Targeting PYK2 Mediates Microenvironment-Specific Myeloma Cell Death

Multiple myeloma (MM) remains an incurable malignancy, at least in part, due to minimal residual disease (MRD). It has been hypothesized that MRD is influenced by the survival promoting aspects of the bone marrow tumor microenvironment (TME) and populations of putative MM cancer stem cells. Therefore, identification of survival determinants specific to the TME and putative MM stem cells is critical for the rational design of therapy and elimination of MRD¹. Within the context of the TME, we have shown that collaborative signaling between β₁ integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation². We have validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in MM patient specimens by flow cytometry. Further characterization of the events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. Western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β₁ integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Although PYK2 has been linked to downstream signaling events of other JAK-dependent cytokine receptors, it has not been associated with a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families^3-6. Assessing downstream gene expression, we also demonstrated that adhesion to FN alone or co-stimulation with FN and IL-6 upregulated mRNA and protein expression of the MM survival-associated gene, deptor⁷, and this enhanced expression was abrogated by RNAi knockdown of STAT3 or PYK2. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Importantly, MM cells co-cultured with patient BMSCs showed similar gp130 and β₁ integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Interestingly, targeting PYK2 with antisense oligonucleotides (ISIS Pharmaceuticals) or with the dual PYK2/FAK (Focal adhesion kinase) tyrosine kinase inhibitors VS-4718, VS-6062, and VS-6063 (Verastem, Inc.) induced preferential MM cell killing in the context of adhesion to patient BMSCs, but not in monoculture or under transwell co-culture conditions. We confirmed that this cell death correlated with inhibition of BMSC-induced PYK2 and STAT3 phosphorylation. Further analysis similarly demonstrated a preferential reduction in colony formation in BMSC-adherent MM cell lines and patient specimens treated with VS-6063 relative to MM cells in the absence of BMSCs. Lastly, we examined the effects of targeting PYK2 with VS-6063 in ALDH+ MM cancer stem cells. We demonstrated that co-culture with BMSCs increased the percentage of ALDH+ MM cancer stems relative to monoculture. VS-6063 decreased the percentage of the ALDH+ cells associated with BMSC to greater degree than MM cells without BMSC. These data identify a novel PYK2-mediated survival pathway activated within the context of multiple microenvironmental cues, suggesting that PYK2 is a putative TME-specific therapeutic target. Further, putative MM cancer stem cells are sensitive to PYK2 inhibition by the FAK/PYK2 inhibitor VS-6063. As such, these data indicate that PYK2 may be critical target for the eradication of MRD by targeting both MM cells in the context of TME and MM cancer stem cells. These results suggest that MM represents a compelling clinical development path for FAK/PYK2 inhibitors, such as VS-4718 and VS-6063, in clinical trials focusing on eradication of MRD.