Fig. 5.

JAK2^V617F and TNFR2 cooperate to increase NF-κB signaling and reduce apoptosis in MF cells. a Human CD34⁺ cells isolated from MF patient (n = 3) or normal BM (n = 3) were treated with birinapant at 10 or 100 nM for 72 h in liquid culture and then plated in clonogenic assays with continued treatment. Colony inhibition was significantly different for normal vs. MF samples at 10 nM, while 100 nM birinapant inhibited both. b Human CD34⁺ cells isolated from MF patient (n = 3) or normal BM (n = 3) were infected with an NF-κB luciferase reporter construct 72 h prior to evaluation. Cells were treated with TNFR1 or TNFR2 BA (10 μg/mL) prior to stimulation with TNF (1 ng/mL). The fold increase in reporter activity was significantly higher in MF cells at 4 h post stimulation and over the complete time course (P = 0.03). Both TNFR1 and TNFR2 BAs reduced TNF stimulated NF-κB activity in MF and normal BM cells. c Annexin V was measured in MF CD34⁺ cells (n = 4) 72 h after infection with XIAP, MAPK8 or vector control expression constructs. Ectopic expression of XIAP or MAPK8 significantly increased Annexin V staining relative to vector control. d Downregulation of MAPK8 by JAK2^V617F and TNFR2 inhibits apoptotic signaling through TNFR1 by preventing MAPK8 from promoting the active form of TNFR1 Complex II. Downregulation of XIAP by JAK2^V617F and TNFR2 is associated with increased cIAP protein levels. Either TNFR1 (with TRADD) or TNFR2 can form a signaling complex through association with TRAF2 and cIAP to activate NF-κB transcription of pro-survival and inflammation-associated genes. These combined effects favor survival of JAK2^V617F cells. *P < 0.05, **P < 0.005