Intrinsic Resistance to JAK2 Inhibition in Myelofibrosis

Abstract 2825

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders with a variable clinical course: essential thrombocytosis (ET) and polycythemia vera (PV) are associated with excess clotting and bleeding but an overall normal or near-normal life expectancy, while myelofibrosis has a variable but generally poor prognosis. The disorders are unified by the constitutive activation of the JAK2 pathway, most commonly by a point mutation in the pseudokinase domain of JAK2 (JAK2 V617F). Small molecule tyrosine kinase inhibitors have been shown to be active in both preclinical and late-phase clinical testing, and yet notably pharmacodynamic validation of target (JAK2) inhibition has never been rigorously demonstrated in clinical studies. Clinical investigation of JAK2 inhibitors has focused on myelofibrosis, with some unexpected results: while reduction in spleen size and amelioration of constitutional symptoms are frequent, only modest changes in mutant clonal burden have been observed and complete responses by any criteria are exceedingly rare. In order to develop a feasible and informative pharmacodynamic assay for measuring JAK2 signaling, we used phospho-specific flow cytometry on whole peripheral blood to measure phosphorylation of STAT3 and STAT5, the canonical targets of JAK2 in hematopoietic cells. Treating whole blood with tyrosine kinase inhibitors currently in clinical development, we observed a dose-dependent decrease in phospho-STAT5 and STAT3 in granulocytes treated ex vivo in cells from normal donors, patients with ET, PV and to a lesser extent from patients with myelofibrosis. We found that phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis were significantly less inhibited than in PV and ET as well as normal donors. Using quantitative RT-PCR, we found that sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. To determine whether extracellular determinants conferred resistance, mixing studies showed that plasma from patients with myelofibrosis did not transfer resistance. Plasma cytokines that signal through JAK2 and/or are known to be altered in myelofibrosis were measured and no single cytokine appeared to account for the observed pattern of resistance. Taken together these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.