USP28 inhibition upregulates CD20 via c-myc suppression and synergizes with anti-CD20 immunotherapy in B-cell lymphomas

Abstract Introduction Anti-CD20 monoclonal antibodies are widely used in the treatment of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). These therapies often lead to good initial responses, but many patients eventually relapse. One known cause of resistance is the decreased or lost expression of CD20 on tumor cells. Notably, suppression of the oncogenic transcription factor c-Myc has been associated with increased CD20 expression [1]. USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that stabilizes multiple oncogenic substrates, including c-Myc, by preventing their proteasomal degradation. Our team has developed CT1113, a selective USP28 inhibitor, which has been evaluated in a Phase I clinical trial for patients with acute myeloid leukemia [2]. This study aims to determine whether USP28 inhibition can suppress c-Myc, upregulate CD20 expression, and enhance the efficacy of anti-CD20 immunotherapy. Materials and Methods Human DLBCL and BL cell lines (OCI-Ly1, OCI-Ly10, Raji, and Daudi) were treated with CT1113 or subjected to USP28 knockdown via shRNA. CD20 mRNA and protein expression levels were assessed using quantitative RT-PCR, flow cytometry, and western blotting. For in vivo studies, a xenograft model was established by intravenous injection of luciferase-expressing Daudi cells into NCG (NOD/ShiLtJGptPrkdcem26Cd52Il2rgem26Cd22/Gpt) mice. Tumor burden was monitored using the IVIS Imaging System. Mice received vehicle, CT1113, Rituximab, or combination treatment. Results CT1113 treatment and USP28 knockdown both resulted in significant downregulation of c-Myc protein and upregulation of CD20 mRNA and protein expression in BL and DLBCL cell lines. Proteomic analyses further confirmed a robust increase in CD20 expression following CT1113 treatment. In vivo, combination therapy with CT1113 and Rituximab significantly suppressed tumor burden in the spleen and bone marrow, demonstrating superior antitumor activity compared to monotherapy. These data suggest that USP28 inhibition upregulates CD20 through c-Myc suppression and enhances anti-CD20 therapeutic efficacy. Conclusion These findings demonstrate that targeting of USP28 leads to c-Myc suppression and CD20 upregulation, thereby enhancing the response to anti-CD20 immunotherapy in c-Myc–driven B-cell lymphomas. The observed synergistic antitumor activity supports further clinical investigation of CT1113 in combination with Rituximab in patients with relapsed or refractory disease. References Torun, Anna et al. “Potassium/sodium cation carriers robustly upregulate CD20 antigen by targeting MYC, and synergize with anti-CD20 immunotherapies to eliminate malignant B cells.” Haematologica vol. 110,7 (2025): 1555-1572. Peng, Jin et al. “Identification of a class of potent USP25/28 inhibitors with broad-spectrum anti-cancer activity.” Signal transduction and targeted therapy vol. 7,1 393. 8 Dec. 2022.