Mark B. Meads

Abstract The bone marrow microenvironment facilitates the survival, differentiation, and proliferation of hematopoietic cells. These cells are supported by fibroblast-like bone marrow stromal cells, osteoblasts, and osteoclasts which secrete soluble factors and extracellular matrix proteins that mediate these functions. This rich environment serves as a safe haven not only for normal and malignant hematopoietic cells, but also for epithelial tumor cells that metastasize to bone, offering protection from chemotherapeutic agents by common mechanisms. Soluble factors produced in the bone marrow, such as stromal cell–derived factor-1 and interleukin-6, mediate homing, survival, and proliferation of tumor cells, and integrin-mediated adhesion sequesters tumor cells to this protective niche. Environment-mediated drug resistance includes a combination of soluble factors and adhesion, and can be subdivided into soluble factor–mediated drug resistance and cell adhesion–mediated drug resistance. Because it is induced immediately by the microenvironment and is independent of epigenetic or genetic changes caused by the selective pressure of drug exposure, environment-mediated drug resistance is a form of de novo drug resistance. In this form of drug resistance, tumor cells are transiently and reversibly protected from apoptosis induced by both chemotherapy and physiologic mediators of cell death. This protection allows tumor cells to survive the insult of chemotherapy, leading to minimal residual disease, and thereby increases the probability for the development of acquired drug resistance.

The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of multiple myeloma (MM) cells; however, in vivo, these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via beta1 integrins, stimulated with IL-6, or treated with the two combined. Although G(1)-S cell cycle arrest associated with FN adhesion was overcome when IL-6 was added, the cell adhesion-mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in signal transducers and activators of transcription 3 (STAT3) phosphorylation, nuclear translocation, and DNA binding, compared with either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN before stimulation with IL-6, relative to nonadherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by beta1 integrin and gp130 confers an increased survival advantage to MM cells.

The novel Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-β1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies.

// Joel G. Turner 1 , Trinayan Kashyap 2 , Jana L. Dawson 1 , Juan Gomez 1 , Alexis A. Bauer 1 , Steven Grant 3 , Yun Dai 3 , Kenneth H. Shain 1, 4 , Mark Meads 1 , Yosef Landesman 2 , Daniel M. Sullivan 1, 5 1 Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Karyopharm Therapeutics, Natick, MA, USA 3 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA 4 Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 5 Department of Blood & Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Correspondence to: Daniel M. Sullivan, email: dan.sullivan@moffitt.org Keywords: XPO1, bortezomib, carfilzomib, multiple myeloma, acquired drug resistance Received: July 26, 2016      Accepted: October 12, 2016      Published: October 28, 2016 ABSTRACT Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.