Next generation sting agonist in combination with DNA methyltransferase inhibitors induce an apoptotic cell death mechanism driven by apoptosis in TP53-mutated Acute Myeloid Leukemia

Abstract 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.