Preclinical Study of a Novel USP28 Inhibitor for the Treatment of Burkitt Lymphoma
Authors: Xu Jieyu , Sun Shu , Gao Wen , Zhang Pumin , Yang Haiyan , Zhu Honghu 
Published: December 2024

Burkitt lymphoma (BL) is a highly aggressive non-Hodgkin lymphoma characterized by IGH/MYC gene translocation, high expression of c-Myc and large lipid droplets in the cytoplasm. The therapeutic options for adult patients with BL remain limited, with intensive multiagent chemotherapy serving as the primary treatment modality. Despite this, the cure rate remains suboptimal. Consequently, the identification and development of novel targeted therapies are imperative to enhance the prognosis of this historically challenging disease. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase that has been reported to facilitate the evasion of ubiquitination and subsequent proteasomal degradation for lots of oncogenic substrates, with c-Myc being one of the identified substrates. Furthermore, our previous studies have shown that USP28 can modulate the SREBP1-mediated lipid synthesis pathway. Our team has developed a small molecule inhibitor, CT1113, which effectively targets USP28 and has been under evaluation in a phase I clinical trial involving patients with acute myeloid leukemia (AML) since 2022. Consequently, it is imperative to investigate the cytotoxic effects of CT1113 on BL cells through experiments and elucidate its underlying mechanisms. This research aims to provide novel therapeutic avenues for the clinical treatment of BL.

We first found that compared with diffuse large B-cell lymphoma (DLBCL), BL had up-regulated levels of c-Myc and SREBP1 proteins, and activated lipid synthesis pathways. To investigate the role of USP28 in BL, we conducted loss-of-function experiments in BL cell lines (Raji and Daudi). We observed that the knockdown of USP28 resulted in a substantial reduction in endogenous c-Myc and SREBP1 protein levels, significantly slowed cell proliferation, and induced apoptosis in both Raji and Daudi cells.

Subsequently, we investigated the effects of pharmacologic inhibition of USP28 using the inhibitor CT1113 in Raji and Daudi cells. Treatment with CT1113 was found to decrease cell viability, induce apoptosis, and markedly reduce the protein levels of USP28, c-Myc, and SREBP1. Furthermore, BODIPY staining experiments revealed that CT1113 treatment significantly diminished the neutral lipid droplets within the cell plasma.

To assess the in vivo efficacy of CT1113, a human xenograft model was established using Raji cells. Luciferase-expressing Raji cells were intravenously injected into NCG mice, which were randomized into two groups: one receiving vehicle treatment and the other receiving CT1113 (20 mg/kg body weight, administered twice daily via oral gavage). CT1113 demonstrated significant suppression of tumorigenesis in the spleen and bone marrow. Notably, CT1113 exhibited dose-dependent cytotoxicity in primary BL cells isolated from the bone marrow of two BL patients.

To investigate the potential role of SREBP1 in BL, we administered the SREBP1 inhibitor fatostatin to Raji and Daudi cell lines. The findings indicated that fatostatin reduced cell viability and induced apoptosis in a dose-dependent manner. Furthermore, bioimaging data obtained following the xenograft procedure demonstrated that fatostatin significantly impeded tumor progression in a CDX model established with luciferase-expressing Raji cells. These experimental results suggest that SREBP1-mediated lipid synthesis plays a crucial role in BL, and that pharmacological inhibition of SREBP1 exerts cytotoxic effects on BL cell lines.

In summary, our findings demonstrate that CT1113 exhibits significant efficacy against BL. Our preclinical studies of CT1113 indicate that targeting USP28 represents a highly promising and potential clinical strategy for the treatment of BL patients.