TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

Abstract

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

Figure 1

TNFα mRNA correlates with JAK2 kinase activity. (A) TNFα plasma concentration was evaluated in 48 MPN patients (16 PV, 7 ET, 25 MF) and 10 normal controls. Each data point represents a patient. Median TNFα concentration is shown as a bar. Detailed patient information including JAK2^V617F status is provided in supplemental Table 1 (available on the Blood Web site, see the Supplemental Materials link at the online article). (B) qRT-PCR analysis of median TNFα mRNA expression of Peripheral blood leukocytes from 43 PV patients and 21 normal controls. Median TNFα expression in cells from normal controls was normalized to 1. (C) Correlation between JAK2^V617F allele burden and TNF plasma concentration. Allele burden was determined in 20 JAK2^V617F-positive patients by real-time quanitative PCR and correlated with TNF plasma concentration. (D) TNFα mRNA expression was analyzed by qRT-PCR in human hematopoietic cell lines and normalized to GAPDH. The TNFα expression in HEL cells was arbitrarily set to 1 and results for the other lines are shown in comparison. (E-F) TNFα mRNA expression after pharmacologic inhibition of JAK2, (E) HEL (left) and HL60 (right) cells treated with JAK2 inhibitor CYT387, (F) primary human blood MNCs treated with CYT387.

Figure 2

MPN mononuclear cells and CD34⁺ cells are resistant to suppression of colony formation by TNFα. (A) Effect of TNFα on colony formation by progenitor cells from MNC. MNC from MPN patients (n = 14) and normal controls (n = 5) were plated in methylcellulose media supplemented with SCF, IL-3, with 5 U/mL (left) or 0.5 U/mL (right) EPO and graded concentrations of TNFα (in triplicate). Colonies were enumerated at day 12. Colony formation in 0 ng/mL TNFα is normalized to 100%. (B) Effect of TNFα on colony formation from CD34⁺ cells. CD34⁺ cells from MPN (n = 16 for 10 ng/mL TNFα, n = 3 for 1 ng/mL TNFα) and normal controls (n = 6 for 10 ng/mL TNFα, n = 3 for 1 ng/mL TNFα) were plated in methylcellulose media supplemented with SCF, IL-3 and 5 U/mL EPO, with graded concentrations of TNFα (in triplicate).

Figure 3

TNF selects for JAK2^V617F+ colony formation. Percentage of JAK2^V617F mutant colonies in the absence or presence of TNFα. Colonies were plucked from (A) 6 MNC starting material and (B) 6 CD34⁺ starting material from plates with 0 ng/mL and 10 ng/mL TNFα (at least 30 individual colonies per condition) and JAK2 mutational status was determined. Panels A and B represent different patients and are numbered arbitrarily. Mutational analysis of separated CFU-GM and BFU-E from these patients is provided in supplemental Figure 3.

Figure 4

Preneoplastic (JAK2^WT) CD34⁺ cells from JAK2^V617F-positive MPN patients are hypersensitive to TNFα. The genotyping data in Figure 3 was used to calculate the effect specifically on the JAK2^WT or JAK2^V617F CD34⁺ compartment of JAK2^V617F+ patients. CFU-GM and BFU-E colony numbers were combined for analysis.

Figure 5

Ectopic expression of JAK2^V617F attenuates TNFα-mediated suppression of myeloid colony formation. JAK2^V617F or empty MIG vector was retrovirally expressed in hematopoietic progenitors (lineage^neg c-kit⁺ Sca-1⁻) from the bone marrow of (A) wild-type and (B) Fancc^−/− C57B/6J mice. Transduced (GFP⁺) cells were plated in methycellulose supplemented with 50 ng/mL mSCF, 10 ng/mL mIL-3 and 3 U/mL hEPO, either with or without TNFα (20 ng/mL for wild-type 10 ng/mL for Fancc^−/−). Colony number was enumerated at day 12, with colony formation in conditions without TNFα set to 100%.

Figure 6

TNFα is not required for development of MPN but promotes expansion of JAK2^V617F cells in a murine transplantation model. Bone marrow from 5-FU treated TNFα^+/+ (C57B/6) or TNFα^−/− (B6.129S-Tnf^tm1Gkl/J) mice was infected with JAK2^V617F retrovirus by spinoculation and injected into syngeneic lethally irradiated hosts (n = 5 for each group). (A) Percentage of GFP⁺ cells in peripheral blood (B) spleen and liver weights at time of sacrifice, (C) percentages of GFP⁺ cells in bone marrow and spleen at time of sacrifice, and (D) H&E-stained histologic sections of representative TNFα^+/+ and TNFα^−/− bone marrow, spleen, and liver. Large panels represent 10× magnification, panel inserts represent 40× magnification of the same area. Images were captured with a Leica DC300 camera running IM50 Image Manager Version 5 software. (E) Model of TNFα-induced JAK2^V617F clonal evolution in MPN. In a TNFα-sensitive stem cell pool JAK2^V617F induced TNF resistance provides a strong selective advantage, allowing for the expansion of the mutant clone and development of clinical disease. Maintenance of a high TNFα environment by JAK2^V617F cells further enhances the selective advantage for the JAK2^V617F clone.