Olivia Barbara Farag

Though 100 kinase inhibitors are approved for clinical use, most kinases in the genome remain poorly characterized. Moreover, though kinase inhibitors have revolutionized the care of selected cancers, most others respond transiently or not at all to these drugs, typically directed against signal-transduction pathways. Given cellular regulation at every step by phosphorylation events, we hypothesized that phenotypic screening and machine-learning analysis can reveal novel kinase targets. Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, responds poorly to signal transduction-directed kinase inhibitors. We applied a library of probes to DLBCL systems broadly representing its pathogenic heterogeneity and subjected results to iterative target-deconvolution analysis aimed at tumor selectivity (idTRAX). These efforts identified the cyclin G-associated kinase (GAK) as a tumor-selective target. Mechanistic studies showed GAK kinase activity is required for mitotic progression, with inhibition leading to G2/M arrest and cell death and increased efficacy in the absence of the retinoblastoma (RB) tumor suppressor. GAK-directed kinase inhibitors, including repurposed clinical compounds, showed promising single-agent anti-DLBCL activity in vivo. GAK kinase function is therefore a targetable dependency, opening novel treatment strategies suitable for rapid clinical translation. Moreover, we establish phenotypic screening with target deconvolution as a generalizable strategy to reveal tumor-selective kinase dependencies.

Cancers co-opt stress response pathways to drive oncogenesis, dodge immune surveillance, and resist cytotoxic therapies. Several of these pathways also provide protection from ferroptosis, an iron-dependent oxidative cell death pathway triggered by clinically available drugs, including chemotherapies, rheumatologic agents, and novel ferroptosis inducers under evaluation in clinical trials. In this study, we found that disrupting cap-dependent translation initiation in diffuse large B-cell lymphoma (DLBCL) sensitizes cells to ferroptosis. Specifically, the eIF4A1 inhibitor zotatifin synergized with pharmacologic ferroptosis inducers primarily through suppression of glutathione production, which protects polyunsaturated fatty acids from ferroptotic oxidation. Loss of nuclear factor erythroid 2–related factor 2 (NRF2) translation, a master regulator of antioxidant genes, was a key consequence of rocaglates, including zotatifin, and other disruptors of cap-dependent initiation. Although NRF2 loss alone was insufficient to trigger ferroptosis, it lowered the antioxidant threshold, sensitizing cells to lipid peroxidation and ferroptotic death under additional oxidative stress. In vivo, combining zotatifin with the optimized ferroptosis inducer imidazole ketone erastin significantly reduced tumor burden in DLBCL patient-derived xenografts. Treatment with zotatifin in combination with chimeric antigen receptor (CAR) T cells, a vital treatment modality for patients with DLBCL, revealed that zotatifin preexposure sensitized DLBCL tumors to CD19-directed CAR T cells in vitro and extended survival of CAR T-cell–treated immunocompetent mice bearing syngeneic DLBCL tumors in vivo. Overall, eIF4A1 inhibition–induced translational disruption provides opportunities to leverage the therapeutic impacts of ferroptosis inducers, including cytotoxic immunotherapies.Significance:Translational disruption causes NRF2 loss that sensitizes lymphomas to ferroptosis and enhances CAR T-cell and drug efficacy, highlighting eIF4A1 targeting as a promising therapeutic strategy for treating cancer.

Transcriptomic analysis of zotatifin-treated DLBCL cells including RNA-seq heatmap, GSEA pathway enrichment, and NF-κB signaling readouts.

Immunoblot analysis of apoptosis and ferroptosis markers in DLBCL cells treated with zotatifin in combination with RSL3 or DMF.

Supplementary Table S1 from Blocking NRF2 Translation by Inhibition of Cap-Dependent Initiation Sensitizes Lymphoma Cells to Ferroptosis and CAR T-cell Immunotherapy

Integrated analysis of translation and transcription upon zotatifin treatment, including immunoblots, TMT-pSILAC data, and GSEA enrichment.

Supplementary Table S2 from Blocking NRF2 Translation by Inhibition of Cap-Dependent Initiation Sensitizes Lymphoma Cells to Ferroptosis and CAR T-cell Immunotherapy

Antibodies used for flow cytometry staining

Tumor imaging and immunohistochemistry analysis of cleaved caspase 3 and 4-HNE in vivo following zotatifin, IKE, or combination treatment.

Zotatifin sensitizes cells to ferroptosis; includes lipid peroxidation, ROS analysis, and Bliss synergy plots.