Defective Necroptosis Mediates Chemotherapy Resistance in AML

Abstract

Acute myeloid leukemia (AML) is a hematopoietic malignancy that can affect individuals of any age and, if left untreated, is always fatal. Standard induction chemotherapy, known as “7+3,” using drugs like cytarabine (Ara-C) and an anthracycline (daunorubicin or idarubicin), is typically given to young and fit older patients. While this treatment regimen has shown positive outcomes in patients who respond to it, relapse and refractory disease remain the primary causes of death. Prognostic classification, which combines cytogenetic and mutational status, can estimate overall and event-free survival but is not effective in predicting induction failure. Despite decades of research and treatment with 7+3 chemotherapy, the reasons behind variable treatment responses in AML patients remain unclear, especially for those who experience induction failure. Understanding these mechanisms could aid in risk-adapted treatment decisions and the identification of new therapeutic targets. Recent research has investigated gene expression and signaling patterns in leukemic cells at the time of diagnosis to categorize patients according to their response to chemotherapy. Notably, increased NF-kB signaling at diagnosis has been linked to chemotherapy resistance. NF-kB is a family of transcription factors involved in regulating genes linked to cancer cell survival, proliferation, and therapy resistance. However, the exact mechanism by which NF-kB activation contributes to chemotherapy failure and resistance in AML is yet to be determined. Using publicly available datasets, we found that AML patients undergoing induction failure exhibited elevated expression of TNFAIP3/A20, which is driven by NF-kB signaling and leads to a worse prognosis for patients undergoing 7+3 chemotherapy. In experiments with primary AML samples, it was observed that A20-High AML are resistant to Doxorubicin treatment, whereas A20-Low AML samples are sensitive to Doxorubicin. Knockdown of A20 in A20-High AML samples resulted in increased cell sensitivity to Doxorubicin, suggesting that elevated A20 expression reduces AML cells' response to the Doxorubicin both in vitro and in vivo. To understand why A20 is overexpressed in AML, we investigated its requirement on AML cell function in various models, including human AML cell lines, patient-derived samples, and mouse models of AML. Deletion or knockdown of A20 led to reduced leukemic progenitor function, cell viability, and AML development in vitro and in vivo. A20 is a dual ubiquitin-editing protein that regulates signaling pathways by acting either as a deubiquitinase to modulate NF-kB signaling or as an E3 ligase to control necroptosis. Further analyses revealed that A20 prevents necroptosis by targeting the necroptosis effector, RIPK1, for degradation through the proteasome in AML. Preventing necroptosis, either pharmacologically or genetically, restored the viability and clonogenicity of A20-deficient AML cells in vitro and in vivo. Notably, Doxorubicin, but not AraC, induced necroptosis in AML cells, which is suppressed in leukemic clones expressing A20. Examination of single-cell RNA-sequencing data from AML patients before and after Doxorubicin treatment confirmed the persistence of A20-expressing clones during treatment. These findings demonstrate that NF-kB-driven overexpression of A20 contributes to failed induction chemotherapy in AML, highlighting the therapeutic potential of targeting an alternative cell death pathway in AML.