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

Article metrics

Abstract

The identification of somatic activating mutations in JAK2 (refs 1–4) and in the thrombopoietin receptor gene (MPL)5 in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors6,7. 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Generation of JAK2-inhibitor-persistent cells.
Figure 2: Inhibitor-persistent cells and granulocytes from INCB18424-treated patients show continual JAK–STAT signalling and JAK2 activation through transphosphorylation by JAK1 and TYK2.
Figure 3: JAK2 inhibitor persistence is reversible and JAK2 levels correlate with persistence and resensitization.
Figure 4: Transphosphorylation of JAK2 by JAK1/TYK2 contributes to persistence, and persistent cells can be targeted with type II JAK2 inhibitors or Hsp90 inhibition.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data are deposited in the Gene Expression Omnibus under accession number GSE38335.

References

  1. 1

    James, C. et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434, 1144–1148 (2005)

  2. 2

    Kralovics, R. et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N. Engl. J. Med. 352, 1779–1790 (2005)

  3. 3

    Baxter, E. J. et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365, 1054–1061 (2005)

  4. 4

    Zhao, R. et al. Identification of an acquired JAK2 mutation in polycythemia vera. J. Biol. Chem. 280, 22788–22792 (2005)

  5. 5

    Pikman, Y. et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 3, e270 (2006)

  6. 6

    Verstovsek, S. et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N. Engl. J. Med. 363, 1117–1127 (2010)

  7. 7

    Pardanani, A. et al. Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J. Clin. Oncol. 29, 789–796 (2011)

  8. 8

    Druker, B. J. et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med. 344, 1031–1037 (2001)

  9. 9

    Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010)

  10. 10

    Rosell, R. et al. Screening for epidermal growth factor receptor mutations in lung cancer. N. Engl. J. Med. 361, 958–967 (2009)

  11. 11

    Mok, T. S. et al. Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma. N. Engl. J. Med. 361, 947–957 (2009)

  12. 12

    Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 352, 786–792 (2005)

  13. 13

    Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005)

  14. 14

    Gorre, M. E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001)

  15. 15

    Engelman, J. A. et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007)

  16. 16

    Pao, W. et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med. 2, e17 (2005)

  17. 17

    Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010)

  18. 18

    Nazarian, R. et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973–977 (2010)

  19. 19

    Azam, M., Latek, R. R. & Daley, G. Q. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003)

  20. 20

    Shah, N. P. et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2, 117–125 (2002)

  21. 21

    Sharma, S. V. et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141, 69–80 (2010)

  22. 22

    Parganas, E. et al. Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93, 385–395 (1998)

  23. 23

    Ihle, J. N. & Gilliland, D. G. Jak2: normal function and role in hematopoietic disorders. Curr. Opin. Genet. Dev. 17, 8–14 (2007)

  24. 24

    Mullighan, C. G. et al. JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc. Natl Acad. Sci. USA 106, 9414–9418 (2009)

  25. 25

    Marubayashi, S. et al. HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans. J. Clin. Invest. 120, 3578–3593 (2010)

  26. 26

    Koppikar, P. et al. Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis. Blood 115, 2919–2927 (2010)

  27. 27

    Rider, L., Shatrova, A., Feener, E. P., Webb, L. & Diakonova, M. JAK2 tyrosine kinase phosphorylates PAK1 and regulates PAK1 activity and functions. J. Biol. Chem. 282, 30985–30996 (2007)

  28. 28

    Andraos, R. et al. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent. Cancer Discov. 2, 512–523 (2012)

  29. 29

    Wang, Y. et al. Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells. Blood 114, 5024–5033 (2009)

  30. 30

    Guerini, V. et al. The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2V617F. Leukemia 22, 740–747 (2007)

  31. 31

    He, H. et al. Identification of potent water-soluble purine-scaffold inhibitors of the heat shock protein 90. J. Med. Chem. 49, 381–390 (2006)

  32. 32

    Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007)

Download references

Acknowledgements

We thank C. Sawyers, J. Licht, P. Poulikakos and N. Rosen for advice and suggestions; P. Bhatt for his help in the saturation mutagenesis screen; T. Taldone for synthesis of PU-H71; L. Staudt and T. Look for shRNA constructs against JAK2 and TYK2, respectively; and T. Radimerski and P. Manley for providing BBT-594. We are grateful to the Genomics Core Laboratories at Memorial Sloan-Kettering Cancer Center and the Geoffrey Beene Core for their assistance with 454 sequencing. This work was supported in part by National Cancer Institute grant 1R01CA151949-01 to R.L.L., by a grant from the Leukemia and Lymphoma Society to R.L.L. and by a grant from the Myeloproliferative Neoplasms Foundation and the Starr Cancer Consortium to R.L.L., B.E.B. and B.L.E. B.E.B. is a Howard Hughes Medical Institute Early Career Scientist.

Author information

P.K. and R.L.L. conceived the project. P.K., N.B., O.K. and R.L.L. designed experiments. P.K., N.B., O.K., T.M., M.A., F.L., O.A.W., L.L., A.W., S.M. and A.G. performed experiments. P.K., N.B., T.H., M.G. and M.A. analysed data. L.M.S., A.M., B.L.E. and G.C. provided reagents. Z.E. and S.V. provided patient samples. P.K., N.B. and R.L.L. wrote the paper with input from S.V., Z.E., O.K., B.L.E., B.E.B. and S.D.N.

Correspondence to Ross L. Levine.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-17 and Supplementary Tables 1-3. (PDF 2799 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.