The Flavonoid Brusatol Induces Apoptosis in Aggressive Lymphoma Cells and Exhibits Synergistic Effect with Venetoclax (2022)

The most common lymphoid malignancies in adults are aggressive lymphomas, including Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL). Despite the available chemoimmunotherapy R-CHOP, one third of DLBCL patients still have primary refractory disease or relapse, and the incidence of these lymphomas continuously increases. Together, there is the great need for further development of new treatment strategies for aggressive lymphoma. One step forward seems to be Venetoclax (an FDA-approved Bcl-2 inhibitor), which showed an overall response rate of 44% in a phase 1 trial. Here, we evaluated a flavonoid - Brusatol, which showed anti-tumour activity in various preclinical cancer models, to improve anti-lymphoma therapies. We investigated the potential of Brusatol in the treatment of aggressive lymphoma cells as a single agent or in combination with other drugs, including Venetoclax. Seven cell lines representing different types of lymphoma (BL2, Raji (Burkitt lymphoma), SuDHL4, Karpas422 (GCB-DLBCL), RI-1, U2932 (ABC-DLBCL) and Jurkat (T-cell leukemia)) were treated with increasing concentrations of Brusatol (up to 10µM) for 72h to determine the IC50 values. Based on these results, 50nM and 250nM Brusatol were selected for further experiments. Cells were treated with Brusatol for 24h and 48h and apoptosis induction (Annexin V staining, Caspase-3- and PARP-cleavage) and changes in cell cycle (PI staining) were assessed. Moreover, samples from untreated and Brusatol-treated cells were collected for Western blot analysis. To investigate potential effects of Brusatol on protein synthesis in lymphoma cells, four cell lines (BL2, SuDHL4, RI-1 and U2932) were treated for 4h, and then the nascent protein synthesis was evaluated by detection of an incorporated methionine analogue using click-chemistry. Furthermore, co-treatment of Brusatol with inhibitors of Bcl-2 (Venetoclax), Bcl-XL (A-1331852), Mcl-1 (S63845), BTK (Ibrutinib), PI3K (Idealisib), Myc (EN4, JQ1), respectively, was performed in the same four cell lines and Annexin V levels were measured after 24h. Finally, the effect of combining Brusatol with Venetoclax was determined using the Bliss independence model for SuDHL4 and U2932 after 24h treatment via TMRE staining. In all investigated cell lines, Brusatol caused cell growth inhibition in a concentration-dependent manner. However, based on the results of the apoptosis assays, they could be grouped into cell lines more (BL2, Jurkat, Raji, SuDHL4) and less (Karpas422, RI-1, U2932) sensitive to Brusatol. Moreover, cell cycle analysis revealed an increase of cells in the sub-G1 phase in the more sensitive cell lines, indicating the induction of cell death. Western blot analysis of more sensitive cell lines showed decreased levels of the prosurvival proteins Bcl-2, Bcl-XL and Mcl-1. Additionally, we detected reduced p53 and Myc protein expression in cell lines more sensitive to Brusatol after 24h treatment. Notably, the protein expression profile of untreated cells indicates that cell lines with higher Myc levels are more sensitive to Brusatol treatment. Furthermore, mRNA expression analysis revealed that the reduction of affected proteins mainly occurred on protein level. Therefore, we investigated the effect of Brusatol on protein biosynthesis and observed protein translation inhibition in all tested cell lines. Interestingly, increased induction of apoptosis was apparent in all cell lines upon co-treatment with Brusatol and Bcl-XL inhibitor, in three of the four cell lines treated with the combination of Brusatol and Bcl-2 inhibitor, and in two cell lines when Brusatol was combined with Mcl-1 inhibitor. In contrast, combining Brusatol had no beneficial effect in combination with all other drugs tested. Finally, analysis of the Bliss independent model showed that the combination of Brusatol and Venetoclax synergistically enhanced the killing of lymphoma cells. Our data indicate that Brusatol efficiently induces cell death in aggressive lymphoma cells by reducing the expression of prosurvival proteins. Interestingly, cells with higher Myc levels were especially sensitive to Brusatol. Additionally, the combination of Brusatol with Venetoclax results in enhanced induction of apoptosis. Thus, our study suggests that Brusatol, alone or in combination with Venetoclax, represents a very interesting agent for further development of novel anti-lymphoma therapies.