Multiple myeloma remains an incurable bone marrow resident malignancy, in part, due to the eventual development of drug resistance 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 . 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 multivariant 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 . 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 . 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 under multivariant 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 338 differentially tyrosine phosphorylated peptides. Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Proline-rich tYrosine Kinase 2 (Pyk2/FAK2) was highly phosphorylated at Y597 in FN-adhered RPMI8226 myeloma cells. Eighty-three phosphorylation events were up-regulated by adhesion, including paxillin at pY118 and p130CAS at pY249 indicating focal adhesion formation. 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 . In addition to enhanced phosphorylation of Pyk2 at Y579 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). 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 used proximal ligation assay (PLA). Confocal imaging of the PLA reaction with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of patient myeloma cells to FN, but not in those grown in suspension. 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). Critically, the protective advantage afforded myeloma cells in coculture was attenuated only by dual knockdown of STAT3 and Pyk2 using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value 0.027). These data demonstrate a novel Pyk2-mediated JAK1/STAT3 signaling cascade within the context of multivariant stimulation. Moreover, these data suggest that strategies targeting of both lateral and vertical signaling (Pyk2 & STAT3) may be required to overcome therapy resistance conferred to tumor cells by the multivariant bone marrow niche.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.