Targeting the DNA Damage Response through TBL1X in Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is an aggressive, incurable B-cell Non-Hodgkin's lymphoma (NHL). MCL patients who progress on targeted therapies (e.g. BTK inhibitors or CAR-T immunotherapy) have short survival, highlighting the urgent need for novel therapeutic strategies. Upregulation of adaptor protein transducin β-like protein 1 X-linked (TBL1X) has been identified in multiple types of cancer and linked to aggressive tumor behavior and poor clinical outcomes. In diffuse large B-cell lymphoma, our group showed that TBL1X stabilizes key oncoproteins, including PLK1 and c-MYC, via interaction with a SKP1-CUL1-F-box protein supercomplex. As TBL1X biology was previously unexplored in MCL, we sought to characterize the expression and oncogenic role of TBL1X in this disease.

Methods: Herein, we characterize the expression and essential function of TBL1X in MCL in maintaining genomic stability and cell viability in preclinical models, including cell lines (MCL-CL), primary patient samples (MCL-PPS), and a murine model of MCL. Pharmacologic targeting of TBL1X was performed with selective, first-in-class small molecule Tegavivint (Iterion) and genetic targeting of TBL1X via shRNA knockdown (KD). Confirmation of KD and mRNA quantification was achieved via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression was evaluated via standard immunoblotting. For cytoxicity assays, cell viability assessed via flow cytometry and Annexin-V/propidium iodide staining.

Results: Immunoblot analyses revealed abundant levels of TBL1X in MCL cells, including MCL-CL (n=9) and MCL-PPS (n=10) of variable genetic backgrounds, compared to normal donor B cells. In vitro, TBL1X KD in MCL-CL and tegavivint treatment of MCL-CL and -PPS induced significant cell death. Tegavivint-induced cytotoxicity was dose-dependent with 24-hour IC50s <200nM (range = 56-197nM) in all MCL-CL. In vivo compared to vehicle control, tegavivint treatment (30mg/kg, intravenous, twice weekly) significantly reduced tumor growth (mean volume at day 13 post-engraftment = 512 mm³ vs 73 mm³, respectively, p=0.032) and prolonged survival (median overall survival = 16 vs 20 days, respectively, p=0.0230) in a murine MCL-CL (FCMCL) subcutaneous tumor model. Mechanistically, we found that targeting TBL1X in MCL-CL and -PPS induced significant DNA damage with robust serine 139 phosphorylation of histone H2A.X (γH2AX, an established marker of double-strand breaks) and depletion of key DNA damage response (DDR) protein, homologous recombination mediator RAD51. Despite loss of RAD51 by immunoblot, qRT-PCR showed no significant changes at the transcript level. Moreover, proteasomal inhibitor MG132 restored RAD51 protein levels when combined with tegavivint suggesting that TBL1X is involved in controlling RAD51 protein stability rather than its transcription. Validating the importance of the TBL1X-RAD51 axis in the MCL DDR, we found that targeting RAD51 pharmacologically (small molecule inhibitor B02, range = 1-10uM) and genetically (shRNA KD) resulted in DNA damage, γH2AX formation, and cell death in MCL-CL.

Conclusion: These studies demonstrate that TBL1X plays an essential role in MCL cell survival by maintaining genomic stability and DDR efficacy via RAD51 stabilization. Ongoing work is focused on delineation of the mechanism through which TBL1X controls RAD51 stability. We believe these findings support further exploration of targeting of TBL1X in MCL with tegavivint. To leverage this finding of TBL1X-mediated DDR regulation, we are currently exploring novel drug combinations targeting genomic instability to maximize the therapeutic potential of tegavivint in this disease.

Chan:Xellera Therapeutics: Consultancy; Cellperior Process LLC: Current equity holder in private company. Baiocchi:Codiak Biosciences: Research Funding; Prelude Therapeutics: Other: Advisory Board, Research Funding; Viracta Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Other: Advisory Board; Agenus: Other: Involved in supply of drug (vaccine) and product development; ATARABio: Consultancy, Other: Advisory Board.