Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia

DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p<0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p<0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.

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

DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML

The Mevalonate Pathway Is a Therapeutic Target in TP53 Mutant Acute Myeloid Leukemia

Immune and Epigenetic Landscape of TP53-mutated Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes (HR-MDS)

Activation of Cgas-Sting Pathway By Hypomethylating Agent in <i>TP53</i> Mutated MDS/AML

DNA Methyltransferase Inhibitors Promote Homologous Recombination Deficiency through Induction of Immune Signaling, Sensitizing Acute Myeloid Leukemia Cells to PARP Inhibitors

Targeting the Mevalonate Pathway in the Mitochondria-Dependent Chemotherapy Resistance of TP53 Mutant Acute Myeloid Leukemia

Synergistic Effect of Trifluridine and PARPi Combination in Targeting <i>TP53</i>-Mutated AML

Inflammatory and metabolic gene signatures predict overall survival and response to venetoclax-based therapy in TP53 mutant AML

Objective Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 1‐year overall survival of &lt;5%. Effective treatment options are limited and current standard‐of‐care includes the hypomethylating agents (HMA) decitabine and azacytidine. While inhibition of kinases involved in cell cycle regulation has been shown to induce synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP‐0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, CHEK1/2, and other cell cycle regulators (Jeon JY et al. JCI Insight 2020), thus providing scientific rationale to evaluate TP‐0903 activity in TP53 mutant AML. Methods To generate an in vitro model of TP53 mutant AML, we isolated single‐cell clones containing mutant (R248W) or wild‐type (WT) TP53 from the established MV4‐11 AML cell line; regulation of p53 targets (MDM2, p21) following gamma irradiation and inhibition of p‐AURKA and p‐CHEK1 by TP‐0903 were assessed by immunoblot. Using these and additional TP53 mutant AML cell lines (Kasumi‐1, HL‐60), i n vitro efficacy of TP‐0903 alone and in combination with HMA was assessed in viability (MTT) and apoptosis (Annexin V) assays. In vivo efficacy studies were conducted in NSG mice following intravenous injection of HL‐60 or luciferase‐tagged MV4‐11/ TP53 ‐R248W cells. Mice (5‐10 per treatment cohort) were treated with vehicle, TP‐0903 (50 mg/kg orally; 5 days on/2 days off), decitabine (0.2‐0.4 mg/kg i.p.; 4 days on/10 days off) or the combination. Whole body bioluminescence imaging was performed weekly and median survival was determined. Results Compared to the clone with WT TP53 , we observed a lack of MDM2 and p21 induction in MV4‐11/ TP53 ‐R248W cells following gamma irradiation. In vitro , TP‐0903 inhibited pAURKA and pCHEK1 at 50 nM, inhibited cell viability (IC 50 values, 12‐40nM), and induced apoptosis at 20‐50nM. The combination of TP‐0903 with HMA was additive to synergistic in all AML cell lines evaluated. In the HL‐60 xenograft model, the TP‐0903/decitabine combination prolonged median survival (75 days) compared to cohorts of mice treated with TP‐0903 (63 days), decitabine (55 days), or vehicle (46 days) (P&lt;0.0001). In the MV4‐11/ TP53 ‐R248W xenograft model, bioluminescence imaging showed that TP‐0903 alone or in combination with decitabine was more effective in suppressing the outgrowth of leukemia cells compared to mice treated with vehicle or decitabine alone (P&lt;0.05); survival analysis is ongoing. Conclusions TP‐0903 was effective in all evaluated preclinical models of TP53 mutant AML. Together, these results provide scientific premise for the initiation of a Phase 1b/2 trial of TP‐0903 in combination with decitabine in TP53 mutant/complex karyotype AML under the umbrella Beat AML Master Trial.

Gene Expression Analysis Reveals a Subset of TP53mutant-like AML with Wild Type TP53 and Poor Prognosis

Prognosis for FLT3-ITD positive acute myeloid leukemia with high allelic ratio (>0.5) is poor, particularly in relapse, refractory to or unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. The combined use of compounds targeting both the mutated FLT3 receptor and cellular p53 inhibitors might be a promising treatment option for this poor risk leukemia subset. We therefore assessed MDM2 and FLT3 inhibitors as well as cytotoxic compounds used for conventional induction treatment as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. Acute myeloid leukemia cells represented all major morphologic and molecular subtypes with normal karyotype, including FLT3-ITD (>0.5) and FLT3 wild type, NPM1 mutant and NPM1 wild type, as well as TP53 mutant and TP53 wild type cell lines. Acute myeloid leukemia cells with mutated or deleted TP53 were resistant to MDM2- and FLT3-inhibitors. FLT3-ITD positive TP53 wild type acute myeloid leukemia cells were significantly more susceptible to FLT3-inhibitors than FLT3-ITD negative TP53 wild type cells. The presence of a NPM1 mutation reduced the susceptibility of TP53 wild type acute myeloid leukemia cells to the MDM2 inhibitor NVP-HDM201. Moreover, the combined use of MDM2- and FLT3-inhibitors was superior to single agent treatment, and the combination of midostaurin and NVP-HDM201 was as specific and effective against FLT3-ITD positive TP53 wild type cells as the combination of midostaurin with conventional induction therapy. In summary, the combined use of the MDM2 inhibitor NVP-HDM201 and the FLT3 inhibitor midostaurin was a most effective and specific treatment to target TP53 and NPM1 wild type acute myeloid leukemia cells with high allelic FLT3-ITD ratio. These data suggest that the combined use of NVP-HDM201 and midostaurin might be a promising treatment option particularly in FLT3-ITD positive acute myeloid leukemia relapsed or refractory to conventional therapy.

TP53 is the most commonly mutated gene in human cancers and was the first tumor suppressor gene to be discovered in the history of medical science. Mutations in the TP53 gene occur at various genetic locations and exhibit significant heterogeneity among patients. Mutations occurring primarily within the DNA-binding domain of TP53 result in the loss of the p53 protein's DNA-binding capability. However, a complex phenotypic landscape often combines gain-of-function, dominant negative, or altered specificity features. This complexity poses a significant challenge in developing an effective treatment strategy, which eradicates TP53-mutated cancer clones. This review summarizes the current understanding of TP53 mutations in AML and their implications. TP53 mutation in AML: In patients with acute myeloid leukemia (AML), six hotspot mutations (R175H, G245S, R248Q/W, R249S, R273H/S, and R282W) within the DNA-binding domain are common. TP53 mutations are frequently associated with a complex karyotype and subgroups of therapy-related or secondary AML. The presence of TP53 mutation is considered as a poor prognostic factor. TP53-mutated AML is even classified as a distinct subgroup of AML by itself, as TP53-mutated AML exhibits a significantly distinct landscape in terms of co-mutation and gene expression profiles compared with wildtype (WT)-TP53 AML. To better predict the prognosis in cancer patients with different TP53 mutations, several predictive scoring systems have been proposed based on screening experiments, to assess the aggressiveness of TP53-mutated cancer cells. Among those scoring systems, a relative fitness score (RFS) could be applied to AML patients with TP53 mutations in terms of overall survival (OS) and event-free survival (EFS). The current standard treatment, which includes cytotoxic chemotherapy and allogeneic hematopoietic stem cell transplantation, is largely ineffective for patients with TP53-mutated AML. Consequently, most patients with TP53-mutated AML succumb to leukemia within several months, despite active anticancer treatment. Decitabine, a hypomethylating agent, is known to be relatively effective in patients with AML. Numerous trials are ongoing to investigate the effects of novel drugs combined with hypomethylating agents, TP53-targeting agents or immunologic agents. Developing an effective treatment strategy for TP53-mutated AML through innovative and multidisciplinary research is an urgent task. Directly targeting mutated TP53 holds promise as an approach to combating TP53-mutated AML, and recent developments in immunologic agents for AML offer hope in this field.

Therapy-related myeloid neoplasms following treatment with PARP inhibitors: new molecular insights