Abstract 5865: Novel allosteric STING agonists in combination with DNA methyltransferase inhibitors induce an interferon-driven apoptotic response in <i>TP53</i>-mutated acute myeloid leukemia

Abstract 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 &amp;lt;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. Citation Format: 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.