PYK2 Inhibitors Sensitize Hypoxia-Induced Drug Resistant Multiple Myeloma Cell
Authors: Muz Barbara , Buggio Maurizio , Azab Feda , Trombino Anthony F. , Padval Mahesh , Azab Abdel Kareem 
Published: October 2019

Abstract
Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and hyperproduction of immunoglobulin IgG. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance and micro-residual disease attributed to cancer stem cells. The hypoxic nature of the BM plays a critical role in MM cells acquiring a stem cell-like phenotype, and together with cellular and acellular components of the BM microenvironment contributes to drug resistance leading to minimal-residual disease. In this study, we tested inhibition of proline-rich tyrosine kinase 2 (PYK2), using VS-4718 and VS-6063, on reversing the hypoxia-inducible stem cell-like phenotype of MM cells and sensitizing them to therapy both in vitro and in vivo.
Methods: VS-4718 and VS-6063 FAK/PYK2 inhibitors were obtained from Verastem, Inc. MM cell lines (MM1s, H929 and RPMI8226) were treated with bortezomib or carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the FAK/PYK2 inhibitors VS-4718 or VS-6063. MM cells were analyzed for cell proliferation by MTT assay; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with cell proliferation and apoptosis was analyzed by western blotting.
Furthermore, we have tested the effect of the PYK2 inhibitors on sensitization of bortezomib-resistant MM cells in two models in vivo. In the first model, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumors were allowed to grow for four weeks; animals were then treated with a low-dose of bortezomib (0.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were divided into 6 groups treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) VS-6063 (50mg/kg) alone (4) bortezomib alone, (5) combination of VS-4718 and bortezomib, and (6) combination of VS-6063 and bortezomib, where VS-4718 and VS-6063 were administered by oral gavage twice a day every day. Mice survival was then followed for four more weeks.
In the second model, H929 cells were injected subcutaneously into SCID mice. When tumors reached a mean of ~128 mm3 mice were randomized into five groups and treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) bortezomib (1mg/kg) alone, (4) combination of VS-4718 and bortezomib (concurrently), and combination of VS-4718 and bortezomib (sequentially to simulate minimum residual disease). VS-4718 was administered by oral gavage twice a day and tumor growth was measured twice weekly using calipers.
Results: We found that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells in both hypoxia and normoxia as single agents. Moreover, we found that hypoxia induced drug resistance to bortezomib and carfilzomib in MM cells. The combination of the two PYK2 inhibitors with either of the proteasome inhibitors reversed the hypoxia-induced resistance in MM cells and induced a synergistic effect with proteasome inhibitors on inhibition of cell proliferation, apoptosis, and cell signaling related to proliferation and apoptosis. In vivo study revealed that mice that developed MM disease with resistance to bortezomib were resensitized by treatment with the PYK2 inhibitors and showed significantly extended survival compared to mice treated with bortezomib alone. Furthermore, in mice treated with bortezomib only, tumors progressively re-grew after bortezomib treatment ended. While, in the bortezomib and VS-4718 combination treated mice and mice treated with VS-4718 in minimal residual setting (MRD) tumor grow-back (relapse) was prevented after cessation of bortezomib treatment.
Conclusions: We report that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells as single agents. Hypoxia induced resistance to proteasome inhibitors and the PYK2 inhibitors resensitized MM cell to therapy in vitro and in vivo. Moreover, FAK/PYK2 inhibitor VS-4718 was able to prevent relapse in in vivo MM model simulating minimal residual disease. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by FAK/PYK2 inhibitors and their use to reduce relapse post front line treatment in an MRD setting.

Disclosures
Trombino: Verastem: Employment. Padval:Verastem: Employment. Azab:Verastem: Research Funding.