Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Abstract

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). In a mouse model of JAK2V617F(+) MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Figure 1

INCB018424 potently and selectively inhibits JAK2V617F-mediated signaling and proliferation. Ba/F3-EpoR-JAK2V617F (A) or HEL (B) cells were treated with increasing concentrations of INCB018424 for 2.5 hours, and extracts from these cells were subjected to immunoblot analysis for phosphorylated (p) or total forms of proteins associated with the JAK/STAT signaling pathway. JAK1 phosphorylation was assessed in primary MF patient samples (C) by immunobloting after immunoprecipitation of total JAK1. The numbers below describe the mutant JAK2V617F allele burden in patient samples. The effect of INCB018424 on viable cell number (D) was assessed after a 48-hour treatment period in cells expressing JAK2V617F (Ba/F3-EpoR-JAK2V617F and HEL) or, to confirm the selective nature of INCB018424, BCR-ABL (TF-1-BCR-ABL and Ba/F3-BCR-ABL).

Figure 2

Cells expressing JAK2V617F undergo apoptosis when treated with a selective JAK inhibitor. Ba/F3-EpoR-JAK2V617F cells were treated with various concentrations of INCB018424 for approximately 24 hours and analyzed for hallmarks of apoptosis by annexin V/propidium iodide staining (A) or mitochondrial membrane depolarization (B). Cells treated with INCB018424 were stained with a combination of fluorescein isothiocyanate-annexin V and propidium iodide and analyzed using flow cytometry to determine the percentages of viable (bottom left), early apoptotic (bottom right), and late apoptotic or dead (top right) cells. The effects of JAK inhibition on mitochondrial membrane potential were determined by flow cytometry using JC-1 as a molecular probe (B). Total and relative percentages of cells with depolarized mitochondria are shown.

Figure 3

INCB018424 treatment improves viability and splenomegaly in a JAK2V617F-driven model of malignant disease. (A) Kaplan-Meier analysis of mice inoculated intravenously with Ba/F3-EpoR-JAK2V617F cells and treated with INCB018424 or vehicle (orally, twice a day). (B) Survival was significantly improved with INCB018424 treatment (n = 14 mice/group; P < .001, log-rank test). Effects of JAK inhibition on spleen weights were assessed in a separate cohort of animals treated for 2 weeks with vehicle or INCB018424 (n = 6 /group; P < .001; 2-sided t test). Group means are represented by horizontal bars. (C) Quantitative genomic PCR analysis demonstrated a preferential diminution of cells expressing JAK2V617F after treatment with INCB018424 for 2 weeks (P < .01, 2-sided t test). (D) Phospho-STAT3 assay was performed on spleen lysates collected 4 hours after dosing using the Milliplex MAP system (Millipore), normalized to total protein, and read on a Luminex 200 instrument (Luminex).

Figure 4

Macroscopic and microscopic effects of INCB018424 on spleens from mice inoculated with Ba/F3-EpoR-JAK2V617F cells. Massive splenomegaly is observed in vehicle-treated mice as assessed by gross appearance (A top) or hematoxylin and eosin-stained transverse sections collected at the midline (A bottom), although markedly reduced with INCB018424 treatment. (B) Microscopic examination (original magnification ×20) of tissue sections shows the massive accumulation of malignant cells in the vehicle group with an abundance of mitotic figures and multinucleated cells present. Treatment with INCB018424 dramatically reduces the malignant cell burden and restores normal lymphoid architecture.

Figure 5

INCB018424 does not affect normal hematologic parameters. Mice (n = 5/group) were treated with INCB018424 or vehicle for 4 weeks at a total daily dose of 180 mg/kg (A). Data are reported for total white blood cell counts (WBC, 10³/μL), absolute neutrophil counts (ANEU, 10³/μL), absolute lymphocyte counts (ALYM, 10³/μL), red blood cells (RBC, 10⁶/μL), reticulocytes (RET, 10³/μL), and platelets (PLT, 10³/μL). No significant differences were found between treatment groups. For comparison, a subset of parameters was measured in mice treated with cyclophosphamide for 2 weeks (CTX, B). All parameters were significantly decreased by CTX (P < .01). Statistical significance was determined using Dunnett test.