous, have multiple signaling pathway alterations, and are highly metastatic with a dense tumor microenvironment (TME). Hence, PDAC remains one of the most lethal malignancies with a 5-year survival rate below 10%.The KRAS mutation is a hallmark of PDAC, occurring early in 95% of pancreatic neoplasms. The mutant KRAS protein constitutively activates the mitogen-activated protein kinase (MAPK) signaling pathway to sustain PDAC pathogenesis. Although the mutant KRAS gene is the initiating molecular alteration, chronic inflammation accelerates PDAC progression to a highly aggressive and therapy-resistant cancer. Our study shows that MAPK and the nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) inflammatory signaling pathway synergize to promote PDAC progression. Live cell imaging analysis of PDAC cell lines after the simultaneous hyperactivation of MAPK and NF-κB signaling by epidermal growth factor (EGF) and tumor necrosis factor alpha respectively (TNF alpha), showed significant increases in cell migration. An integrated genome-wide epigenetic and transcriptomic analysis (ChIPseq and RNAseq) after the simultaneous stimulation of both pathways revealed significant increases in the occupancy of the active mark, H3K27ac. This was observed around transcription start sites of a subset of genes involved in cell polarity, migration, and subtype switch, processes known to be important for tumor aggressiveness. Epigenome mapping of the transcription factors FOSL1 and RELA, which are downstream targets of the MAPK and NF-κB signaling pathways respectively, show their co-occupancy in the H3K27ac-enriched regions. Furthermore, loss-of-function approaches show the synergistic effect of both MAPK and NF-κB hyperactivation on the migration capacity in vitro and in vivo. Based on these observations, we aim to delineate the molecular mechanism involved in the synergism, investigate the role of the TME, and decipher the signaling interactions between PDAC tumor cells and cells of the TME. This study is the first comprehensive analysis of the synergism between MAPK and NF-κB signaling and will provide novel insights into the development of mechanism-based therapeutic approaches. Citation Format: Joana E. Aggrey-Fynn, Feda H. Hamdan, Alexander Q. Wixom, Thomas L. Ekstrom, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1481.

Objective Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. Design We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. Results iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. Conclusion Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

Abstract PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. Significance: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Abstract Tumors with defective homologous recombination (HR) DNA repair are more sensitive to chemotherapies that induce lesions repaired by HR as well as PARP inhibitors (PARPis). However, these therapies have limited activity in HR-proficient cells. Accordingly, agents that disrupt HR may be a means to augment the activities of these therapies in HR-proficient tumors. Here we show that VLX600, a small molecule that has been in a phase I clinical trial, disrupts HR and synergizes with PARPis and platinum compounds in ovarian cancer cells. We further found that VLX600 and other iron chelators disrupt HR, in part, by inhibiting iron-dependent histone lysine demethylases (KDM) family members, thus blocking recruitment of HR repair proteins, including RAD51, to double-strand DNA breaks. Collectively, these findings suggest that pharmacologically targeting KDM family members with VLX600 may be a potential novel strategy to therapeutically induce HR defects in ovarian cancers and correspondingly sensitize them to platinum agents and PARPis, two standard-of-care therapies for ovarian cancer.

Abstract BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.