PYK2 Mediates Microenvironment-Specific Myeloma Survival

Multiple myeloma remains an incurable bone marrow resident malignancy due to the eventual development of drug resistance and minimal residual disease linked to both acquired genetic changes and the dynamic influences of 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]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow, myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariate microenvironment, we 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 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced by beta1 integrin mediated adhesion and under multivariate conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 179 differentially tyrosine phosphorylated peptides among four conditions (Suspension or FN, +/- IL-6). Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Among 67 peptides upregulated by adhesion to FN alone, proline-rich tYrosine Kinase 2 (PYK2/FAK2) was highly phosphorylated at Y580 in RPMI8226 myeloma cells, and focal adhesion formation in adhered cells was validated by phosphorylation of paxillin at pY118 and p130CAS at pY128/pY249. PYK2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation [3]. PYK2 has also been linked to downstream signaling events of JAK-dependent cytokine receptors, but not to a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families [4-7]. As such, we hypothesized that PYK2 may be an important modulator of the enhanced STAT3 activation following multivariate signaling between beta1 integrin and the IL-6 signal transducer, gp130. In addition to enhanced phosphorylation of PYK2 at Y580 identified by proteomic screening, western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting PYK2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. PYK2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). The protective advantage afforded myeloma cells in coculture was attenuated knockdown of PYK2 or STAT3 expression in myeloma cell lines and patient specimens using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value < 0.05). Critically, inhibition of PYK2 with nanomolar amounts of the focal adhesion kinase inhibitor PF562.271 had a similar impact only on myeloma cells directly adhered to BMS, but did not impact BMS viability. These data demonstrate a novel PYK2-mediated JAK1/STAT3 signaling cascade within the context of multivariate stimulation. Moreover these data identify a novel PYK2-mediated survival pathway activated only within the context of multivariate stimulation in the bone marrow microenvironment, and suggests that PYK2 represents a MRD-specific target.