Published OnlineFirst April 21, 2011; DOI: 10.1158/0008-5472.CAN-10-4002

Cancer Molecular and Cellular Pathobiology Research

Bone Marrow –Secreted Protect JAK2V617F-Mutated Cells from the Effects of a JAK2 Inhibitor

Taghi Manshouri1, Zeev Estrov1, Alfonso Quintas-Cardama 1, Jan Burger1, Ying Zhang1, Ana Livun1, Liza Knez1, David Harris1, Chad J. Creighton2, Hagop M. Kantarjian1, and Srdan Verstovsek1

Abstract Signals emanating from the microenvironment, such as stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative (MPN). To examine this hypothesis, we established a coculture platform [cells cocultured directly (cell-on-cell) or indirectly (separated by micropore membrane)] designed to interrogate the interplay between Janus activated kinase 2- V617F (JAK2V617F)–positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET-2) and mouse (FDCP-EpoR) JAK2V617F-positive cells as well as primary blood or bone marrow mononuclear cells from patients with polycythemia vera; however, these effects were attenuated when any of these cell types were cocultured (cell-on-cell) with human marrow stromal cell lines (e.g., HS5, NK.tert, TM-R1). Coculture with stromal cells hampered the ability of atiprimod to inhibit phosphorylation of JAK2 and the downstream STAT3 and STAT5 pathways. This protective effect was maintained in noncontact coculture assays (JAK2V617F-positive cells separated by 0.4-mm-thick micropore membranes from stromal cells), indicating a paracrine effect. profiling of supernatants from non- contact coculture assays detected distinctly high levels of (IL-6), (FGF), and chemokine C-X-C-motif ligand 10 (CXCL-10)/IFN-g–inducible 10-kD protein (IP-10). Anti-IL-6, -FGF, or -CXCL- 10/IP-10 neutralizing ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2V617F-positive cells. Therefore, our results indicate that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, thus underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes. Res; 71(11); 3831–40. 2011 AACR.

Introduction and in approximately 50% of patients with ET or PMF (1–5). JAK2V617F activates several signaling pathways crucial for The myeloproliferative neoplasms (MPN)—polycythemia cellular survival and proliferation. The putative role of vera (PV), essential thrombocythemia (ET), and primary mye- JAK2V617F in the pathogenesis of MPNs provided the rationale lofibrosis (PMF)—originate from the clonal transformation of for the development of JAK2 inhibitors for the treatment of hematopoietic stem cells (HSC)/hematopoietic progenitors patients with MPNs. Clinical trials testing the activity of (HP), which gives rise to abnormal proliferation of one or several JAK2 inhibitors are ongoing, particularly in myelofi- several hematopoietic lineages driven by hypersensitivity to brosis (6, 7). Although preliminary results show significant regulatory growth factors (1). Deregulation of kinase activity clinical benefit of therapy, these agents have shown no activity has emerged as a major mechanism by which cancer cells in correcting the fibrosis, osteosclerosis, and neoangiogenesis evade normal physiologic constraints on growth and survival. that characterize the bone marrow of patients with myelofi- In MPNs, the gain-of-function Janus activated kinase 2-V617F brosis, and, furthermore, there has been no elimination of V617F (JAK2 ) mutation is present in almost all patients with PV malignant clone, as evaluated by the continuous presence of JAK2V617F-positive cells in patients on therapy. Several lines of evidence suggest that, in myelofibrosis, Authors' Affiliations: 1Department of Leukemia, the University of Texas stromal cells are primed by the malignant hematopoietic MD Anderson Cancer Center; and 2Division of Biostatistics, Dan L. Duncan clone, which, in turn, conditions the stroma to create a Cancer Center, Baylor College of Medicine, Houston, Texas "favorable" pathologic microenvironment that nurtures and Note: Supplementary data for this article are available at Cancer Research protects the malignant cells. In myelofibrosis, both cellular Online (http://cancerres.aacrjournals.org/). and extracellular levels of various fibrogenic and angiogenic Corresponding Author: Srdan Verstovsek, Department of Leukemia, The cytokines are increased, thus supporting the notion that the Univeristy of Texas MD Anderson Cancer Center, Unit 428, 1515 Hol- combe Blvd., Houston, TX 77030. Phone: 713-745-3429; Fax: 713-794- bone marrow histologic changes that characterize myelofi- 4297; E-mail: [email protected] brosis are reactive and mediated by cytokines such as TGFb, doi: 10.1158/0008-5472.CAN-10-4002 platelet-derived growth factor (PDGF), basic fibroblast growth 2011 American Association for Cancer Research. factor (bFGF), and VEGF (8). The net result is a tumor niche

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Manshouri et al.

that provides environmental cues contributing to the prolif- rabbit anti-total STAT3 (06-596), and rabbit anti-total STAT5 eration, maintenance, and (potentially) resistance to therapy (05–533); all obtained from Upstate Biotechnology. Goat anti- of the malignant clone. Indeed, marrow stromal cells have human-interleukin 6 (IL-6; AF-206-NA), -chemokine C-X-C- been shown to protect chronic lymphocytic leukemia (CLL) motif ligand 10 [CXCL-10/IFN-g–inducible 10-kD protein (IP- cells from spontaneous or drug-induced apoptosis in vitro and 10); AF-266-NA], and -FGF basic/2 (FGF-2; AF233-NA) were to confer resistance to therapy in CLL and other B-cell obtained from R&D Systems (Minneapolis, MN). Mouse anti- malignancies, such as acute lymphoblastic leukemia (ALL; b-actin (A5441) was purchased from Sigma-Aldrich. Conju- refs. 9–11). Understanding the mechanisms of information gated horseradish peroxidase sheep anti-mouse antibodies exchange between the malignant clone and the bone marrow (NA931V) from GE Healthcare, and horseradish peroxidase milieu may illuminate methods to eliminate malignant MPN donkey anti-rabbit antibodies (711-035-15) from Jakson cells that reside in a protective stromal niche within the ImmunoReseach were obtained. Cytokines used include marrow. In this article, we present evidence supporting a recombinant human anti-IL8 (208-IL010), -FGF (hFGF; 233- protective effect of the stromal bone marrow niche against FA-025), and -VEGF (hVEGF; 4644-VS/CF) from R&D Systems. JAK2 inhibitor therapy via stromal cell–secreted humoral The JAK2 inhibitor atiprimod (Callisto Pharmaceutical) was factors. The manipulation of these contextual cues might dissolved in PBS (Gibco BRL) to a final concentration of 8 be potentially exploited in therapeutic applications for the mmol/L. The stock solution was stored at 4C and further eradication of JAK2V617F-positive clones. diluted in tissue-cultured medium just before use.

Materials and Methods Growth-inhibition MTT assay MTT assays were conducted as previously described (13, Cells, monoclonal antibodies, and chemicals 14). Results shown represent the average SD of 3 independent Murine FDCP (factor-dependent cell Patersen) cells trans- experiments, each done in sextuplets. fected with the