Characterizing the Role of Interferon-γ Signaling in Natural Killer Cell Dysfunction in Venetoclax-Resistant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is associated with a dismal prognosis. Venetoclax is a BCL2 inhibitor currently approved as front-line therapy in combination with hypomethylating agents (HMAs) for adults with AML who are unfit for high-intensity chemotherapy. Although venetoclax-based therapy has response rates as high as 70%, a significant proportion of patients experience upfront resistance or relapse with few viable subsequent treatment options. Thus, effective strategies for treating venetoclax-resistant (VR) relapsed/refractory AML remain an unmet clinical need. Natural killer (NK) cells are cytolytic lymphocytes of the innate and adaptive immune system and have been shown to have potent anti-tumor effects as a cellular therapy product for hematologic malignancies. Our group recently showed, however, that venetoclax-resistant AML blasts are less susceptible to NK cell-mediated killing, and the mechanisms underlying this phenomenon remain poorly understood. Increased interferon-gamma (IFN-γ) signaling is associated with venetoclax resistance (Wang et al., 2024), but its connection to NK cell dysfunction in VR AML has not been fully explored. In this study, we seek to characterize the immunophenotypic alterations induced by IFN-γ on AML blasts and explore the IFN-γ signaling pathway as a therapeutic target in VR AML.

We investigated the sensitivity of AML blasts to NK cell cytotoxicity through in vitro co-culture assays with human AML cell lines and NK cells derived from a healthy human donor. We found that the venetoclax-resistant versions of the Molm14 and OCI-AML2 cell lines were less susceptible to NK cell killing compared to their wild-type (WT) counterparts. To confirm the correlation between IFN-γ and VR AML, we quantified IFN-γ levels using the Olink proximity extension assay in 8 paired plasma samples from 4 AML patients pre- and post-HMA plus venetoclax treatment. All 4 patients had received treatment with either decitabine or azacitidine plus venetoclax at the time of residual disease (N = 3) or relapsed disease (N =1). Treatment resistance developed after 1 cycle in 1 patient and after ≥ 2 cycles in 3 patients. European LeukemiaNet (ELN) risk categorization ranged from intermediate (N = 2) to adverse (N = 2) risk. We found increased normalized protein expression of IFN-γ in 3 out of 4 patients post-HMA plus venetoclax treatment (corresponding to the time of relapsed or refractory disease). In order to characterize the immunophenotypic alterations induced by IFN-γ that could lead to NK cell resistance, we stimulated WT Molm14 cells with IFN-γ (100 IU/mL for 24 hours) and performed flow cytometry to evaluate the expression of NK cell activating and inhibitory ligands. We found that not only did IFN-γ lead to upregulation of inhibitory classical major histocompatibility complex (MHC) class I and class II molecules and the non-classical human leukocyte antigen-E (HLA-E), but it also induced decreased NK cell mediated lysis compared to vehicle control (20:1 effector to target ratio; 54% vs. 21%, p < 0.0001).

Given that IFN-γ activates the Janus kinase (JAK) pathway to mediate downstream regulation of immunomodulatory genes, we investigated the potential of the JAK1/JAK2 inhibitor, ruxolitinib, to reverse the effects of IFN-γ on altering AML immunophenotype and inducing NK cell resistance. We found that ruxolitinib given at 2 μM for 72 hours not only abrogated the IFN-γ induced upregulation of classical MHC class I and II molecules and HLA-E in WT Molm14 AML cells, but it also rescued IFN-γ mediated decreased NK cell killing (20:1 effector to target ratio; 20% vs. 53%, p < 0.0001).

These findings reveal that IFN-γ is upregulated in VR AML and associated with a distinct immunophenotype, and that the IFN-γ signaling axis is a potential therapeutic target for enhancing NK cell responses in AML.