ATR Inhibitor Elimusertib Suppresses Drug Persister Clones in TP53-Mutated Acute Myeloid Leukemia Via CGAS-Sting-Mediated Cell Death

Acute myeloid leukemia (AML) with tumor protein p53 (TP53) mutations, comprising 7-8% of all AML cases, is a highly malignant subtype, exhibiting poor response to initial treatment and a higher relapse rate. Despite this, studies on treatment resistance in TP53-mutated AML and the development of optimized treatment strategies remain insufficient. This study proposes a novel drug combination strategy to inhibit treatment-resistant clones in AML with TP53 mutations.

To investigate drug resistance in TP53-mutated AML, we used AML cell lines with TP53 missense mutations (mut) in the TP53 DNA-binding domain (DBD) (Kasumi-1, Mono-Mac-1) and TP53 wild-type (wt) cell lines (OCI-AML2, MV4-11). Drug-tolerant persister cells (DTPCs) resistant to the AML first-line treatment drugs cytarabine and idarubicin were isolated from these cell lines (Melat T. Gebru et al., Blood, 2020). Under combined treatment in an optimal ratio showing a synergistic cytotoxic effect, TP53 mut cells showed higher survival rates than TP53 wt cells, and the surviving TP53 mut DTPCs could be further cultured post-treatment. Moreover, TP53 mut AML DTPCs showed significant resistant to repeated treatment and exhibited transient G2/M phase cell cycle arrest, with increased tumorigenicity.

Next, we sought to discover clinically approved or clinical trial drugs (Phase I/II) predicted to be effective against TP53 mut AML DTPCs. Forty-eight drugs were selected to test cytotoxicity on TP53 mut AML DTPCs, including 12 potential inhibitors extracted from the CMAP (Connectivity Map) database using TCGA (The Cancer Genome Atlas) transcriptome data of TP53-mutated AML patients, 5 TP53-targeted drugs, 19 leukemic stem cell (LSC)-targeted drugs, and 12 anti-cancer drugs for hematologic malignancies. Among these, the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor elimusertib (BAY1895344) effectively inhibited TP53 mut AML DTPCs at low doses, similar to known inhibitors the CHK1 inhibitor (SRA737) and the Wee1 inhibitor (Adavosertib). Elimusertib treatment increased the apoptotic population (subG1) of TP53 mut AML DTPCs while significantly decreasing the G2/M phase cell cycle-arrested populations. Furthermore, the triple combination treatment of cytarabine, idarubicin, and elimusertib effectively suppressed DTPC formation in TP53 mut AML cell lines.

In TP53 mut AML cells, combined treatment of cytarabine and idarubicin increased the levels of p53 and p21 proteins and upregulated G2/M phase cell cycle checkpoint regulators, cdc25c, phosphorylated-CDK1 (p-CDK1), and Cyclin B. However, treatment with elimusertib suppressed these cell cycle regulators-cdc25c, p-CDK1, and Cyclin B-by inhibiting the ATR-CHK1 pathway. Additionally, blocking the ATR-CHK1 pathway significantly decreased the accumulation of γH2AX and increased the activation of the STING. These results demonstrate that elimusertib treatment reactivates the cell cycle in TP53 mut AML DTPCs arrested in the G2/M phase, leading to an accumulation of DNA replication stress.

To further investigate elimusertib-induced DNA replication stress, we performed RNA sequencing using TP53 mut AML PC, DTPCs, and elimusertib-treated DTPCs. As a result, TP53 mut AML DTPCs exhibited activation of the Leukocyte Migration signaling pathway (NES=1.49, q=0.00266), indicative of AML aggressiveness. In DTPCs, elimusertib treatment significantly activated Type I and II interferon (IFN) response signaling (NES=1.927, q=1.99E-08 and NES=1.735, q=9.43E-06, respectively) and upregulated the expression of STAT1, NF-kB, PARP9, and CXCL10, including 6 ISGs (SIGLEC1, IFI27, IFI44L, IFIT1, ISG15, and RSAD2) that are associated with cGAS-STING-Interferon pathway, indicating DNA replication stress and genomic instability.

Collectively, elimusertib effectively inhibits drug-tolerant TP53-mutant AML cells by reactivating the cell cycle. This consequently results in cGAS-STING-mediated cell death following irreparable DNA replication stress accompanied by p53 dysfunction. Our findings provide a basis for developing optimized treatment strategies for patients with TP53-mutant AML using the ATR inhibitor elimusertib.

Koh:Sanofi Genzyme: Research Funding; Amgen: Speakers Bureau; Tomocube: Current holder of stock options in a privately-held company; DeppMetrics: Current equity holder in private company; Proteina: Current holder of stock options in a privately-held company; GC Cell: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Johnson & Johnson - Janssen: Consultancy; Celltrion: Honoraria, Speakers Bureau; Curocell: Current equity holder in publicly-traded company; NOBO medicine: Current equity holder in private company; GSK: Consultancy. Yoon:Janssen, Novartis, F. Hoffmann-La Roche Ltd, Genentech, Inc.: Consultancy; Janssen: Honoraria; F. Hoffmann-La Roche Ltd, Genentech, Inc.: Research Funding. Shin:Boryung Pharmaceuticals, Abbvie: Research Funding.