Synergistic Co-Targeting of BTK and BCL2 in Mantle Cell Lymphoma

            Mantle cell lymphoma (MCL), a B-cell non-Hodgkin lymphoma, remains incurable with current treatment modalities. Bruton tyrosine kinase (BTK), a key component of the early BCR signaling pathway, has emerged as a promising therapeutic target. Ibrutinib, a specific inhibitor that binds covalently to the active site of BTK at cysteine 481, has been approved for the treatment of MCL. In a recent Phase II study, treatment for relapsed or refractory MCL with Ibrutinib alone achieved a response rate of 68%. Although this response rate is striking, approximately one third of patients show primary resistance, and acquired resistance with a C481S mutation in BTK can also develop. To overcome primary and acquired resistance to BTK inhibition, a combinatory strategy that targets multiple pathways is needed.

           Here, we focused on the BCL2 anti-apoptotic pathway. Our previous study established FBXO10 as the E3 ubiquitin ligase that targets BCL2 for proteasomal degradation and revealed reduced levels of FBXO10 expression in MCL cell lines. To examine BCL2 and FBXO10 expression in patient samples, we performed immunohistochemical (IHC) analysis in a tissue microarray (TMA) that contained 62 MCL cases.  Our data showed high levels of BCL2 expression but no or low FBXO10 expression in MCL cases. Based on this initial expression analysis, we hypothesized that a defect in FBXO10-mediated proteasomal degradation contributes to high BCL2 expression in MCL. To test this hypothesis, we genetically manipulated FBXO10 expression in MCL cell lines. Flow cytometric analysis revealed that overexpression of FBXO10 resulted in reduced BCL2 expression. Inversely, we silenced endogenous FBXO10 by shRNA. Indeed, after blocking protein synthesis by cycloheximide, the amount of BCL2 protein was sustained in these FBXO10 shRNA-expressing cells. These results indicate that BCL2 is the predominantly expressed anti-apoptotic protein in MCL, due to a specific defect in its proteasomal degradation. Inhibition of BCL2 by the specific inhibitor ABT-199 killed cancer cells in culture and also inhibited xenografted tumor growth.

BCL2 is a target gene of the BTK/canonical NF-κB signaling pathway. We next analyzed BTK expression in the above TMA and found that BTK was highly expressed and positively correlated with BCL2 expression in these cases. BTK inhibition reduced BCL2 expression as shown in the BCR-dependent cell lines Jeko and Mino. RNA-seq analysis confirmed that a set of anti-apoptotic genes (e.g. BCL2, BCL-XL and DAD1) was downregulated by BTK shRNA. The downregulated genes also included those that are critical for B cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Elevated BCL2, however, can also result from other mechanisms, such as amplification. Indeed, this genomic alteration is present in some MCL cell lines, including Granta-519 and Z138 used for this study. Since these cell lines are insensitive or less sensitive to Ibrutinib, we hypothesized that BCL2 upregulation is a mechanism underlying primary resistance to Ibrutinib, and thus targeting BCL2 by ABT-199 increases or restores sensitivity of these cells to Ibrutinib. The results supported this hypothesis and demonstrated a synergistic effect of ABT-199 and Ibrutinib on growth inhibition of Granta-519 and Z138 cells both in vitro and in vivo.

In summary, this study elucidated mechanisms of BCL2 overexpression and association with the BCR/BTK signaling pathway in MCL. Our data provided a mechanistic rationale for co-targeting of these two oncogenic pathways by ABT-199 and Ibrutinib as a new therapeutic strategy in a pre-clinical setting. Most importantly, the findings indicate that this combination has a synergistic effect on the killing of both BCR-dependent and independent MCL cells. Since these two signaling pathways are deregulated in DLBCL and follicular lymphoma, co-targeting by Ibrutinib and ABT-199 may provide an attractive therapeutic strategy for these additional lymphoma patients as well.