Exaggerated Response To Toll-Like Receptor Agonist Contributes To Excessive TNF Production In Myeloproliferative Neoplasm

Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2^V617F neoplastic clone. Because JAK2^V617F cells are TNF resistant, a high TNF environment, as is the case in MPN patients, gives JAK2^V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts, resulting in expansion of the neoplastic clone. Targeting excessive TNF production therapeutically in MPN would reduce the competitive advantage of the JAK2^V617F neoplastic clone and lead to its contraction. To efficiently target TNF production therapeutically in MPN it is necessary to first identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Upon TLR stimulation a signaling transduction cascade ensues, culminating in the production of inflammatory cytokines including TNF. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls.

We compared the response to the TLR 7/8 agonist R848 in peripheral blood monocytes from MPN versus normal controls. After stimulation with R848 for 24 hours, CD14⁺ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (0.5, 1, 3, 5µM, p<0.05). The percentage of TNF⁺ CD14⁺ monocytes (detected by intracellular flow cytometery) after stimulation with R848 was not different in MPN (n=16) versus normal controls (n=8). This demonstrates that the excessive TNF production in MPN is not due to an increased fraction of monocytes producing TNF but by exaggerated TNF production in MPN on a per cell basis. Without stimulation, however, MPN patients have an increased percentage of TNF⁺ CD14⁺ monocytes as compared to normal controls (4.6% vs 1.6% respectively, p<0.05), suggestive of a hyper-inflammatory state at baseline. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with R848. At early time points (15min) following stimulation with R848 (5µM), MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05). These data suggest that the excessive production of TNF in MPN patients may be due either to persistent activation of signaling following TLR stimulation or failure to repress the activation state of these two proteins. Targeting the TLR pathway therapeutically in MPN may serve to reduce TNF production and neutralize the selective advantage of the JAK2^V617F neoplastic clone, ultimately leading to reduction in the JAK2^V617F allele burden.