TP53 -mutated acute myeloid leukemia (AML) has dismal outcomes with current treatments and represents a critical unmet need. TP53 -mutated AML is proposed to be susceptible to immunotherapeutic approaches but, to date, there is no established immunotherapy for this sub-group. Expression of stimulator of interferon genes (STING), a key innate immune driver that activates interferon (IFN) signaling, is decreased by epigenetic silencing or mutation in many cancers, including those with TP53 mutations. Here, we report that response to the next-generation synthetic STING agonist C92 is potentiated in AML cell lines and primary cells with TP53- mutated versus wild-type (WT) cells, representing a previously undescribed vulnerability of these leukemia cells to STING small molecule therapies. Moreover, combining treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC), significantly increases STING activation, with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes. Cell death in TP53 KO versus WT AML is specifically dependent on innate immune zinc finger NFX1-type containing 1 (ZNFX1) and Z-DNA-binding protein 1 (ZBP1) driving increased cleavage and activation of Receptor-Interacting-Serine/Threonine-Protein Kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), suggesting mechanisms of necroptosis. Finally, C92 and DAC combination significantly reduces leukemia burden in humanized AML mouse models, accompanied by increased immune responses, including cytokines and cytotoxic T lymphocytes in the leukemia microenvironment. These results support development of clinical trial strategies combining STING agonists with DNMTis for patients with TP53 -mutated AML. Summary:TP53-mutated AML potentiates effects of novel next-generation STING agonist C92, with unique allosteric and non-cyclic dinucleotide (non-CD) mechanism of action, inducing increased STING activation and cytokine release STING agonists and DNMTis, synergistically increase STING activation with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes in TP53 -mutated AML STING agonists induce necroptosis via a STING-ZNFX1-ZBP1-necroptosis axis in TP53 -mutated AML. This drug combination reduces leukemia burden, activates immune responses in AML models and supports translation for high-risk AML patients. Statement of Translational Relevance:This pre-clinical study identifies a novel therapeutic vulnerability in ( TP53 )-mutated acute myeloid leukemia (AML), a poor prognosis subtype with a critical unmet need. Novel next-generation STING agonist C92, with unique allosteric and non-cyclic dinucleotide (non-CD) mechanism of action, induces increased STING activation and cytokine release, compared with WT TP53 in AML cell lines and primary cells, and has superior STING activity with respect to several STING agonists currently in clinical studies. Combining C92 treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) synergistically increases STING activation, with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes, driving ZNFX1-driven inflammatory necroptotic cell death. Utilizing humanized mouse models, C92 in combination with DAC significantly reduces leukemia burden and enhances cytotoxic T-cell responses in the tumor microenvironment, supporting clinical translation for high-risk AML patients.

Abstract Triple-negative breast cancer (TNBC) has a higher rate of metastasis; a poor prognosis and survival compared with other breast cancer types. Poly-ADP ribose polymerase inhibitors (PARPis) are used to treat TNBC patients that harbor germline BRCA1/2 mutations, inducing synthetic lethality, but responses are not durable. We previously reported that PARPis in combination with DNA methyltransferase inhibitors (DNMTis) exert synergistic cytotoxicity in TNBC, independent of BRCA mutations, but the effects of these drugs on metastasis and stemness, which are associated with poor survival outcomes, are not known. Aberrant Wnt/β-catenin signaling in TNBC is known to drive cancer stemness, metastasis, and resistance to apoptosis and chemotherapy. Genome-wide transcriptomic analysis in TNBC cell line MDA MB 231 demonstrated that combining DNMTis azacytidine (AZA) and PARPis talazoparib (TAL) down-regulated cancer stemness and metastases pathways, and key leading-edge genes including those involved in Wnt/β-catenin signaling and tenasin-C (TNC), a multimodular glycoprotein that promotes the migration of cancer cells, were decreased. The effects of this drug combination on cell migration were functionally validated using scratch and transwell migration and invasion assays in multiple TNBC cell lines, including MDA MB231 and SUM159, and patient-derived organoids (N=3). Xenograft studies of MDA MB 231 by tail vein and SC injection showed decreased metastasis to the lung with this drug combination. Notably, stem cell assays, including spheroid assays and stem cell markers, SOX2, ALDH1a1 and CD44, were also decreased with AZA and TAL treatment. Notably, we show for the first time in TNBC that Beta-catenin/TCF12 transcriptionally regulates TNC by binding to its promoter region and that inhibition or KD of WNT/Beta catenin or TNC expression decreases the cell migration, metastasis and stemness, mimicking the effects of the drug combination in TNBC cells. Taken together, our results show for the first time that PARPi and DNMTi combination therapy targets WNT/Beta-catenin signaling and TNC regulation in driving aggressive disease, metastasis, stemness and poor survival in TNBC. Citation Format: Lora Stojanovic, Kaushlendra Tripathi, Zahra Gohari, Julia L. Rutherford, Saranya Rajendran, Tara X. Metcalfe, Shu Zhang, Stephen B. Baylin, Michael Topper, Kenneth P. Nephew, Feyruz V. Rassool. DNMTi in combination with PARPi inhibits aberrant Wnt/β-catenin signaling and tenasin-C pathways, cancer stemness and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2245.

BACKGROUND:Poly(adenosine diphosphate ribose) polymerase (PARP) is recruited to DNA damage sites along with epigenetic factors such as DNA methyltransferase 1 (DNMT1). Inhibitors of DNMT modulate reactive oxygen species (ROS)-cyclic adenosine monophosphate (cAMP)/Protein Kinase A signaling and induce a "BRCAness phenotype" that further sensitizes cells to PARPi. In preclinical studies, combined DNMTi + PARPi therapy was effective in both triple-negative (TNBC) and hormone resistant (HRBC) models with intact BRCA. METHODS:The authors conducted a phase 1 study combining the oral DNMTi ASTX727 with the PARPi talazoparib in patients with previously treated TNBC or HRBC. Patients with deleterious mutations of BRCA were excluded. A classical 3+3 design guided dose escalation/de-escalation, and 28 days constituted each cycle. Serial peripheral blood mononuclear cells (PBMCs) were analyzed for changes in methylation using the Infinium Methylation EPIC BeadChip and LINE1 sequencing. RESULTS:Thirty-four evaluable patients were enrolled and treated in eight dose cohorts. Myelosuppression was common with grade >3 neutropenia in 42% and grade 3 anemia and thrombocytopenia in 13%. Dose-limiting toxicity was limited to neutropenia. Efficacy was assessed in 29 patients. There were no objective responses, six patients had stable disease persisting for >4 months in three patients. LINE1 demethylation ranged from ∼2%-10% and immune-specific CpGs (methylation in immune cells) changed 1%-5% at day 15. Methylation changes were not dose-dependent. CONCLUSIONS:ASTX727 plus talazoparib produces significant myelosuppression without other adverse events. Modest methylation changes in PBMCs were detected. There were no objective responses, but some heavily pretreated patients had stable disease for >4 months despite the attenuated doses.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a 5-year survival rate of 13%. In those with unresectable disease, standard chemotherapy offers limited benefit, with a median survival between 8 and 12 months. Approximately 17% of patients with PDAC carry a germline or somatic mutation in the BRCA or PALB2 genes. These patients may benefit from poly (ADP-ribose) polymerase inhibitors (PARPi), which induce synthetic lethality in tumors with homologous recombination deficiency. The POLO trial showed that maintenance olaparib - a PARPi - doubled progression-free survival in patients with germline BRCA mutations and metastatic PDAC. We’ve previously reported that DNA methyltransferase inhibitors (DNMTi) can induce a “BRCAness” phenotype in other types of cancer, thus sensitizing BRCA-proficient cancer cells to PARPi. Here we explore the synergistic potential of PARPi talazoparib (TAL) and DNMTi decitabine (DAC) in pancreatic cancer cell lines and patient-derived pancreatic tumor organoids (PTOs). Synergy studies with TAL and DAC were performed in two murine (Panc02 and KPC) and three human (Capan1, SW1990, and MIA PaCa-2) pancreatic cancer cell lines with diverse genetic profiles. Of note, Capan1 harbored a BRCA2 mutation. Combination TAL+DAC treatment was performed in a dose-dependent and fixed ratio scheme, and the various degrees of synergy were determined using Chou-Talalay mathematical modeling (Compusyn). Clonogenic assays were performed with murine cell lines. Three patient-derived PTOs were treated with single agent (TAL or DAC) or combination (TAL+DAC) regimens and response was determined by MTS assay. Methylomic profiles including gene level methylation and hydroxymethylation fraction in treated and untreated PTOs were acquired using Biomodal’s Duet Multiomics Assay. Statistical analyses for clonogenic and MTS assays were performed using GraphPad Prism. A synergistic inhibitory effect was observed in all pancreatic cancer cell lines when treated with TAL+DAC. The Capan1 cell line was more sensitive to TAL, as expected. Clonogenic survival assays showed a statistically significant decrease in colony numbers after treatment with the combination regimen compared to single-agent treatment. All PTOs exhibited growth inhibition when treated with DAC alone, whereas TAL alone had limited effects. The combination of TAL and DAC at low doses was more effective than either agent alone. Methylomic analysis revealed decreased methylation of key genes in DAC-treated PTOs, validating DAC’s mechanistic effects. The combination of TAL and DAC demonstrates synergistic cytotoxicity in pancreatic cancer cell lines and tumor organoids, suggesting potential for this treatment strategy in BRCA-mutated and BRCA-proficient PDAC. Zachery Keepers, Brandon Carter-Cooper, Hurley Ryan, Stephen B. Baylin, Rena Lapidus, Feyruz V. Rassool, Hem D. Shukla, Aaron T. Ciner. Synergy with decitabine expands the utility of talazoparib: A novel therapeutic strategy for BRCA-proficient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1698.

High-grade serous ovarian carcinoma (HGSOC), accounting for 70% of ovarian carcinoma cases, is often diagnosed at an advanced stage and associated with a poor prognosis. A significant clinical challenge is the lack of effective treatments for platinum-resistant OC and non-BRCA-mutated, homologous recombination proficient (HRP) tumors, which represent over 50% of HGSOC. Furthermore, although cancer immunotherapies can produce complete and durable responses, response rates in OC are modest with most patients experiencing only transient therapeutic benefit, underlining the urgent need for novel approaches to enhance antitumor immunity. Our prior research demonstrated that DNMT inhibitors (DNMTis) and PARP inhibitors upregulate ZNFX1, a zinc finger protein that facilitates STING-dependent interferon signaling and antitumor immune responses through a mechanism of pathogen mimicry. We hypothesize that in addition to upregulating ZNFX1 and immune responses, DNMTis restore STING through epigenetic reprogramming, leading to tumor cell intrinsic STING activation mediating anti-tumor response. In this study, we investigated the role of STING in HGSOC and the mechanisms underlying STING pathway activation by DNMTi. Analysis of the TCGA and GTEX database revealed reduced basal STING expression in HGSOC patient tumors, predominantly in TP53 mutant HGSOC. Furthermore, higher STING expression was linked to improved overall and progression free survival. Analysis of STING methylation in normal and HGSOC samples from TCGA database identified 6 hypermethylated sites near the promoter region/transcriptional start site. Our Infinium MethylationEPIC Array v2.0 BeadChip profiling of the HGSOC cell line OVCAR3 showed hypermethylation of these 6 sites. To determine whether this promoter-driven silencing could be reversed, we treated HRP and HR-deficient (HRD) HGSOC cell lines with increasing concentrations of DNMTi (0-100 nM decitabine, DAC). This treatment increased (P<0.01) STING expression in a dose-dependent manner and upregulated (P<0.001) downstream STING targets, CXCL10, TNFα, IL6. To further evaluate STING agonist could restore functional activation of STING signaling in addition to demethylation, HGSOC cells were treated with DAC (10nM), STING agonist (CRD3874; 1μM) and combination and evaluated for downstream STING targets by western and qPCR. The DAC-STING agonist combination increased pSTING1, pTBK1, and pIRF3 and apoptosis-dependent cell death. Additionally, combination treatment markedly reduced proliferation, colony and spheroid formation compared to single drug treatments. Together, these results demonstrate epigenetic regulation of STING expression in HGSOC, regardless of HRD status. Combining a DNMTi with a STING agonist has the potential to enhance the antitumor immune response and represents a promising therapeutic strategy for HGSOC patients. Saranya Rajendran, Elnaz Abbasi Farid, Kaushlendra Tripathi, Ishani Chattopadhayay, Shu Zhang, Stephen B. Baylin, Feyruz V. Rassool, Kenneth P. Nephew. Epigenetic reprogramming of the STING pathway as a combination strategy in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4844.

Women harboring deleterious BRCA mutations are at greatly increased risk of developing high grade serous ovarian cancer (HGSC). BRCA1 and BRCA2 genes are crucial for repairing DNA double-strand breaks via homologous recombination, maintaining genomic stability. Recent findings suggest BRCA1 is also localized to the mitochondrial (mt) membrane, where it helps maintain mt metabolic stability. Mutations in BRCA1 and BRCA2 promote both genomic instability and mt dysfunction leading to an increase in cancer susceptibility. We recently reported that viral mimicry and therapeutic agents—poly (ADP-ribose) polymerase inhibitors (PARPis) and DNA methyltransferase inhibitors (DNMTis)—activate a cytosolic nucleic acid sensor, zinc finger NFX1-type containing 1 protein (ZNFX1) which is a master regulator of mt dysfunction and Stimulator of Interferon Genes (STING)-dependent interferon (IFN) and inflammasome signaling. However, the role of BRCA1/2 proteins in regulating ZNFX1-induced mt dysfunction and STING-dependent immune signaling remains unclear. To test whether BRCA1/2 proteins influence ZNFX1 expression, binding and STING activation, we used multiple isogenic mice and human HGSC cell lines with wild-type (WT) or CRISPR knock-out (KO) BRCA1. We showed for the first time that ZNFX1 colocalized with BRCA1 but had reduced binding to the mitochondrial antiviral-signaling protein (MAVS) in BRCA1 WT cells, using proximity ligation assays. In contrast, in BRCA1 KO cells, BRCA1–ZNFX1 binding was lost, and ZNFX1–MAVS colocalization was significantly increased, suggesting that BRCA1 may have acted as a negative regulator of ZNFX1 activity. Consistent with these findings, BRCA1 KO cells showed significantly increased ZNFX1 expression compared to WT controls, as measured by qPCR. As expected by its known role in genome stability, BRCA1 KO cells demonstrated increased nuclear and cytosolic double-stranded DNA, as well as R-loops, compared with WT cells. Mitochondrial reactive oxygen species (ROS) were also increased in BRCA1 KO vs WT cells, as measured by flow cytometry with MitoSOX dye. Notably, compared with WT cells, STING-dependent IFN signaling was also elevated in BRCA1 KO cells, indicated by increased TBK1, CXCL10, IFI27, TNFα, and IRF3. Treatment with PARPis and DNMTis further enhanced ZNFX1 binding to MAVS and mt ROS in both BRCA1 KO and WT cells, suggesting that these agents could have amplified immune signaling. Validation of above-described signaling is ongoing in HGSC cell lines with naturally occurring BRCA1 mutations (SNU-251) or with BRCA1 methylation (OVCAR8 and A1847). Further studies are evaluating these effects in syngeneic mouse models to assess changes in the tumor microenvironment and in vivo therapeutic responses. This study revealed novel mechanisms linking BRCA1 loss to ZNFX1 activation, viral mimicry, and immune modulation. It highlighted the therapeutic potential of targeting ZNFX1 pathways to enhance treatment efficacy in BRCA1-deficient HGSC. Zahra Gohari, Feyruz V. Rassool, Lora Stojanovic, Stephen B. Baylin, Kenneth P. Nephew. BRCA1 regulates ZNFX1-induced mitochondrial dysfunction and STING activation in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B009.

High-grade serous ovarian carcinoma (HGSC), which accounts for ∼70% of ovarian carcinoma cases, is typically diagnosed at an advanced stage and carries a poor prognosis. Although immunotherapies can induce durable responses, overall response rates in HGSC remain modest, highlighting the need for new strategies to enhance antitumor immunity. Emerging evidence suggests epigenetic mechanisms regulate innate immune pathways, including Stimulator of Interferon Genes (STING), a key mediator of tumor immunosurveillance. Analysis of TCGA and GTEx datasets showed that STING expression is reduced in HGSC, particularly in TP53-mutant tumors, and that higher STING levels correlate with improved overall, progression-free, and disease-free survival. Compared to normal fallopian tube epithelial cells (FT190), HGSC cells exhibited reduced CXCL10 and CCL5 expression and impaired STING pathway activation, as evidenced by lower phospho-TBK1, phospho-IRF3, after dsDNA stimulation. STING knockout confirmed that cytokine production was STING-dependent. Interrogation of TCGA epigenetic data and methylation-specific qPCR revealed hypermethylation of the STING promoter in HGSC samples and cell lines (OVCAR3 and OVCAR5). Based on these findings, we hypothesized that DNA methyltransferase inhibitors (DNMTis) could restore STING expression through epigenetic reprogramming. Indeed, DNMTi treatment significantly reduced promoter methylation and dose-dependently increased STING and downstream chemokine (CXCL10, CCL5) expression. Given that these chemokines attract immune cells, we further analyzed TCGA tumors and observed enrichment of CD4+ and CD8+ T cell infiltration signatures in cases with high STING expression, suggesting that restoring STING may help reprogram the tumor microenvironment and sensitize HGSC to STING agonists. STING agonists have been in clinical trials in solid tumor studies, but responses to date have been suboptimal. Intravenous formulation of next generation STING agonist, CPD1 (Curadev Pharma) has demonstrated high levels of systemic safety in non-human primates and is currently undergoing dose escalation in patients with advanced solid cancers (MSKCC) and our first-in-human AML (UMGCCC). Initial studies with STING agonist CPD1 alone was not effective at inducing pathway activation. We therefore tested whether epigenetic re-activation of STING expression was necessary to restore signaling. We treated HGSC cells with DNMTi, STING agonist, or the combination. Only the combination significantly increased phospho-TBK1, phospho-IRF3, CXCL10 and CCL5 and induced apoptosis-dependent cell death. Combination therapy also markedly suppressed oncogenic phenotypes, including proliferation and colony formation. Together, these results demonstrate that epigenetic silencing contributes to defective STING signaling in HGSC. As inhibiting DNA methylation restores both STING expression and responsiveness to agonist stimulation, combining DNMTi with STING agonists represents a promising therapeutic strategy to enhance antitumor immunity in HGSC. Saranya Rajendran, Elnaz Abassi Farid, Ishani Chattopadhayay, Shu Zhang, Lalitha Holaly Sastry, Qiqi Xie, Jia Shen, Sheng Liu, Jun Wan, Stephen B. Baylin, Feyruz V. Rassool, Kenneth P. Nephew. Epigenetic reprogramming restores STING pathway activation in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A069.

Treatment for acute myeloid leukemia (AML) relapsed after or refractory to (R/R) standard therapies is an unmet clinical need. Efficacy of immunotherapies is being explored. Stimulator of Interferon Genes (STING) is an important component of the human innate immune system which generates a rapid initial inflammatory response to danger signals associated with cellular or tissue damage from pathogens, via effectors such as cytokines and chemokines. Cytosolic DNA is a danger signal that binds to the enzyme cGAS, generating cyclic dinucleotides (CDNs) that trigger the cGAS-STING pathway, leading to activation of the downstream transcription factors IRF3 and NFκB to induce pro-inflammatory Type I interferons and related cytokines. CDN site-targeting STING agonists tested in the clinic thus far have shown limited systemic tolerability and efficacy. CRD3874 is an allosteric, non-cytotoxic, small-molecule human STING agonist with a unique binding mode that generates robust Type I interferon responses while simultaneously blocking STING proton transport activity associated with pyroptosis and autophagy. The compound has demonstrated impressive tumor clearance in a range of pre-clinical models and high levels of systemic safety in non-human primates. (Pre-clinical) Allosteric binding was established using radioligand binding assays. Anti-cancer activity of CRD3874-SI by the intravenous (IV) route was evaluated in human STING knock-in (KI) C57BL/6 mice (genOway, Lyon, France). Inflammasome and autophagy markers were studied in human and mouse cells. (Clinical) Patients with R/R AML are eligible for a Phase 1 clinical trial testing CRD3874-SI administered IV at six sequential dose levels, using a 3+3 design, with pharmacokinetic and pharmacodynamic correlative studies at the University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, Maryland (NCT06626633). Maria R. Baer, Vu H. Duong, Oyinkansola Arasanmi, Devon T. Issac, Sunita Philip, Megan Wagner, Ar'Lena Hassan, Feyruz V. Rassool, Sandip Middya, Ritesh Shrivastava, Anindita Middya, Nagaswamy Mane, Thanilsana Soram, Kavita Puniya, Nidhi Rawat, Debjani Chakraborty, Rubeena R. Mansuri, Anul Singh, Rajib Ghosh, Anuj Gautam, Sourav Basu, Monali Banerjee, Arjun Surya. Intravenous infusion of the allosteric small molecule STING agonist CRD3874-SI in patients with relapsed or refractory acute myeloid leukemia: A phase I trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT191.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are effective in patients with germline BRCA 1/2 and PALB2 mutations but have been largely ineffective as monotherapy in others. PARP interacts with, and is recruited to, DNA damage sites along with epigenetic factors, such as DNA methyltransferase 1 (DNMT1). In addition to increasing PARP-trapping, inhibitors of DNMT modulate ROS-cAMP/PKA signaling and induce a pathogen mimicry, inflammasome signaling response and a ‘BRCAness phenotype’ that further sensitizes cells to PARPi. In preclinical in vitro and in vivo studies, combined DNMTi + PARPi therapy was effective in both triple negative (TNBC) and hormone resistant (HRBC) models with intact BRCA. We conducted a phase I study combining the oral DNMTi ASTX727 with the PARPi talazoparib in patients (pts) with previously treated TNBC or HRBC; pts with deleterious mutations of BRCA were excluded. Pts with TNBC had received at least one prior chemotherapy and pts with HRBC had received prior endocrine therapy with a cyclin-dependent kinase inhibitor for metastatic disease. An ECOG PS 0-1 and adequate organ function was required. A classical 3+3 design guided dose escalation/de-escalation with dose-limiting toxicity (DLT) defined as Grade 4 neutropenia or thrombocytopenia lasting ≥7 days, or clinically significant grade ≥3 non-hematologic toxicity in cycle 1; 28 days constituted each cycle. Serial peripheral blood mononuclear cells (PBMCs) were analyzed for changes in methylation using the Infinium Methylation EPIC BeadChip and LINE1 sequencing. 34 evaluable pts were enrolled and treated in 8 dose cohorts. Median age was 59 years, 12% identified as Black. Myelosuppression was common with grade ≥3 neutropenia in 42% and grade 3 anemia and thrombocytopenia in 13%. DLT was limited to neutropenia. Efficacy was assessed in 29 pts. There were no objective responses, 6 pts had stable disease persisting for > 4 months in 3 pts. LINE1 demethylation ranged from ∼2-10% and immune-specific CpGs (methylation in immune cells) changed 1-5% by differential methylation locus analysis at Day 15. Methylation changes were not dose dependent. ASTX727 plus talazoparib produces significant myelosuppression but is otherwise well tolerated. Low dose ASTX727 (10 mg decitabine: 100 mg cedazuridine) on Days 1,3,5 with talazoparib 0.5 mg daily on Days 1-21 of each 28-day cycle is recommended for phase II trials. Methylation changes in PBMCs were detected and some heavily pre-treated pts had prolonged stable disease despite the attenuated doses. Inhibitors of poly (ADP-ribose) polymerase (PARPi) have significant clinical benefit in patients with advanced breast cancer who harbor deleterious mutations of BRCA1, BRCA2, or PALB2 but have had limited benefit in those without mutations. Similarly, epigenetic therapies such as the DNA methyltransferase inhibitors (DNMTi) slow disease progression in some hematologic malignancies and myelodysplastic syndromes but have not found a role in solid tumors. Despite limited clinical activity as monotherapy, the combination of PARP and DNMTi significantly inhibited tumor growth in several preclinical models with intact BRCA. In the first clinical trial combining talazoparib and ASTX727, myelosuppression limited drug exposure but the combination was otherwise well tolerated. Strategies to reduce myelosuppression with this combination should be explored. Despite the attenuated doses, methylation changes in peripheral blood mononuclear cells (PMBCs) and clinical benefit were observed; neither were clearly dose dependent.

Background: Poly (ADP-ribose) inhibitors (PARPi) are effective in patients (pts) with germline BRCA 1/2 and PALB2 mutations but have been largely ineffective as monotherpy in others. PARP interacts with, and is recruited to, DNA damage sites along with epigenetic factors, such as DNA methyltransferase 1 (DNMT 1). In addition to increasing PARP-trapping, inhibitors of DNMT modulate ROS-cAMO-PKA signaling and induce a pathogen mimicry, inflammasome signaling response and a 'BRCAness phenotype' that further sensitizes cells to PARPi. In preclinical in vitro and in vivo studies. combined DNMTi + PARPi therapy was effective in both triple negative (TNBC) and hormone resistant (HRBC) models with intact BRCA. Methods: We conducted a phase I study combining the oral DNMT1 ASTX727 with the PARPi talazoparib in pts with previously treated TNBC or HRBC; pts with dleterious mutations of BRCA or PALB2 were excluded. Pts. with TNBC had received at least one prior chemotherapy and pts with HRBC had received proir endocrine therapy with a cyclin-dependent kinase inhibitor for metastatic disease. An ECOG PS 0-1 and adequate organ function was required. A classical 3+3 design guided dose escalation/de-escalation with dose limiting toxicity (DLT) defined as Grade 4 neutropenia or thrombocytopenia lasting > 7 days, or clinically significant grade > 3 non-hematologic toxicity in cycle 1; 28 days constituted each cycle. Serial peripheral blood mononuclear cells (PBMCs) were analyzed for changes in methylation using the Infinium MethylationEPIC BeadChip and LINE1 sequencing. Results: 34 evaluable pts were enrolled and treated in 8 dose cohorts (Table). Median age was 59 years, 12% identified as Black. Myelosuppresson was common with grade > 3 neutropenia in 42% and grade 3 anemia and thrombocytopenia each in 13%. DLT was limited to neutropenia. Efficacy was assessed in 29 pts. There were no objective responses, 6 pts had stable disease persisting for > 4 months in 3 pts. LINE1 demethylation ranged from 2-12% and immune-specific CpGs (methylation in immune cells) changed by 1-5% by differential methylation locus analysis at Day 15.Methylation changes were not dose dependent. Conclusions: ASTX727 plus talazoparib produces significant myelosuppression but is otherwise well tolerated. Attenuated dosing identified dose level 1LDseq or 1LDcon for phase II trials. Methylation changes in PBMCs were detected and some heavilty pre-treated pts had prolonged stable disease. Citation Format: Kathy Miller, Alexandra Thomas, Sandra Althouse, Yong Zang, Erin Condor, Ryan Burgos, Bryan Schneider, Tarah Ballinger, Emily Douglas, Katherine Ansley, H Josh Jang, Jean-Pierra Issa, Kenneth P Nephew, Feyruz V Rassool. A Phase I Study of ASTX727 plus Talazoparib in Patients with Triple Negative or Hormone Resistant/HER2-negative Metastatic Breast Cancer and non-mutated BRCA [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-16.

Breast cancers with BRCA1 or BRCA2 mutations are defective in repair of DNA double-strand breaks by homologous recombination, resulting in compensatory error-prone repair that causes genomic instability. Poly(ADP-ribose) polymerase inhibitors (PARPi) are FDA-approved to treat homologous recombination–defective cancers, inducing therapy responses by synthetic lethality. PARPis increase micronuclei formation and cytosolic double-stranded DNA accumulation, activating stimulator of interferon genes (STING). Activation of STING can mediate anticancer innate immune responses by increasing T-cell infiltration into the tumor microenvironment. However, PARPi responses are not durable, and therapy resistance ensues with limited therapeutic options available for these patients. Using PARPi-sensitive and -resistant patient-derived xenografts and mouse-derived allografts, Pedretti and colleagues show in this issue of Cancer Research that the PARPi olaparib in combination with the next-generation STING agonist diABZI can overcome PARPi resistance in a manner dependent on NK cell function in the tumor microenvironment. Their study highlights a novel component of the STING-dependent innate immune response repertoire required for fighting PARPi-resistant cancer. Potent and specific next-generation STING agonists are being tested in the clinic in solid and liquid tumors, indicating a resurgence of these drugs after a long period of modest clinical activity, with a special focus on combination therapy strategies to fight therapy-resistant cancer. See related article by Pedretti et al., p. 1888

Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Although we and others have demonstrated that mitochondria (mt) are an important gateway for antiviral inflammasome signaling, the underlying mechanism in cancer remains to be fully elucidated. In this regard, our studies of the little-known gene, NFX1-type zinc finger–containing 1 (ZNFX1), show that ZNFX1 acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In a panel of ovarian cancer (OC) cell lines, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells further show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, further supported by bioinformatics analysis of RNA seq data, thus defining ZNFX1 as a gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Pathway analysis of ZNFX1 KO vs wild-type RNAseq data, combined with in vitro and in vivo validation assays, demonstrate increased proliferation and epithelial to mesenchymal transition, suggesting that not only is ZNFX1 a master regular of the above mt dynamics but also a new tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, increased tumor growth in vivo. Taken together, this work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Feyruz V Rassool, Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Saranya Rajendran, Colin M Coon, Elnaz Abbasi, Sheng Liu, Jun Wan, Michael J Topper, Stephen B Baylin, Kenneth P Nephew. ZNFX1 is a master regulator and tumor suppressor in epigenetically-induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr PR001.

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

DNA methyltransferase (DNMT) and PARP inhibitors induce a stimulator of IFN gene-dependent pathogen mimicry response (PMR) in ovarian and other cancers. In this study, we showed that combining DNMT and PARP inhibitors upregulates expression of the nucleic acid sensor NFX1-type zinc finger-containing 1 (ZNFX1) protein. ZNFX1 mediated the induction of PMR in mitochondria, serving as a gateway for stimulator of IFN gene-dependent IFN/inflammasome signaling. Loss of ZNFX1 in ovarian cancer cells promoted proliferation and spheroid formation in vitro and tumor growth in vivo. In patient ovarian cancer databases, expression of ZNFX1 was elevated in advanced stage disease, and ZNFX1 expression alone significantly correlated with an increase in overall survival in a phase III trial for patients with therapy-resistant ovarian cancer receiving bevacizumab in combination with chemotherapy. RNA sequencing revealed an association between inflammasome signaling through ZNFX1 and abnormal vasculogenesis. Together, this study identified that ZNFX1 is a tumor suppressor that controls PMR signaling through mitochondria and may serve as a biomarker to facilitate personalized therapy in patients with ovarian cancer.Significance: DNMT and PARP inhibitors induce a nucleic acid sensor, ZNFX1, that serves as a mitochondrial gateway to STING-dependent inflammasome signaling with tumor suppressor properties in ovarian cancer.

DNA methyltransferase and poly(ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon (IFN) genes (STING)-dependent pathogen mimicry response (PMR) in ovarian (OC) and other cancers. We now show that combining DNMTis and PARPis upregulates expression of a little-studied nucleic-acid sensor, NFX1-type zinc finger-containing 1 protein (ZNFX1). We demonstrate that ZNFX1 is a novel master regulator for PMR induction in mitochondria, serving as a gateway for STING-dependent PMR. In patient OC databases, high ZNFX1 expression levels correlate with advanced stage disease. ZNFX1 expression alone significantly correlates with an increase in overall survival in a phase 3 trial for therapy-resistant OC patients receiving bevacizumab in combination with chemotherapy. In correlative RNA-seq data, inflammasome signaling through ZNFX1 correlates with abnormal vasculogenesis. ZNFX1 controls PMR signaling through the mitochondria and may serve as a biomarker to facilitate offering personalized therapy in OC patients, highlighting the strong translational significance of our findings.