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.

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2025
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