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Syk kinase and Shp2 phosphatase inhibition cooperate to reduce FLT3-ITD-induced STAT5 activation and proliferation of acute myeloid leukemia

Leukemia volume 30, pages 2094–2097 (2016)Cite this article

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Acute myeloid leukemia (AML) continues to be a difficult-to-treat disease with a high relapse rate, and individuals presenting with FLT3-ITD+ status exhibit a poor prognosis.1 We demonstrated previously that genetic disruption or pharmacologic inhibition of the protein tyrosine phosphatase, Shp2, reduced FLT3-ITD-induced STAT5 hyperactivation, hyperproliferation and leukemia-induced mortality in vivo.2 We also demonstrated that mutation of duplicated tyrosine (Y) 599, located in the juxtamembrane domain of FLT3 and known to interact with Shp2,3 to phenylalanine (F) reduced FLT3-ITD-induced STAT5 hyperactivation and hyperproliferation.2 As Shp2 is a phosphatase, we anticipated that Shp2 cooperates with a STAT5-activating kinase to promote cellular hyperproliferation. Given that Syk has been found to interact with Y599 on FLT3-ITD and phosphorylates inter-kinase domain Y768,4 that Y768 recruits Grb2, Gab2 and p85α,5 and that Shp2 is reported to participate in multi-protein complexes containing Gab2 and p85α,6, 7 we hypothesized that increased Shp2 recruitment to duplicated Y599 on FLT3-ITD cooperates with Syk to enhance constitutive STAT5 phosphorylation and to promote Y768-dependent Erk and Akt phosphorylation. Consequently, we predicted that mutation of duplicated Y599 or of Y768 to phenylalanine would lead to reduced proliferation in vitro and reduced FLT3-ITD-induced myeloproliferative disease (MPD) in vivo.

We hypothesized that reduction in hyperpoliferation in vitro would prolong latency to onset of MPD in vivo. To investigate disease development, transfected 32D cells were transplanted into syngeneic C3H/HeJ hosts, and animals demonstrating ⩾0.5% enhanced green fluorescent protein (EGFP)-positive cells in peripheral blood at three weeks post transplant were followed for overall survival. Although mice transplanted with N51-FLT3-Y768F cells failed to show reduced MPD or improved survival, mice transplanted with N51-FLT3-Y599F1/2 cells demonstrated prolonged overall survival compared to N51-FLT3 (Figure 1c). In spite of the differential effect of the Y599 and Y768 mutations on overall survival, we found similarly reduced Shp2 interaction with both N51-FLT3-Y599F1/2 and N51-FLT3-Y768F compared to N51-FLT3 in immunoprecipitation assays (Figure 1d, compare lanes 3 and 4 to lane 2). Taken together, these findings suggest duplicated Y599 and Y768 have differential effects on FLT3-ITD-induced MPD, most likely due to varied activation of downstream signaling effectors. In addition, although duplicated Y599 has a more prominent role, it is not the only factor contributing to the disease, as its mutation slows but does not ablate leukemia progression.

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Acknowledgements

This work was supported by the Riley Children’s Foundation and U.S. National Institutes of Health (F31 CA183342 to BMR; RO1 CA134777 to RJC, RK; RO1 HL077177, HL081111 and CA173852 to RK; RO1 CA96202 to Z-YZ; and the Indiana Clinical and Translational Sciences Institute, funded in part by UL1 TR000006 to JDB). We appreciate the technical assistance from Dr Karen Pollok and Tony Sinn in the Indiana University In Vivo Therapeutics Core and from Susan Rice in the Flow Cytometry Resource Facility (supported by P30 CA082709). We gratefully acknowledge the administrative assistance of Marilyn L Wales and Tracy Winkle.

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  1. Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA

    B M Richine, E L Virts, J D Bowling, B Ramdas, R Mali, R Naoye, R Kapur & R J Chan

  2. Department of Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA

    B M Richine, R Kapur & R J Chan

  3. Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA

    E L Virts, B Ramdas, R Mali, R Kapur & R J Chan

  4. Department of Biostatistics, Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN, USA

    Z Liu

  5. Department of Medicinal Chemistry & Molecular Pharmacology, Purdue College of Pharmacy, West Lafayette, IN, USA

    Z-Y Zhang

  6. Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN, USA

    H S Boswell

  7. Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    R Kapur

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Correspondence to R J Chan.

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Richine, B., Virts, E., Bowling, J. et al. Syk kinase and Shp2 phosphatase inhibition cooperate to reduce FLT3-ITD-induced STAT5 activation and proliferation of acute myeloid leukemia. Leukemia 30, 2094–2097 (2016). https://doi.org/10.1038/leu.2016.131

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