Original Article | Published:

Chronic Myeloproliferative Neoplasias

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia

Leukemia volume 29, pages 586597 (2015) | Download Citation

  • An Erratum to this article was published on 03 May 2017

Abstract

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , , , et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355: 2408–2417.

  2. 2.

    , , , , , et al. Imatinib for newly diagnosed patients with chronic myeloid leukemia: incidence of sustained responses in an intention-to-treat analysis. J Clin Oncol 2008; 26: 3358–3363.

  3. 3.

    , , , , , et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001; 293: 876–880.

  4. 4.

    , , , , , 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 2002; 2: 117–125.

  5. 5.

    , , , , , et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell 2009; 16: 401–412.

  6. 6.

    , , , . Pushing the limits of targeted therapy in chronic myeloid leukaemia. Nat Rev Cancer 2012; 12: 513–526.

  7. 7.

    , , , , , . Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest 2011; 121: 396–409.

  8. 8.

    , , , , , et al. Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival. Blood 2012; 119: 1501–1510.

  9. 9.

    , , , , , . Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther 2008; 7: 3169–3175.

  10. 10.

    , , , , , et al. Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors. Leukemia 2012; 26: 1140–1143.

  11. 11.

    , . A high-throughput fluorescence polarization assay for signal transducer and activator of transcription 3. Anal Biochem 2004; 330: 114–118.

  12. 12.

    , , , . New insights about HERG blockade obtained from protein modeling, potential energy mapping, and docking studies. Bioorg Med Chem 2006; 14: 3160–3173.

  13. 13.

    , , , , , et al. Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry. Anal Chem 2012; 84: 3771–3779.

  14. 14.

    , , , , , et al. BMS-214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibitors. Blood 2008; 111: 2843–2853.

  15. 15.

    , , , , . Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor. Blood 1996; 88: 3765–3773.

  16. 16.

    , , . Cytogenetic and molecular monitoring of residual disease in chronic myeloid leukaemia. Acta Haematol 2002; 107: 64–75.

  17. 17.

    , . Overexpression of a dominant negative form of STAT3 selectively impairs hematopoietic stem cell activity. Oncogene 2002; 21: 4778–4787.

  18. 18.

    , , , , , et al. Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth. Neoplasia 2005; 7: 545–555.

  19. 19.

    , , , , , et al. Oncogenic stress induced by acute hyper-activation of Bcr-Abl leads to cell death upon induction of excessive aerobic glycolysis. PLoS One 2011; 6: e25139.

  20. 20.

    , , , , , et al. Disruption of transcriptionally active Stat3 dimers with non-phosphorylated, salicylic acid-based small molecules: potent in vitro and tumor cell activities. Chembiochem 2009; 10: 1959–1964.

  21. 21.

    , , , , , . A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes. Biochem Pharmacol 2010; 79: 1398–1409.

  22. 22.

    , , , , , et al. Antagonism of the Stat3-Stat3 protein dimer with salicylic acid based small molecules. ChemMedChem 2011; 6: 1459–1470.

  23. 23.

    , , , , , et al. BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia. Nat Chem Biol 2012; 8: 285–293.

  24. 24.

    , , , , , et al. Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia. Leukemia 2014; e-pub ahead of print 12 May 2014; doi:10.1038/leu.2014.156.

  25. 25.

    , , , , , et al. Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain. Bioorg Med Chem Lett 2011; 21: 5605–5609.

  26. 26.

    , , , , , et al. Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proc Natl Acad Sci USA 2012; 109: 9623–9628.

  27. 27.

    , , , , , et al. Inhibiting aberrant signal transducer and activator of transcription protein activation with tetrapodal, small molecule Src homology 2 domain binders: promising agents against multiple myeloma. J Med Chem 2013; 56: 7190–7200.

  28. 28.

    , . Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry. FEBS J 2013; 280: 5616–5625.

  29. 29.

    , , , , , . Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia. Blood 1996; 88: 2162–2171.

  30. 30.

    , , , , , et al. BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571. Blood 2003; 101: 690–698.

  31. 31.

    , , , , , et al. Association between imatinib-resistant BCR-ABL mutation-negative leukemia and persistent activation of LYN kinase. J Natl Cancer Inst 2008; 100: 926–939.

  32. 32.

    , . Expression of a Src family kinase in chronic myelogenous leukemia cells induces resistance to imatinib in a kinase-dependent manner. J Biol Chemistry 2010; 285: 21446–21457.

  33. 33.

    , , , , , et al. Longitudinal studies of SRC family kinases in imatinib- and dasatinib-resistant chronic myelogenous leukemia patients. Leuk Res 2011; 35: 38–43.

  34. 34.

    , , , , , et al. Differential contributions of STAT5A and STAT5B to stress protection and tyrosine kinase inhibitor resistance of chronic myeloid leukemia stem/progenitor cells. Cancer Res 2013; 73: 2052–2058.

  35. 35.

    , , , , . BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance. J Hematol Oncol 2011; 4: 6.

  36. 36.

    , , , , , et al. PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells. J Clin Invest 2013; 123: 4144–4157.

  37. 37.

    , , , , , et al. Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling. Blood 2013; 121: 1824–1838.

  38. 38.

    , , , , , et al. A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib. Blood 2010; 115: 315–325.

  39. 39.

    , , . Role of STAT3 in transformation and drug resistance in CML. Front Oncol 2012; 2: 30.

  40. 40.

    , , , , , et al. High STAT5 levels mediate imatinib resistance and indicate disease progression in chronic myeloid leukemia. Blood 2011; 117: 3409–3420.

  41. 41.

    , , , , , et al. Stat3 as an oncogene. Cell 1999; 98: 295–303.

  42. 42.

    , , , , , et al. Tumor-derived mutations in the gene associated with retinoid interferon-induced mortality (GRIM-19) disrupt its anti-signal transducer and activator of transcription 3 (STAT3) activity and promote oncogenesis. J Biol Chem 2013; 288: 7930–7941.

  43. 43.

    , , , , , . Loss of expression and function of SOCS3 is an early event in HNSCC: altered subcellular localization as a possible mechanism involved in proliferation, migration and invasion. PLoS One 2012; 7: e45197.

  44. 44.

    , , , , , et al. HER2 overexpression elicits a proinflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis. Cancer Res 2011; 71: 4380–4391.

  45. 45.

    , , , , , et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012; 366: 1905–1913.

  46. 46.

    , , , , , et al. STAT3 mutations identified in human hematological neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model. Haematologica 2013; 98: 1748–1752.

  47. 47.

    , , , . Activation and association of Stat3 with Src in v-Src-transformed cell lines. Mol Cell Biol 1996; 16: 1595–1603.

  48. 48.

    , , , , , . Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improved treatment of Ph+ leukemia in mice. Proc Natl Acad Sci USA 2006; 103: 16870–16875.

  49. 49.

    , , , , , et al. Jak2 inhibition deactivates Lyn kinase through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukemia patients. Oncogene 2009; 28: 1669–1681.

  50. 50.

    . Transcription factors as targets for cancer therapy. Nat Rev Cancer 2002; 2: 740–749.

  51. 51.

    , . Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002; 3: 651–662.

  52. 52.

    , , , . A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl Acad Sci USA 2005; 102: 4700–4705.

  53. 53.

    , , , , . Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol 2006; 13: 1235–1242.

  54. 54.

    , , , . Stat3 inhibitor Stattic exhibits potent antitumor activity and induces chemo- and radio-sensitivity in nasopharyngeal carcinoma. PLoS One 2013; 8: e54565.

  55. 55.

    , , , , , et al. LLL-3 inhibits STAT3 activity, suppresses glioblastoma cell growth and prolongs survival in a mouse glioblastoma model. Br J Cancer 2009; 100: 106–112.

  56. 56.

    , , , , , et al. A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation. Cancer Res 2013; 73: 1922–1933.

  57. 57.

    , , , , , et al. Selective small molecule Stat3 inhibitor reduces breast cancer tumor-initiating cells and improves recurrence free survival in a human-xenograft model. PLoS One 2012; 7: e30207.

Download references

Acknowledgements

We acknowledge Johanna Estrada, Kevin Gantz, Hannah Redwine, Hillary Finch and Anthony Iovino for technical assistance, and Kimberly Snow and Candice Ott for clerical assistance. We thank Dr Rob C Laister and the Minden group for providing full-length purified STAT3 protein and a STAT3 expression construct. We also thank Dr Il-Hoan Oh, Catholic University of Korea, for providing a dominant-negative STAT3 construct. We acknowledge support of funds in conjunction with grant P30 CA042014 awarded to the Huntsman Cancer Institute, and 5P30CA042014-24 awarded to The University of Utah Flow Cytometry Facility. MWD was supported by grants from the National Institutes of Health (NIH), including HL082978-01, CA046939-23 and R01CA178397, was a Scholar in Clinical Research of the Leukemia & Lymphoma Society (LLS; 7036-01), and is funded by LLS grant SCOR7005-11. AME was supported by a NIH T32 training grant (CA093247), followed by an LLS Career Development Award (5090-12), and is currently funded through a Scholar Award from the American Society of Hematology. AME also acknowledges support from the NIH Loan Repayment Program. This research was supported in part by the LLS Screen-to-Lead Program awarded to MWD, TO and PTG (SLP-8002-14). TO is supported by NIH grant R01CA178397. RB acknowledges a petascale computing Research Award at the Extreme Science and Engineering Discovery Environment (XSEDE) supercomputers (TG-CHE120086). XSEDE is supported by National Science Foundation grant OCI-1053575. RB acknowledges startup funds from the Department of Medicinal Chemistry, and technical support and computing allocations at the Center for High Performance Computing, The University of Utah. RM was supported by grant SFBF47 from the Austrian Science Fund (FWF). PTG and BDGP are supported by the National Sciences and Engineering Research Council. PTG is also supported by the Canadian Breast Cancer Research Foundation. DJW is supported by a Discovery Grant (257588) and by an Ontario Ministry of Research and Innovation Early Researcher Award.

Author information

Author notes

    • A M Eiring
    • , B D G Page
    • , I L Kraft
    • , T O'Hare
    • , P T Gunning
    •  & M W Deininger

    These authors contributed equally to this work.

Affiliations

  1. Huntsman Cancer Institute, The University of Utah, Salt Lake City, UT, USA

    • A M Eiring
    • , I L Kraft
    • , C C Mason
    • , M S Zabriskie
    • , T Y Zhang
    • , J S Khorashad
    • , A J Engar
    • , K R Reynolds
    • , D J Anderson
    • , A Senina
    • , A D Pomicter
    • , W L Heaton
    • , S K Tantravahi
    • , T O'Hare
    •  & M W Deininger
  2. Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada

    • B D G Page
    • , C C Arpin
    • , A Todic
    • , R Colaguori
    •  & P T Gunning
  3. Department of Medicinal Chemistry, College of Pharmacy, The University of Utah, Salt Lake City, UT, USA

    • N A Vellore
    • , S Ahmad
    •  & R Baron
  4. York University Chemistry Department, Toronto, Ontario, Canada

    • D Resetca
    •  & D J Wilson
  5. Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria

    • R Moriggl
  6. Department of Chemistry, Center for Research in Mass Spectrometry, York University, Toronto, Ontario, Canada

    • D J Wilson
  7. Division of Hematology and Hematologic Malignancies, The University of Utah, Salt Lake City, UT, USA

    • T O'Hare
    •  & M W Deininger

Authors

  1. Search for A M Eiring in:

  2. Search for B D G Page in:

  3. Search for I L Kraft in:

  4. Search for C C Mason in:

  5. Search for N A Vellore in:

  6. Search for D Resetca in:

  7. Search for M S Zabriskie in:

  8. Search for T Y Zhang in:

  9. Search for J S Khorashad in:

  10. Search for A J Engar in:

  11. Search for K R Reynolds in:

  12. Search for D J Anderson in:

  13. Search for A Senina in:

  14. Search for A D Pomicter in:

  15. Search for C C Arpin in:

  16. Search for S Ahmad in:

  17. Search for W L Heaton in:

  18. Search for S K Tantravahi in:

  19. Search for A Todic in:

  20. Search for R Colaguori in:

  21. Search for R Moriggl in:

  22. Search for D J Wilson in:

  23. Search for R Baron in:

  24. Search for T O'Hare in:

  25. Search for P T Gunning in:

  26. Search for M W Deininger in:

Competing interests

MWD is a consultant for BMS, Novartis, ARIAD, Pfizer and Incyte. His laboratory receives research funding from BMS and Novartis.

Corresponding author

Correspondence to M W Deininger.

Supplementary information

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/leu.2014.245

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Further reading