Pyk2 Mediates Enhanced STAT3 Phosphorylation Following Collaborative Signaling Between gp130 and beta1 Integrins in Adhered Myeloma and B Cells

Abstract

Abstract 2895Multiple myeloma is the second most common hematologic malignancy and remains incurable due to the eventual development of drug resistance. Therapy resistance has been linked to both acquired genetic changes as well as the dynamic influences of soluble and physical factors present in the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. To date, the influence of these two effectors on drug resistance has been studied separately; however, within the context of the bone marrow myeloma cells are influenced by both soluble and physical effectors of the microenvironment simultaneously. Using a reductionist model of the multivariant microenvironment, we recently demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype[2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. To further characterized the molecular events mediating this unique event we focused on the focal adhesion kinase, Proline-rich tYrosine Kinase 2 (Pyk2/FAK2). Pyk2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation. As such, we hypothesized that Pyk2 may be an important modulator of the enhanced STAT3 activation following multivariant signaling between beta1 integrins and gp130[3]. Our results link this amplification to Pyk2 in myeloma and murine pro-B cell lines. Western blot analysis demonstrated that Pyk2 autophophorylation on tyrosine 402 is induced following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3, JAK1, and gp130 phosphorylation in both myeloma and pro B cell line models. Targeting Pyk2 with RNA interference attenuated the adhesion-associated amplification of STAT3 signaling as well as JAK1 and gp130 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. Further we demonstrated that Pyk2 kinase activity was similarly required for the activation of JAK1/STAT3 signaling under collaborative conditions, but not by IL-6 alone. Consistent with previous results ERK1/2 and Akt signaling were not affected by Pyk2 siRNA or kinase activity. Importantly, Pyk2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). Our previous results demonstrated that that the enhanced STAT3 signaling involved a FN-adhesion specific binding of unphosphorylated STAT3 with gp130 (independent of IL-6 stimulation). To determine if Pyk2 was similarly recruited to gp130 under co-stimulatory conditions we have initially used confocal microscopy. Confocal imaging with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of myeloma cell to FN, but not in those grown in suspension. These data demonstrate that within the context of multivariant stimulation (IL-6 and FN-adhesion) a unique Pyk2-mediated JAK1/STAT3 signaling cascade is associated with unique biologic sequelae. We are currently exploring the biological sequelae of Pyk2-mediated collaborative signaling between integrins and IL-6. Key endpoints currently under investigation include proliferation and drug-response. These findings are significant because they suggests strategies targeting Pyk2 could be used to block cooperative signaling between integrins and IL-6, and inhibit the proliferation and/or therapy resistance conferred to tumor cells by the multivariant bone marrow niche. Disclosures:No relevant conflicts of interest to declare.