Effect of TNFα and RELA inhibition on gene expression in human PDAC cells
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ABSTRACT:
PROVIDER: PRJNA1118415 | ENA |
REPOSITORIES: ENA
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Project description:The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. Here, we characterised previously unknown endogenous RELA-GFP dynamics in PDAC cell lines by live single cell imaging, which revealed rapid, sustained and non-oscillatory nuclear RELA following TNFα stimulation. Using Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. Using RNA-seq, we identified the actin regulators NUAK2 and ARHGAP31 as transcriptionally regulated by RELA. In turn, NUAK2 or ARHGAP31 siRNA depletion downregulates TNFα-stimulated RELA nuclear localisation in PDAC cells, establishing a novel negative feedback loop regulating RELA activation by TNFα. We identify an additional actin-independent feedback loop involving RELB, which suppresses TNFα-mediated RELA nuclear localisation following RELA mediated upregulation of RELB. Taken together, we provide computational and experimental support for interdependence between the F-actin network and RELA translocation dynamics in PDAC.
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Project description:TNFα has an evolutionary conserved role in mediating inflammation via activation of the transcription factor NF-κB. The functions of individual NF-κB binding sites are not well understood. To identify conserved and functionally important NF-κB binding sites in mammals, we performed ChIP-seq to map the genome-wide binding of RELA and select histone modifications in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. The conserved RELA binding sites show strong epigenetic changes in response to TNFα and enrich near genes controlling vascular development and pro-inflammatory responses. Our method identifies novel modes of RELA-chromatin interactions that are conserved in mammals and shared between multiple cell-types. Particularly, genomic regions bound by RELA prior to stimulation are important responders during TNFα stimulation. We use CRISPR/Cas9 genome editing to validate the roles of the conserved RELA pre-bound sites near pro-inflammatory genes such as CCL2 and PLK2. Our evolutionary approach describes new aspects of mammalian NF-κB biology including its role within super-enhancers and relevance in inflammatory disorders.
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Project description:Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to analyze the effect of telomerase inhibition on TNFα-induced genome-wide p65 binding in HeLa cells. By obtaining over 40 million uniquely mappable reads per sample from ChIP-seq, maps for TNFα-induced p65 binding in absence and presence of an hTERT inhibitor, MST-312, were generated. As expected, TNFα treatment significantly increased genome-wide p65 occupancy. Interestingly, when cells were treated with MST-312 prior to TNFα stimulation, the number of p65 binding sites was reduced. In addition, some binding sites, including important p65 targets like IL6 and TNF, showed a reduced p65 occupancy with a minimum fold change of 1.5, after MST-312 exposure. Taken together, our ChIP-seq data indicate that telomerase is required for optimal p65 binding at a small proportion of p65 target sites upon inflammatory stimuli.
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Project description:In pancreatic cancer, classical and basal-like subtypes are identified as two major PDAC subtypes. Our previous data showed that PDAC tumor cells can switch between the two phenotypes through transcriptional reprogramming particularly in response to inflammatory cues. In this study, we were trying to understand TNFα mediated transcription regulatory network in pancreatic cancer. Hence, we performed RNA-seq in TNFα treated classical cells as well as aqua dest control.
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Project description:Pancreatic ductal adenocarcinoma(PDAC) is a highly lethal malignancy with poor response rate to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have been implicated as drivers of resistance to both conventional and immune therapies. As such, targeting RAS/MAPK signaling is an attractive strategy. However, in practice, MAPK inhibition has not yet shown clinical efficacy, likely due to rapid acquisition resistance in PDAC cells. Tumor intrinsic mechanisms of resistance to RAS/MAPK have been studied, however, the unique PDAC TME may also be a key driver in resistance. Previous studies have shown that FAK inhibition can reprogram the PDAC TME and delay PDAC progression in animal models. Herein, we found that long-term FAK inhibitor treatment leads to hyperactivation of the MAPK pathway in both genetically engineered mouse models and in post-treatment PDAC tissues from FAK inhibitor clinical trials. Concomitant inhibition of both FAK (VS4718) and RAF/MEK (V6766) signaling dramatically suppressed tumor cell growth, leading to increased survival across multiple PDAC mouse models. The mechanisms of synergy include both changes in tumor-intrinsic signaling and modulation of tumor/stroma interactions that drive MEK resistance. In the TME, we found that cancer associated fibroblasts (CAFs) can impair the downregulation of cMyc by MEK inhibition in PDAC cells. This results in de-novo resistance to MEK inhibition in fibrotic conditions. By contrast, FAK inhibitors reprogram CAFs to suppress the production of key growth factors, including FGF1, that drives resistance to RAF/MEK inhibition. While combined FAK and RAF/MEK inhibition only leads to disease stasis, the addition of chemotherapy to the combination led to tumor regression and improved long-term survival in PDAC mouse models. Analysis of tumor immunity showed that the combination of FAK and MEK inhibition improved anti-tumor immunity and improved priming of T cell responses, which was further improved with the addition of chemotherapy. These findings led to testing of FAK (Defactinib) plus RAF/MEK (Avutometinib) inhibition in combination with gemcitabine and nab-paclitaxel in advanced pancreatic cancer patients (NCT05669482). Finally, we tested if the addition of immunotherapy could enhance the efficacy of the FAKi/MEKi/chemotherapy and found adding in either PD1 or CTLA4/PD1 blockade led to long term disease control in PDAC animal models. Together, these studies identified novel FAK inhibition as a route to overcome both tumor intrinsic and stromal-derived resistance to MAPK inhibition and showed that this combination can be exploited to increase the efficacy of cytotoxic and immunotherapy approaches.
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Project description:The treatment of chronic mucocutaneous ulceration is challenging and only some patients respond selectively to inhibitors of tumor necrosis factor-alpha (TNF-a). TNF-a activates opposing pathways leading to caspase-8-mediated apoptosis as well as NF-kB-dependent cell survival. We investigated the etiology of autosomal dominant mucocutaneous ulceration in a family whose proband was dependent on anti-TNF-a therapy for sustained remission. A heterozygous mutation in RELA (NM_021975: c.559+1G>A), encoding the NF-kB subunit RelA (p65), segregated with the disease phenotype and resulted in RelA haploinsufficiency. The patients’ fibroblasts exhibited increased apoptosis in response to TNF-a, impaired NF-kB activation, and defective expression of NF-B-dependent anti-apoptotic genes. We show that Rela+/- mice have similarly impaired NF-kB activation, develop cutaneous ulceration from TNF-a exposure, and exhibit severe dextran sodium sulfate-induced colitis ameliorated by TNF-a inhibition. These findings demonstrate an essential contribution of biallelic RELA expression in protecting stromal cells from TNF-a-mediated cell death, thus delineating the mechanisms driving the effectiveness of TNF-a inhibition in this disease.
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