Translatome Reprogramming By Rocaglates in Lymphoma Induces Drug Resistance through up-Regulation of CD98hc, Activating Downstream AKT Survival Signaling

Abstract

Background: Up to 40% of diffuse large B-cell lymphoma (DLBCL) patients do not achieve a cure in response to standard immunochemotherapy R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). In addition, related aggressive lymphomas such as high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (a.k.a. double and triple-hit lymphomas) have persistently inferior outcomes in response to frontline therapy, and no widely accepted alternative exists yet for these high-risk patients. Therefore, there is a strong unmet need to identify novel, effective therapeutics. B-cell lymphomas are driven by deregulation of several oncoproteins that rely on cap-dependent translation, including MYC, MCL1, Cyclin D1, and BCL2. Among strategies to bypass drug-resistance driven by redundant signaling pathways, targeting cap-dependent translation has been shown to be effective. Several studies highlight various types of lymphoma, including the "undruggable” MYC-driven B cell lymphoma, that respond to rocaglates - natural and synthetic translation inhibitors. Among these compounds, eFT226 (zotatifin), is currently in phase I/II evaluation for solid tumors. Despite advancement of rocaglates to clinical evaluation, their mechanisms of action on target molecules remains incompletely defined to advance clinical use for B-cell lymphoma. Methods and Results: Using unbiased proteomic strategies, previous findings from our laboratory in glioblastoma in vitro models have shown that rocaglates induce a far more complex, dynamic process than simple translation inhibition. Indeed, despite translational repression, we discovered significant adaptation that caused active translational synthesis of many targets in the translation machinery (i.e eEF1ε1) and the translatome. We have also shown that some upregulated proteins counteract drug effects (i.e., CD98 heavy chain-CD98hc). Our previous findings and additional data confirmed that rocaglates also induced reprogramming in a DLBCL patient-derived xenograft (PDX) mouse model, and in several DLBCL cell lines. Intriguingly, in DLBCL cell lines we found upregulation of the survival mediators CD98hc and AKT-signaling in persistent cells at 48 hours upon zotatifin treatment via Western Blot and flow cytometry, possibly representing a novel mechanism of resistance driven by CD98hc in response to rocaglate treatment. To further explore the role of the CD98hc counteracting mechanism, we established a doxycycline-inducible (TET-on) CD98hc-overexpressing DLBCL cell line (SU-DHL10), and we consistently found that overexpression of CD98hc was responsible for decreased sensitivity to zotatifin treatment (48h, 72h) by ATP-dependent viability assay. This data was further corroborated by Annexin-V/PI staining via flow cytometry, depicting a significant decrease in cell death upon 12.5 nM zotatifin treatment for 24h (P=0.0074) and 48h (P=0.0010) in induced versus uninduced CD98hc-overexpressing SU-DHL10 cells. For statistical analysis, repeated measures two-way Anova with Sidaks multiple comparison test were used. Conclusions: We define for the first time CD98hc as a new putative driver of rocaglate resistance in B-cell lymphoma. Paradoxically, CD98hc is upregulated as part of an overall cellular translational response to rocaglate exposure, but it provides insights and new potential targets for further drug combinations. This study lays the foundation for new treatment strategies to optimize the use of zotatifin to overcome rocaglate resistance in lymphoma patients.