Abstract Ovarian cancers (OC) are characterized by an immunosuppressive tumor microenvironment and frequent mutations in the TP53 gene, which encodes for the p53 tumor suppressor protein. Low infiltration of tumor-fighting immune cells in these tumors contribute to ovarian cancer’s static 5-year survival rate and poor response to immunotherapy treatment. We and others have shown that using therapies that inhibit DNA methylation (DNMTis) can facilitate immune cell recruitment and activation to fight ovarian cancers. This response is mediated by interferon signaling that is initiated by the derepression of highly repetitive DNA elements called transposable elements (TEs). TEs are stretches of mobile noncoding DNA that transcribe virus-like double stranded RNA (dsRNA) that are recognized by cytosolic sensors and activate the type I interferon pathway. Downstream of type I interferon, tumors treated with DNMTis show increased recruitment of effector T cells, NK cells, and proinflammatory macrophages necessary for tumor-cell killing. Relevant to ovarian cancer, our lab has recently shown that baseline TE expression is altered by the mutational status of the TP53 gene. We showed the baseline expression of TEs is higher in OC cell lines with the p53 DNA binding domain mutation R175H. In addition, isogenic wildtype and R175H mutant p53 CRISPR modified OC cell lines recapitulate these differences in TE transcription. We are now characterizing the direct and indirect effects p53 has on TEs and downstream immune signaling. Western blot analysis of mutant and wildtype p53 OC cell lines show differences in baseline DNMT1 expression, the enzyme responsible for methylating cytosine bases on DNA known to be regulated by p53. Additionally, treatment with a p53 stabilizing drug, Nutlin-3a, shows wildtype p53 decreases DNMT1 expression while mutant p53 does not. The greater DNMT1 expression in the mutant p53 cell lines indicates that DNA methylation is not the driving regulator to TE expression, which we hypothesized was being blocked by increased p53 binding at TE loci. We sought to determine how p53 binds directly to TE loci using Cleavage Under Targets and Release Using Nuclease sequencing (CUT&RUN-seq), a method that precipitates protein-DNA complexes via an antibody guided nuclease system. Our data show that mutant p53 has increased occupancy of both the DNMT1 gene and specific TE classes like HERVH that we previously identified having a higher baseline expression in mutant OC cell lines. These new data support the conclusion that p53 binding activates the transcription of TEs and that “hotspot” mutant p53 creates an altered TE landscape. Future experiments will characterize this epigenetic regulation of TEs in a larger panel of mutant p53 cell lines and study the downstream immune signaling cascade of mutant versus wildtype p53 tumors treated with epigenetic therapies. Citation Format: Reddick R. Walker, Kevin Nestler, Melissa Hadley, Noor Diab, Katherine Chiappinelli. P53 transcriptional regulation of transposable elements and the downstream immune response in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B006.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immu-notherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence -associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbin-din reduces CXCL8 chemokine production and downstream neu-trophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and nonmalignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines’ morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell-adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

Resistance to cancer treatments remains a major barrier in developing cancer cures. While promising combination chemotherapy treatments and novel immunotherapies have improved patient outcomes, resistance to these treatments remains poorly understood. New insights into the dysregulation of the epigenome show how it promotes tumor growth and resistance to therapy. By altering control of gene expression, tumor cells can evade immune cell recognition, ignore apoptotic cues, and reverse DNA damage induced by chemotherapies. In this chapter, we summarize the data on epigenetic remodeling during cancer progression and treatment that enable cancer cell survival and describe how these epigenetic changes are being targeted clinically to overcome resistance.

BackgroundNovel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response.MethodsHuman OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8Trp53-/-mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8Trp53-/-cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.ResultsWe show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.ConclusionIn summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

The physical connections between the cytoskeletal system and the nucleus provide a route for the nucleus to sense the mechanical stress both inside and outside of the cell. Failure to withstand such stress leads to nuclear rupture, which is observed in human diseases. In this review, we will go through the recent findings and our current understandings of nuclear rupture. Starting with the triggers of nuclear rupture, including the aberrant nuclear lamina composition and the elevated actomyosin contractility. We will also discuss the role of ESCRT-III in nuclear rupture repair and the biological consequences of nuclear rupture, including the negative impacts on cellular compartmentalization, DNA damage, and cellular differentiation. Recent studies on nuclear rupture provide further insights into the direct mechanistic link between nuclear rupture and several pathological conditions. Such knowledge can guide us in developing potential therapeutic solutions for the patients.