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

Abstract

Signals emanating from the bone marrow microenvironment, including stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis we established a co-culture platform (cells co-cultured directly [cell-on-cell] or indirectly [separated by micropore membrane]) designed to interrogate the interplay between JAK2V617F–positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET2) and mouse (FDCP-EpoR) JAK2V617F–positive cells, and of primary blood or bone marrow mononuclear cells from patients with polycythemia vera, but these effects were attenuated when any of these cell types were co-cultured (cell-on-cell) with human marrow stromal cell lines (HS5, NK.tert, or TM-R1). Co-culture with stromal cells hampered the ability of atiprimod to inhibit the phosphorylation of JAK2 and the downstream signal transducer and activators of transcription (STAT) 3, and STAT5. This protective effect was maintained in non-contact co-culture assays (JAK2V617F–positive cells separated by 0.4 μm micropore membranes from stromal cells), suggesting a paracrine effect. Cytokine profiling of supernatants from non-contact co-culture assays detected distinctly high levels of IL-6, FGF, and CXCL10/IP10. Anti-IL-6, -FGF, or -CXCL10/IP10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2V617F–positive cells. Thus, our results suggest that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.

Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms

“…Such cytokines are produced by the bone marrow stroma and have been shown to promote the growth of MPN clones, while, in some cases inhibiting the growth of wild-type clones. 93,94 Overexpression of NF-I, which has been described in patients with uniparental disomy (UPD) of chromosome 9, has also been suggested to result in resistance to transforming growth factor-β (TGF-β), which has been demonstrated to have inhibitory effects on the hematopoiesis and on myeloid cell lines. 95,96 The secretion of proteases which disrupt the stromal cell derived factor-1/chemokine receptor 4 (CXCR4) axis, as well as the downregulation of CXCR4 itself by tumor cells, have both been associated with greater mobilization of HSCs, and these mechanisms may contribute to extramedullary hematopoiesis and potentially mediate the loss of HSC quiescence.…”

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms

“…Therefore, it remains to be addressed whether these differences in ST2 expression patterns rely on species-dependent divergences and whether ST2 can be also engaged on human BM stromal cells. This is relevant because humoral factors secreted by BM stromal cells may also support MPN pathogenesis by impairing compound-mediated JAK2 inhibition (11).…”

“…While JAK inhibitors have been successful in treating JAK2-mutated MPNs, elimination of the mutant clone and resistance may pose an important problem, and patients with WT JAK2 MPNs are not amenable to this therapy (7). The abnormal expression and activity of a number of proinflammatory cytokines are associated with BCR-ABL1-negative MPNs (8), possibly initiating clonal evolution (9) or promoting progression to myelofibrosis (10) and resistance against JAK2 inhibitors (11). Thus, cytokines and growth factors support the aberrant hematopoiesis associated with MPNs and provide mechanisms for drug resistance.…”

Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells

“…23 A phase 2 study of INC424 in patients with relapsed/ refractory AML showed good tolerance and modest antileukemic activity. 24 Fifteen of 38 patients studied showed decreased or stabilized blasts in blood and marrow with complete remission achieved in 3 patients with prior myeloproliferative neoplasms.…”