Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Apr 8;115(14):2919-27.
doi: 10.1182/blood-2009-04-218842. Epub 2010 Feb 12.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

Priya Koppikar  1 , Omar Abdel-Wahab, Cyrus Hedvat, Sachie Marubayashi, Jay Patel, Aviva Goel, Nicole Kucine, Jeffrey R Gardner, Andrew P Combs, Kris Vaddi, Patrick J Haley, Timothy C Burn, Mark Rupar, Jacqueline F Bromberg, Mark L Heaney, Elisa de Stanchina, Jordan S Fridman, Ross L Levine
Affiliations

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

Priya Koppikar et al. Blood. .

Abstract

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Viability curves, immunoprecipitation analysis, and Western blots revealing viability and down-modulation of signaling intermediates downstream of JAK2 with various concentrations of INCB16562 in Ba/F3 isogenic cell lines bearing mutant MPN alleles. (A) Cells bearing mutations which result in constitutive activation of the JAK-STAT signaling pathway (JAK2V617F, JAK2K539L, and MPLW515L) have a lower IC50 compared with Ba/F3 cells bearing BCR-Abl. \E Error bars denote SEM. (B) Immunoprecipitation analysis demonstrated decrease in JAK2 phosphorylation in Ba/F3 isogenic cell lines with INCB16562 treatment. A total of 20 million cells were incubated with either DMSO or INCB16562 for 4 hours, followed by immunoprecipitation as described previously. (C) Western blots revealing a dose-dependent down-modulation of signaling intermediates in the JAK-STAT pathway after treatment with INCB16562.
Figure 2
Figure 2
Phenotypic characteristics of hMPLW515L mice treated with vehicle versus INCB16562 at varying doses over time. (A)Treatment with INCB16562 resulted in significant increase in survival at 60 mg/kg and 120 mg/kg doses compared with vehicle as shown by Kaplan-Meier survival curve (P < .001, log-rank test). (B) Mice treated with INCB16562 regained weight lost after tail vein injections compared with vehicle-treated mice, which continued to lose weight. This difference in weights were statistically significant starting at 17 days of treatment with 120 mg/kg per day (P = .007, Mann-Whitney U test) and after 18 days of treatment with 60 mg/kg per day (P = .004, Mann-Whitney U test). (C) Measurement of hematologic parameters at various time points in INCB16562 treatment reveal significant improvement in WBC and platelet counts with 60 mg/kg and 120 mg/kg of INCB16562 at day 14 after treatment initiation. (D) Treatment with INCB16562 resulted in decreased hepatosplenomegaly as measured in organ weight at 12 days of treatment. *P < .05 compared with vehicle-treated mice. (B-D) Error bars denote SD.
Figure 3
Figure 3
H+E staining of bone marrow, spleen, and liver, and reticulin of hMPLWT mice and hMPLW515L mice treated with vehicle or INCB16562 for 12 days. Hematoxylin and eosin (H+E) stains demonstrate reduced cellularity in the (A) bone marrow and (B) reduced myeloproliferation in the spleen of INCB16562-treated W515L mice. Enumeration of megakaryocytes per ×20 field in 5 fields from the spleens of 2 to 3 mice killed on day 12 after beginning treatment reveals significant dose-dependent decrease in number of megakaryocytes with 60 mg/kg and 120 mg/kg INCB16562 (F). (C) Liver histology reveals greatly reduced extramedullary hematopoeisis in the liver with INCB16562 treatment at either 60 mg/kg or 120 mg/kg W515L mice. Reticulin staining of marrow (D) and spleen (E) reveals a decrease in myelofibrosis with 12 days of INCB16562 treatment. *P < .05 compared with vehicle-treated mice. hMPLWT mice on the other hand, show normal bone marrow (A left panel) and splenic (B left panel) architecture with minimal fibrosis (D-E left panels), and a normal hepatic structure (D left panel). (F) Error bars denote SD.
Figure 4
Figure 4
Effect of INCB16562 treatment on serum cytokine levels in hMPLW515L mice. Displayed are log2 fold changes in serum cytokine levels in vehicle-treated hMPLW515L mice after 9 days of treatment compared with levels in normal BALB/c mice as well as serum cytokine levels in hMPLW515L in mice treated with 9 days of 60 mg/kg or 120 mg/kg INCB16562 relative to vehicle-treated mice. Treatment with INCB16562 led to a greater than 2 log2–fold decrease in inflammatory cytokines such as IL-1β, IL-9, IL-6, and TNF-α.
Figure 5
Figure 5
Inhibition of JAK-STAT signaling with INCB16562 treatment of primary cells from hMPLW515L mice. (A) Western blotting of JAK-STAT signaling intermediates in hMPLW515L splenocytes after 12 days of INCB16562 treatment reveals abrogation of phosphorylation of STAT3, STAT5, and MAPK with INCB16562 treatment compared with vehicle. (B) Phosphoprotein-specific flow cytometry of CD11b+ and CD61+ myeloid cells in hMPLW1515L bone marrow cells treated ex vivo with INCB16562 at 1μM or with vehicle for 2 hours. Treatment with INCB16562 greatly decreased phosphorylation of STAT3 and STAT5 in response to 10-minute ex vivo stimulation with rhGCSF (375 ng/mL) and rhTPO (1250 ng/mL). (C) Immunohistochemical staining (×40) of pSTAT3Y705 in mouse spleen after 12 days of treatment with vehicle or INCB16552 at 60 mg/kg or 120 mg/kg reveals a decrease in pSTAT3 in treated versus control mice tissue.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med. 2006;355(23):2452–2466. - PubMed
    1. Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood. 2007;110(4):1092–1097. - PubMed
    1. Ma X, Vanasse G, Cartmel B, Wang Y, Selinger HA. Prevalence of polycythemia vera and essential thrombocythemia. Am J Hematol. 2008;83(5):359–362. - PubMed
    1. Tefferi A. Essential thrombocythemia, polycythemia vera, and myelofibrosis: current management and the prospect of targeted therapy. Am J Hematol. 2008;83(6):491–497. - PubMed
    1. Deeg HJ, Gooley TA, Flowers ME, et al. Allogeneic hematopoietic stem cell transplantation for myelofibrosis. Blood. 2003;102(12):3912–3918. - PubMed

Publication types

MeSH terms