TNFR2 Signaling Promotes Myeloproliferative Disease Progression: A Role for MAPK8?

The majority of patients diagnosed with Philadelphia negative (Ph-) myeloproliferative neoplasms (MPNs) harbor somatic, gain-of-function mutations in JAK2, CALR or MPL. All of these mutations are associated with constitutive JAK/STAT signaling which confers a proliferative advantage to MPN cells and leads to malignant myeloid expansion at the expense of normal hematopoiesis. The bone marrow (BM) microenvironment in MPNs, particularly myelofibrosis (MF), is characterized by high concentrations of inflammatory cytokines, and we have previously shown that MF cells generate tumor necrosis factor alpha (TNF) in a JAK2-dependent manner. Elevated TNF promotes the survival of JAK2V617F mutant cells over their JAK2WT counterparts, creating a feedback loop in which the mutant cells enhance the inflammatory environment that supports their survival and expansion (Fleischman et al. Blood. 2011 Dec 8;118(24):6392-8). To determine which hematopoietic lineages contribute to increased levels of TNF in MPN, we measured intracellular TNF expression in immunophenotypically defined white blood or BM cells from MF patients and normal controls. TNF expression was relatively low in unstimulated cells. However, lipopolysaccharide (LPS) treatment induced a 16-fold greater increase of TNF expression in hematopoietic stem cells (HSCs) from MF patients relative to normal controls (p TNF signaling is mediated by two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). To evaluate the contribution of each receptor to the TNF-mediated survival of MPN cells, we cultured CD34+ cells from MF patients or normal controls for 72 hours ± TNFR1 or TNFR2 blocking antibodies (BA), then plated in clonogenic assays. While TNFR1 BA had no effect, TNFR2 BA reduced colony numbers in MF specimens by 26% (p To identify TNF signaling networks that differentiate the TNF responsiveness of cells expressing JAK2V617F compared to JAK2WT, BM cells were harvested from JAK2V617F MPN mice and cultured for 16 hours ± TNFR1 or TNFR2 BA. Lin-Kit+ cells were sorted based on GFP (JAK2V617F) expression and 3 independent experiments were tested with Affymetrix Mouse Genome 430 2.0 Arrays. We focused our analysis on genes with differential regulation between JAK2V617F and JAK2WT, whoseexpression was equalized with TNFR2 BA. We then prioritized genes with reported involvement in TNF signaling. The top candidate was Mapk8 (Jnk1) whose expression was strongly downregulated in untreated JAK2V617F relative to JAK2WT expressing cells (3.4-fold; p Disclosures Deininger: Gilead: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.