Combination of ibrutinib with ABT‐199: synergistic effects on proliferation inhibition and apoptosis in mantle cell lymphoma cells through perturbation of BTK, AKT and BCL2 pathways (2014)

Mantle cell lymphoma (MCL) is considered incurable with a shorter survival than indolent lymphomas. Current standard therapeutic regimens have high initial response rates, however, even with high dose therapy, drug resistance is common and recurrence is anticipated (Garraway & Janne, 2012). Adaptive resistance often results in progression to more clinically aggressive disease. Most patients eventually relapse with a median overall survival of 4–6 years. Thus, more effective therapies are needed. Abnormal activation of Bruton tyrosine kinase (BTK) mediated B-cell-receptor (BCR) signalling pathway contributes to the pathogenesis of B-cell malignancies (Davis et al, 2010). Signals mediated by BTK trigger cell survival pathways such as NFκB, RAS/RAF/MEK/ERK and PI3K/AKT. BTK is therefore an attractive target for inhibition of B-cell growth. A clinical trial suggested Ibrutinib is a promising agent in patients with relapsed or refractory MCL (Wang et al, 2013). However, considering that genetic mutations cause resistance to ibrutinib in chronic lymphocytic leukaemia (CLL) patients and altered signalling pathways are common mechanisms of resistance to single agents, Ibrutinib monotherapy is not expected to cure MCL (Zucca & Bertoni, 2013). The BCL2 family proteins play a critical role in regulating apoptosis and lymphoid malignancies frequently have high BCL2 expression. The BH3-only mimetic ABT-199 selectively inactivates BCL2 and is a promising drug for treatment of BCL2-dependent cancers. However, acquired mutations could cause resistance to ABT-199 in lymphoma cells (Fresquet et al, 2014). These observations indicate potential clinical challenges of applying ABT-199 as a single agent. Combination of ABT-199 with other agents may be a strategy to overcome acquired resistance. The effectiveness of ABT-199 as a single agent or in combination is largely unexplored in MCL. Given the presumed non-overlapping pathways, we hypothesized that ibrutinib/ABT-199 combination may overcome the resistance observed with single agents alone. To test this, a new MCL cell line CCMCL1 (Zhao et al, 2013) was examined for the response to these agents. Synergistic inhibition of proliferation was confirmed in 22 of 24 tested combinations, based on combination index (CI) values <1, in which 18 combinations had CI values <0·7, suggesting strong synergy (Fig 1A). Synergistic apoptosis induction was observed in all combinations (Fig 1B). The same assay with four additional MCL cell lines, Jeko-1, Mino, JVM2 and Rec-1, showed strong synergistic effects, as evidenced by CI values <0·7 with a majority being <0·2 (Fig 1C, D). Testing with primary cells from two cases of recurrent MCL also displayed robust synergy of apoptosis induction (Figure S1). Although these two cases displayed different degrees of response to single agents, synergy was observed for both. Another noteworthy finding of this study is molecular mechanisms underlying the interaction of ibrutinib/ABT-199 in MCL cells. In CCMCL1 cells, ibrutinib caused dephosphorylation of BTK(Y223). ABT-199 had no detectable effect on this target but there was a larger decrease of p-BTK(Y223) upon cells co-treated with ibrutinib/ABT-199. A similar effect was observed on p-AKT(S473) (Fig 2A). Immunoblotting of four other MCL cell lines confirmed that this combination enhanced dephosphorylation of the above signalling molecules (Figure S2), which was associated with survival/proliferation of malignant B-cells. The impact of ibrutinib and/or ABT-199 on BCL2 family proteins (BCL2, MCL1 and BCL2L1) was more cell-line dependent. In CCMCL1 cells, ibrutinib alone down-regulated MCL1. Each single agent had little effect on BCL2 and BCL2L1, but the combination down-regulated both of these proteins (Fig 2B). Co-treatment of other MCL cell lines with ibrutinib/ABT-199 resulted in decrease of at least one BCL2 family protein (Figure S3). Ibrutinib/ABT-199 co-treatment also more effectively triggered reduced mitochondrial membrane potential compared to single agent, and more poly (ADP-ribose) polymerase (PARP) cleavage (Fig 2C, D) suggested caspase activation. In summary, we report the therapeutic potential of ibrutinib/ABT-199 combination in MCL cells. This combination displayed strongly synergistic effects in all tested cell lines and primary cells from recurrent MCL patients. These cell lines represent different types of MCL, including aggressive MYC-translocated CCMCL1 (X. Zhao, S. Shetty, M. R. Smith, J. Bodo, E. D. Hsi unpublished data). Mechanistically, ibrutinib/ABT-199 interaction caused synergistic effects in MCL cells through perturbation of p-BTK and p-AKT mediated survival signals and of BCL2 family proteins (Fig 2E). Ibrutinib was developed as a BTK inhibitor to block BCR signalling. To date, few studies described alterations of BCR signalling causing any effects on expression or function of BCL2 family proteins. However, BTK is necessary for BCR-induced phosphorylation of cAMP-response element-binding protein (CREB) (Blois et al, 2004). In mature B cells, phosphorylation of CREB induced BCL2 protein expression upon cross-linking of surface immunoglobulin, which leads to rescue cells from apoptosis (Wilson et al, 1996). Such mechanism may apply in malignant B-cells and help to explain the effects of inactivation of BTK in tested MCL cells. High levels of BCL2 family proteins are implicated in resistance of cells to many anticancer drugs. In this study, at least one, and often two, BCL2 family protein(s) were down-regulated by ibrutinib/ABT-199 co-treatment. For example, ibrutinib alone or in combination with ABT-199 caused a substantial decrease of MCL1 protein in CCMCL1 cells. It was reported that cyclin-dependent kinase inhibitor potentiate BCL2 antagonist ABT-737 induced apoptosis through down regulation of MCL1. Such a synergistic drug interaction provides a mechanistic basis for simultaneous targeting of MCL1 and BCL2/BCL2L1 in leukaemia/lymphoma cells (Chen et al, 2007). Our data support a mechanism by which enhanced apoptotic induction by ibrutinib/ABT-199 combination is at least in partl due to simultaneous targeting of BCL2 family proteins. While performing our studies, a combinational drug screening study reported that ibrutinib in combination with a range of agents, including ABT-199, had synergy in inhibiting proliferation of MCL cell lines (Axelrod et al, 2014). Our study provides data about changes in signalling and other cellular molecules that can potentially serve as biomarkers for activity and resistance. Further, this knowledge can be exploited in developing multi-agent combination therapy. Our mechanistic studies demonstrate that the synergy is not merely due to these two agents affecting separate pathways, but, rather unexpectedly, that each agent enhances the predicted effects of the other agent on its known targets. Overall, our study supports further investigation of this combined therapeutic strategy for MCL. This work was partially supported by Pilot Project Award from Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic to XZ. XZ and EDH are the principal investigators and take primary responsibility for the paper. XZ, JB, DS, LD and JL performed laboratory work for this study. XZ, JB, MRS and EDH coordinated the research and wrote the paper. Authors have no conflict of interest to disclosure. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.