BTK inhibitor PCI-32765 targets endogenous and microenvironment-mediated B-cell receptor signaling to suppress lymphoma cell growth and is highly synergistic with Bortezmib against non-Hodgkin B-cell lymphomas

B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of B-cell malignancies and Bruton tyrosine kinase(Btk) is essential for BCR signaling and function. PCI-32765, a specific and irreversible small-molecule Btk inhibitor, has recently been reported to display a significant clinical activity against non-Hodgkin B-cell lymphomas (NHL) especially chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL). In this study we set to explore 1) the role of Btk in NHL cell apoptosis and proliferation, 2) the role of BtK in bone marrow strom-mediated lymphoma cell survival and 3) to test if PCI-32765 as a therapeutic agent in single or in combination with Bortezomib for NHL therapy.

B-cell lymphoma cell lines including mantle cell lymphoma lines (Jeko-1 and HBL-2), Burkitt lymphoma cell line (Raji) and transformed large cell lymphoma cell line (SUDHL-10) as well as primary lymphoma cells from various NHL samples were used for the experiments. These cells were cultured in the presence or absence of bone marrow mesenchymal stromal cells (MSC). The endogenous and MSC-induced Btk and its signaling activation such as BtK, ERK1/2 and AKT expression and phosphorylation status as well as its inhibition by were examined PCI-32765 by Western blot. The effects of PCI-32765 on lymphoma cell growth and appotosis were analyzed by using MTT, DAPI stain and flow cytometric annexin V/PI staining. Furthermore, the combined effect of PCI-32765 and Bortezomib on lymphoma cell growth and apoptosis was analyzed using the CalcuSyn software program in search for a synergistic or additive effect.

We found constitutive expression and activation of Btk and its downstream signaling in most of these cell lines and primary lymphoma cells. Furthermore, co-culture with MSC cells further enhanced the phosphoration of Btk and AKT in these cells. Incubation of Jeko-1, Raji, HBL-2 and SUDHL-10 cell lines with PCI-32765 induced cell growth inhibitory effects. We found that PCI-32765 exhibited a significant dose-dependent induction of cytotoxicity in these cells at various time points as measured by MTT. We also found significant apoptosis in these cells treated with PCI-32765. In addition, PCI-32765 significantly inhibited phpsphorylation of AKT and Btk, confirming the block of BCK signal pathways in these cells. Finally, MTT assays indicated that combined PCI-32765 with Bortezomib induced a synergistic cytotoxicity against these NHL cells (CI<1).

Our studies therefore highlight the biological significance of Btk in B-cell lymphoma cell growth and survival. PCI-32765 effectively antagonizes B-cell survival provided by bone marrow stromal cells and synergistically in combination with Bortezomib eliminates lymphoma cells. This study provide rational for targeting BCR and Btk as a novel therapeutic approach for NHL.

No relevant conflicts of interest to declare.