Tumor Necrosis Factor-Alpha (TNF) Expression Is Elevated in Myelo-Proliferative Neoplasms (MPN) and Modulated by Inhibition of JAK2 V617F.

Myeloproliferative neoplasms (MPN) are hematopoietic stem cell diseases encompassing chronic myeloid leukemia (CML), essential thrombocythemia (ET), idiopathic myelofibrosis (IMF), and polycythemia vera (PV). More than 95% of all patients with PV and some 50% of patients with ET and IMF have an activating mutation of JAK2 (JAK2 V617F). Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine produced by multiple cell types, including macrophages, NK, T and B cells. TNF plays a key regulatory role in immune and inflammatory responses. There is also evidence that TNF is involved in the regulation of erythropoiesis. We have previously reported that TNF levels are elevated in mice with retrovirally induced JAK2 V617F-positive PV-like MPN and that lack of TNF attenuates MPN in this model.

We studied the expression of TNF in white blood cells from PV patients (n=43) and normal controls (n=21), using qPCR. PV patients expressed on average 2.5-fold higher levels of TNF than controls (p<0.0004). Upon examination of a panel of human leukemia lines (n=8) we found the highest TNF expression in HEL cells, which are homozygous for JAK2 V617F, suggesting that JAK2 V617F may upregulate TNF expression. Consistent with this, we found 6.9-fold higher TNF expression in Ba/F3 cells constitutively co-expressing EPOR and JAK2 V617F compared to parental cells. Exposure of HEL cells or Ba/F3 EPOR JAK2 V617F cells to the JAK2 inhibitor CYT387 (2 μM) resulted in a time dependent 3.3 to 7.5-fold decrease of TNF mRNA as assessed by qPCR. By contrast, TNF mRNA expression was not found to be down-regulated by CYT387 in HL60 cells lacking mutated JAK2. CYT387 was also found to down-regulate TNF mRNA expression in MNC derived from MPN patients. To assess differential effects of TNF on normal versus MPN progenitor cells, we performed clonogenic assays of peripheral blood MNC from normal donors (n=4) and MNC from MPN patients (n=9) in the presence of graded concentrations of EPO (0, 0.05, 0.5 and 5 IU/mL) and TNF (1, 10, 100 ng/mL). Intermediate and high TNF (10, 100 ng/mL) caused a dose-dependent reduction of BFU-E and CFU-GM compared to controls. However, in all conditions colony survival in MPN samples was higher compared to normal controls. Low TNF (1 ng/mL) in cultures supported by EPO (0.5 or 5.0 IU/mL) increased BFU-E formation by MPN cells to 142% of controls, but reduced BFU-E formation by normal MNC to 65%.

Our data indicate that JAK2 V617F upregulates TNF expression in cell lines and primary MPN cells. Compared to normal progenitor cells, MPN progenitor cells are less sensitive to or even stimulated by TNF. These data suggest that JAK2 V617F-induced TNF may contribute to MPN pathogenesis by conferring a growth advantage to MPN over normal cells.

Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Burns:Cytopia: Employment. Deininger:Genzyme: Research Funding; BMS: Consultancy; Novartis: Consultancy, Honoraria; Ariad : Research Funding.