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.

Supplementary Figure 8: PD-L1 levels in tumor or tumor microenvironment does not associate with tumor progression or control.

Background TP53 mutations in acute myeloid leukemia (AML) are associated with resistance to chemotherapy and poor prognosis. Patients with TP53-mutated AML respond to DNA methyltransferase inhibitors (DNMTis) but responses are brief. Immunotherapeutic approaches are being explored. Stimulator of interferon genes (STING) is a key innate immune driver that activates interferon (IFN) and nuclear factor Kappa B (NFkB)/tumor necrosis factor-alpha (TNF) signaling. STING is regulated epigenetically, and we previously reported that DNMTi treatment increases basal STING transcripts and epigenetically-silenced endogenous retroviruses (ERVs), increasing cytosolic dsRNA and IFN signaling through a mechanism known as viral mimicry. Thus, increasing basal STING expression with DNMTI treatment should increase substrate for activation by STING agonists, making this a potentially attractive novel combination therapy in AML. STING activation was also recently shown to trigger p53-independent apoptosis, and STING agonists have shown a synergistic effect with BH3-mimetics in TP53-mutated AML cells. Here we studied the next-generation allosteric STING agonist C92 from Curadev Pharma, which potently binds and activates all human STING variants, in combination with the DNMTi decitabine (DAC) in TP53-mutated AML. Methods To test the hypothesis that DNMTis synergize with STING agonists, we treated AML cell lines with wild-type (WT) (MOLM-14, OCI AML1) and mutated TP53 (KG1, KASUMI, U937) and patient samples (N=5-10) with C92 in combination with decitabine (DAC). We also utilized CRISPR-Cas9 gene editing to knock out TP53 in MOLM-14 and OCI AML1 in order to confirm observed results from mutated cell lines. To assess synergy of the two drugs, we utilized MTS assays and the Chou-Talalay combination index. To identify the molecular pathways and genes activated by the drug combination in TP53-mutated vs -WT AML, we performed ribosomal depleted RNA-seq after C92 and/or DAC treatment. We validated gene expression using qPCR of RNA, Western blotting of proteins and cytokine (including CXCL10, IFN a, b g, TNFa) release by ELISA assays. To investigate apoptosis in TP53 KO vs WT, we measured Annexin V labeling by flow cytometry pre and post treatment with the 2 drugs, administered alone and in combination in the presence of the pan-caspase apoptosis inhibitor Z-VAD-FMK, the JAK/STAT inhibitor ruxilitinib and the TBK1 inhibitor amlexanox. Finally, to test efficacy of the combination therapy in vivo, we injected a humanized AML mouse model with MOLM-14 cells expressing luciferase, and measured leukemia burden by non-invasive luciferin imaging. To measure cytokines, T cells and myeloid in the leukemia microenvironment (LME), we performed IHC in spleen and bone marrow, pre and post treatment. We also quantitated cytokines in the plasma extracted from peripheral blood using ELISA assays. Results The STING agonist C92 was highly synergistic with DAC in AML cells, with combination index of <1 in all AML cell lines, ranging from 0.1-0.8. This drug combination induces an interferon signature in TP53-mutated AML cells, compared with TP53-WT, with increased expression (p<0.05) of Th1 type chemokines (CXCL10, CXCL11), RNA detection- (DHX58, EIFAK2, IFIH1, OASL, PARP9, RIGI), and interferon cell death-associated genes (IFI27, IFI44L, IFIT2, MX1, OAS1, OAS2, OAS3, and TNFSF10). TP53-mutated AML cells, compared with TP53-WT, induce interferon-driven cell death by apoptosis, as demonstrated by Z-VAD-FMK, ruxilitinib and amlexanox treatment (p<0.05). Finally, C92 and DAC combination decreased the AML burden in vivo and increased cytokines and T cells in the LME, compared to single-agent therapy and vehicle controls (p<0.05). Conclusion Our results demonstrate that next-generation STING agonist C92 in combination with DNA methyltransferase inhibitor induces interferon-driven apoptosis in TP53-mutated acute myeloid leukemia, compared with TP53-WT AML. The more advanced analogue CRD3874-SI is currently in a Phase I clinical trial in relapsed/refractory AML at UMGCCC, and our data here support development of a clinical trial combining CRD3874-SI with DAC for patients with TP53- mutated AML.

Supplementary Figure 2: Modest treatment-induced DNA methylation changes in UC tumors.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide, with 5-year survival rates of less than 30%. Therefore, there is a critical need for novel combinatorial strategies to enhance the efficacy of current approaches. A current approach for the treatment of advanced NSCLC was developed in our lab and others utilizing DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi) to sensitize patients to immunotherapy. However, most patients do not benefit durably from epigenetic therapy alone or in combination with other treatments. We and others have shown that STAT3 activation is increased after epigenetic therapy. Because persistent STAT3 signaling is associated with poor prognosis and promotes cancer cell survival in multiple cancer types, including NSCLC, we treated a panel of 19 human NSCLC cell lines with the STAT3 inhibitor, C188-9, and epigenetic therapy and identified 12 cell lines demonstrating a synergistic reduction in cancer cell proliferation. We further demonstrated a durable reduction in anchorage-dependent and -independent colony formation two weeks post-treatment with STAT3i+epigenetic therapy. Interestingly, cell lines with Ras and KRAS+STK11 mutations were positively associated with response. In addition to phenotypic characterization, we defined the transcriptome consequences of our novel therapeutic paradigm by RNA sequencing. These data showed that STAT3i+epigenetic therapy suppressed cell cycle and activated apoptotic pathways, which we further validated by immunoblotting and flow cytometry. Notably, we observed significant perturbation of gene sets involved in key bioenergetic pathways, namely oxidative phosphorylation, glycolysis, and glutamine metabolism. When considered as a whole, these pathway-level data indicated that glutamine metabolism might be significantly perturbed, and so exogenous glutamine might be conditionally essential in combination-treated cells. Utilizing a glutaminase inhibitor, we confirmed sensitization to glutamate reduction in STAT3i+epigenetic therapy treated cells. Additionally, we observed significant induction of IL-24 in STAT3i-treated groups, with the highest induction (>2000 fold) in the STAT3i+epigenetic therapy group. IL-24, silenced in most cancer cells, is a tumor-suppressive cytokine shown to induce cancer cell death, cancer stem cell differentiation, and sensitization to cytotoxic therapies. Therefore, we hypothesized that the anti-cancer effects of STAT3i+epigenetic therapy are mediated through IL-24 induction. To test this hypothesis, we treated NSCLC cells with exogenous IL-24 and confirmed epigenetic therapy-induced sensitization to IL-24. In summary, we define STAT3 as an actionable target that synergizes with epigenetic therapy to diminish the ability of NSCLC cells to proliferate and survive in anchorage-dependent and -independent conditions, associated with significant induction of IL-24. Julia An, Calvin Chen, Ying-Yu Chen, Jina Park, Stephen Baylin, Michael Topper. Targeting STAT3 in combination with epigenetic therapy induces tumor suppressive IL-24 in non-small cell lung cancer [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 LB421.

Supplementary Figure 3: Copy number variation detected by SeSAMe from DNA methylation data.

TP53-mutated acute myeloid leukemia (AML) has dismal outcomes; new therapies are needed, and immunotherapeutic approaches are being explored. STimulator of INterferon Genes (STING) is a key innate immune driver that activates interferon (IFN) and nuclear factor Kappa B (NFκB)/tumor necrosis factor-alpha (TNFα) signaling. We previously reported that phospho (activated)-STING expression is higher in TP53-mutated AML and that DNA methyltransferase inhibitors (DNMTis) demethylate epigenetically silenced endogenous retroviruses (ERVs), increasing cytosolic dsRNA and IFN signaling in a viral mimicry mechanism. Moreover DNMTis upregulate STING transcription. Thus STING agonists in combination with DNMTis could be an attractive therapy in TP53-mutated AML. The next-generation allosteric STING agonists CPD 1 and 2 potently bind and activate all human STING variants. While CPD 1 has been extensively used for in vitro cellular studies, the more advanced analogue CPD 2 (CRD3874-SI) is currently in Phase I clinical trials in patients with solid cancers (MSKCC) and AML (UMGCCC). We perfomed MTS assays in TP53-mutated and -WT AML cell lines and primary cells (N=4) treated with CPD 1 and DNMTi decitabine (DAC) combination in vitro. Decreased viability was particularly noted in TP53-mutated cells, and the drug combination was synergistic, with combination index <1. To test the transcriptome-wide effects of CPD 1 and DAC, we performed ribosomal depleted RNA-seq on WT and TP53 CRISPR KO MOLM-14 AML cells after treatment for 3 days. Differentially expressed genes (DEGs) were markedly increased in TP53 CRISPR KO MOLM-14 treated with CPD 1 and CPD 1 + DAC. Moreover, increased repetitive element (RE) expression was largely restricted to TP53-KO cells treated with CPD 1 and CPD 1 + DAC. Pathway analyses for both CPD 1- and CPD 1 + DAC-treated TP53-KO cells showed conserved positive enrichment of gene sets associated with Type I/II IFN, TNFα, and apoptosis. Drug combination-specific augmented genes resided in critical interferon gene categories such as Th1 type chemokines (CXCL10, CXCL11), RNA detection (DHX58, EIFAK2, IFIH1, OASL, PARP9, RIGI), and IFN signaling-induced cell death (IFI27, IFI44L, IFIT2, MX1, OAS1, OAS2, OAS3, and TNFSF10), thus potentially combining substrate, sensors, and effectors in a suicidal interferon-driven apoptosis response in TP53 KO cells. qPCR and apoptosis detection by Annexin V staining validated these findings in TP53-mutated vs -WT AML cell lines and patient samples. Treatment with the IFN inhibitor ruxolitinib or the pan-caspase inhibitor ZVAD rescued IFN-driven apoptosis. Finally CPD 1 in combination with DAC significantly reduced leukemia burden in humanized AML mouse models. These results support development of a clinical trial combining CRD3874-SI with DAC for patients with TP53-mutated AML. Kaushlendra Tripathi, Lora Stojanovic, Monali BanerJee, Sandip Middya, Ritesh Srivastava, Arjun surya, Stephen B. Baylin, Michael Topper, Maria R. Baer, Fervez Rassool. Novel allosteric STING agonists in combination with DNA methyltransferase inhibitors induce an interferon-driven apoptotic response in TP53-mutated acute myeloid leukemia [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 5865.

Supplementary Figure 10: Peripheral immune cell dynamics induced by combination therapy.

Supplementary Figure 12: HLA-DR and NKG2D abundance on peripheral lymphocytes associate with longer progression free survival.

Supplementary TablesSupplementary Table 1: Patient and tumor sample informationSupplementary Table 2: Tumor mutation burdenSupplementary Table 3: Unfiltered whole exome sequencing mutation analysisSupplementary Table 4: Filtered whole exome sequencing mutation analysisSupplementary Table 5: Immunohistochemistry scores of tumorsSupplementary Table 6: FACS panel for PBMC analysisSupplementary Table 7: Geometric mean fluorescence intensity (GMFI) from PBMC FACSSupplementary Table 8: Representativeness of Study Participants

Supplementary Figure 7: Representative IHC staining of additional PD and SD tumors in pre-treatment and post-treatment timepoint from additional patients.

Despite much progress in targeting the MYC oncoprotein, combination treatment strategies are needed to exploit this molecular vulnerability. To this end, we interrogated transcriptome data from cancer cell lines treated with MYC inhibitors and identified HDAC5 and HDAC9, both class IIa histone deacetylases (HDACs), as potential therapeutic targets. Notably, these therapeutically actionable HDAC isoforms are known augmenters of several hallmarks of cancer. Dual targeting of MYC and class IIa HDACs induces a significant reduction in viability for non-small cell lung cancer (NSCLC) cell lines with high MYC and mitochondrial activity. Additionally, combination treatment induces a robust MYC suppression with mitochondrial reactive oxygen species (ROS) elevation, which has a causal relationship with therapeutic efficacy. Confirmation of in vivo efficacy was pursued in several animal models, with subsequent molecular-correlate derivation confirming the importance of MYC depletion and mitochondrial dysfunction in drug efficacy. Ultimately, we define a therapeutic approach combining MYC- and class IIa HDAC-inhibition to potentiate anti-tumor efficacy in NSCLC.

Supplementary Figure 4: Weak correlation of TE expression induction with DNA methylation change and viral mimicry activation.

Supplementary Figure 1: Patient recruitment and sample collection overview.

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.

Supplementary Figure 5: Overall tumor mutation burden and frequency of mutations in cancer-associated genes in UC tumors.

Supplementary Figure 6: Gene Set Enrichment Analysis reveals immune-related pathways discriminate PD and SD tumors.

Supplementary Figure 9: CD8 infiltration in tumor is associated with survival.

Supplementary Figure 11: Peripheral immune cells are activated mid-treatment cycle.