Abstract
Background. Proline-rich tyrosine kinase (Pyk2) is a non-receptor tyrosine kinase which belongs to the focal adhesion kinase (FAK) family. It is known to facilitate the TNFα induced EMT process in solid tumors, but this has not been investigated in the field of hematologic malignancies. We therefore dissected the role of PyK2 in multiple myeloma (MM) by looking at its ability to modulate MM biology both in vitro and in vivo.
Methods. Lentiviral packaged small hairpin RNA (shRNA), overexpression plasmid, related scrambled shRNA probe and empty vector were introduced into the MM1.S (GFP+/Luc+) cell line, to generate stable Pyk2 knock down (K.D.; #A2 and #A4), Pyk2 over-expression (Pyk2+), and control cells, respectively. The efficiency of K.D. or over-expression was validated by qPCR and immunoblotting. Cell viability and cell proliferation were detected by using CellTiter-Glo® luminescent assay and thymidine uptake, respectively. Gain- and loss-of function studies were also performed on MM cells in the presence of primary bone marrow stromal cells isolated from MM patients (MM-BMSCs). Adhesion of Pyk2 stable cells to fibronectin was measured by using an ECM cell adhesion assay kit. The synergistic effects of Pyk2 with Bortezomib were determined through calculating the DNA synthesis of Pyk2 K.D. cells treated with Bortezomib (2.5-5 µM), using Calcusyn software and Chou-Talalay method. Pyk2 K.D. stable cells were intravenously injected into SCID-Biege mice to generate a xenograft model. In vivo tumor growth was monitored by Bioluminescent Imaging. Pyk2-dependent-modulation of the Wnt/β-catenin pathway signaling was assessed using immunoblotting. The FAK/Pyk2 kinase inhibitor, VS-4718, was tested in vitro using both MM cell lines and primary bone marrow-derived MM cells; and in vivo using bioluminescence imaging.
Results. Knockdown of Pyk2 in MM cells significantly repressed cell viability and proliferation, as well as their adhesive ability to BMSCs, compared to scrambled shRNA control cells. Moreover, Pyk2 knockdown induced de-adhesion of MM cells from BMSCs thus inducing chemosensitivity of tumor cells to Bortezomib. We next corroborated our findings by studying Pyk2+ MM cells, and showed that stably upregulated Pyk2 expression promoted MM cell growth as measured by either ATP quantitation or DNA synthesis. Upregulation of Pyk2 expression also stabilized the adhesion of MM cells to BMSCs, leading to drug resistance of MM cells to Bortezomib, compared with vector control cells. Pyk2-regulated tumor growth was further validated by establishing a xenograft mouse model. By using bioluminescence imaging, we found a significantly lower tumor burden in mice injected with Pyk2 K.D. cells, compared to control mice (injected with scrambled shRNA cells). We next dissected the effect of Pyk2 in modulation of cellular signaling in MM cells by using immunoblotting, and demonstrated that Pyk2 played an important role in regulating β-catenin signaling. Indeed, knockdown of Pyk2 induced GSK3β phosphorylation, leading to increased β-catenin phosphorylation, thus resulting in β-catenin degradation and inhibited translocation to the nucleus. Importantly, Pyk2 K.D. cells presented with reduced expression of c-myc and cyclin D1 at the protein level. Conversely, Pyk2 overexpression enhanced β-catenin expression together with c-myc and cyclin D1 up-regulation, thus confirming the role of Pyk2 in modulating Wnt/β-catenin signaling activity in MM. We finally determined that VS-4718, a potent FAK/Pyk2 kinase inhibitor currently in a phase I clinical study, induced apoptosis in MM cell lines and primary MM cells, and inhibited MM tumor growth in vivo.
Conclusion. These findings indicate that Pyk2 exhibits pro-oncogenic properties in MM through modulation of Wnt/β-catenin signaling. The findings that the FAK/Pyk2 kinase inhibitor VS-4718 exerts anti-MM activity in cellular and in vivo models support MM as a potential clinical direction for this agent.
Disclosures
Ring: Verastem: Employment. Tam:Verastem: Employment. Xu:Verastem: Employment. Pachter:Verastem Inc.: Employment, Equity Ownership. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.
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