Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy

Abstract

The identification of somatic activating mutations in JAK2 (refs 1–4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

Figure 1. Generation of JAK2 inhibitor-persistent cells

a) Proliferation of naïve and persistent SET-2 (i) and WL (ii) cells with JAK2 inhibitors. Data are from wells plated in triplicate (S.D.), and are representative of 3 independent experiments. b) IC₅₀ values of SET-2 IN^Per and WL IN^Per cells exposed to INCB18424, TG101348, and JAK Inhibitor I.

Figure 2. Inhibitor-persistent cells and INCB18424 treated patient granulocytes show continual JAK-STAT signaling and JAK2 activation via transphosphorylation by JAK1/TYK2

a) SET-2 and SET-2 IN^Per cells were washed and incubated with increasing concentrations of INCB18424 for 4 hours and western blotted. b) Granulocytes from naïve and INCB18424-treated patients were incubated ex vivo with DMSO or 150 nM of INCB18424 for 6 hours and western blotted. c) Increased phosphorylation of JAK1 in persistent cells and constitutive TYK2 phosphorylation in both naïve and persistent cells. d) Increased association between phosphoJAK2 and both TYK2/JAK1 in SET-2 JAK^Per cells and increased association between JAK2 and both TYK2/JAK1 in WL JAK^Per cells. e) JAK1/TYK2 association with phosphoJAK2 in granulocytes from 3 INCB18424 treated patients, which is not observed in INCB18424 naïve MPN samples.

Figure 3. JAK2-Inhibitor persistence is reversible and JAK2 levels correlate with persistence/resensitization

a) Percent viability of SET-2 IN^Per and SET-2 IN^Resens cells at 0.25μM JAK Inhibitor I, 0.25μM INCB18424 and 2μM TG101348, data are from wells plated in triplicate (S.D.), and are representative of 3 independent experiments. b) Loss of JAK1/TYK2 association with phosphoJAK2 in SET-2 and WL IN^Resens cells. c) Reversible changes in JAK2 levels in IN^Per cells compared to naïve and IN^Resens SET-2 and WL cells. d) ChIP-PCR of JAK2 locus shows increased H3K4me3 and decreased H3K9me3 marks in SET-2 IN^Per cells. e) PhosphoJAK2 and total JAK2 levels are degraded upon cycloheximide treatment (500 μg/ml for 2, 4 and 6 hours) in naïve and resensitized WL cells, but not in IN^Per cells. f) Higher JAK2 levels in INCB18424 treated MPN granulocytes by qRT-PCR compared to those in a small cohort of best responders.

Figure 4. Transphosphorylation of JAK2 by JAK1/TYK2 contributes to persistence and persistent cells can be targeted using Type II JAK2 inhibitors or HSP90 inhibition

a) SET-2 cells were transfected with scrambled pLKO or JAK2 shRNA. Viability after 10 days of puromycin selection relative to cell numbers on day 1 is shown, results are from 3 biologic replicates, S.E.M. is shown. b) JAK2 knockdown inhibits signaling in puromycin selected sensitive and persistent SET-2 cells. c) IN^Per SET-2 cells were partially resensitized to INCB18424 following loss of JAK1 or JAK1+TYK2 using siRNA. Data are from wells plated in triplicate (S.D.), and are representative of 3 independent experiments. d) Naïve and persistent SET-2 cells are inhibited by PU-H71, data are from wells plated in triplicate (S.D.), and are representative of 3 independent experiments. e) PU-H71 degrades JAK2, inhibits signaling in SET-2 cells. Cells were treated with DMSO or 2 μM PU-H71 (SET-2) and 1 μM PU-H71 (WL) for 16 hours. f) 4 hour treatment with BBT-594 inhibits signaling in naïve and persistent SET-2 cells.