Abstract A027: ADAR1 loss in combination with PKR activation leads to anti-tumor effects in TP53-Mut AML

Abstract Among the various subtypes of acute myeloid leukemia (AML), TP53-mutated (TP53-Mut) AML is one of the most aggressive in both adults and children due to its resistance to available therapies. Our goal is to identify vulnerabilities that these cancer cells exhibit in order to exploit them as potential therapeutic targets. Using the Cancer Dependency Map (DepMap), we stratified the 26 AML cell lines into TP53-Mut and TP53-wildtype (TP53-WT) categories and identified adenosine deaminase (ADAR1) as the top selective dependency in TP53-Mut AML. ADAR1 is an enzyme that catalyzes adenosine-to-inosine (A-to-I) editing of double stranded RNA (dsRNA), thereby marking it for degradation. Loss of ADAR1 therefore leads to an accumulation of dsRNAs which activates cytoplasmic dsRNA sensors that trigger increased interferon (IFN) signaling and activation of PKR, leading to translation inhibition and ultimately cell death. To validate this dependency, we generated ADAR1 knockout (KO) cells in TP53-Mut and TP53-WT cell lines. Our results showed that TP53-Mut AML cells are sensitive to ADAR1 loss in vitro and in vivo. Due to the role of ADAR1 in dsRNA editing, we hypothesized that TP53 negatively regulates the expression of dsRNA, which renders TP53-Mut AML cells more reliant on ADAR1 editing. Notably, we found through immunofluorescent and flow staining of dsRNA that TP53-Mut AML cells have higher basal levels of dsRNA compared to TP53-WT cells. After loss of ADAR1, the amount of dsRNA increases above the activation threshold of PKR and activates it in a TP53-Mut dependent manner.To identify the molecular mechanism behind the selective sensitivity of TP53-Mut AML cells to ADAR1, we performed a genome-wide CRISPR screen in TP53-KO MV4-11 AML cells. The screen revealed that KO of IFN pathway activators (e.g., IRF9, STAT2, and IFNAR1) conferred resistance to ADAR1 loss, while KO of genes that inhibit inflammation (e.g., ISG15, USP18), enhanced sensitivity to ADAR1 loss. Notably, KO of IRF9 rescued the cells from ADAR1 loss and decreased the levels of PKR activation. In contrast, loss of USP18 further enhanced ADAR sensitivity and increased the levels of PKR activation. This suggests that PKR activation is a biomarker for sensitivity to ADAR1 loss. Overall, a high inflammatory environment present in TP53-AML cells leads to increased PKR expression, which primes them to sensitivity to the loss of ADAR1. Thus, we hypothesized that activating PKR in combination with ADAR1 loss would exacerbate cell death in TP53-Mut AML cells. Two FDA-approved small molecules that activate PKR were successful in producing anti-proliferative effects in TP53-Mut AML cells when combined with ADAR1 loss compared to TP53-WT AML cells in vitro. Our findings show that TP53-Mut AML cells rely on the ADAR-1/PKR pathway for survival and provide a new therapeutic target that can be combined with FDA-approved drugs as a promising basis for treating an aggressive subtype of AML. Citation Format: Audrey Taillon, Yara Rodriguez, Gabriela Alexe, Allen Basanthakumar, Lucy Merickel, Silvi Salhotra, Olasunkanmi Olaoye, Constanze Schneider, Oivin Guicherit, Paige Stocker, Haley Curtis, Kimberly Stegmaier. ADAR1 loss in combination with PKR activation leads to anti-tumor effects in TP53-Mut AML [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A027.