Pak and Rac GTPases promote oncogenic KIT–induced neoplasms
Holly Martin, Raghuveer Singh Mali, Peilin Ma, Anindya Chatterjee, Baskar Ramdas, Emily Sims, Veerendra Munugalavadla, Joydeep Ghosh, Ray R. Mattingly, Valeria Visconte, Ramon V. Tiu, Cornelis P. Vlaar, Suranganie Dharmawardhane, Reuben Kapur
Holly Martin, Raghuveer Singh Mali, Peilin Ma, Anindya Chatterjee, Baskar Ramdas, Emily Sims, Veerendra Munugalavadla, Joydeep Ghosh, Ray R. Mattingly, Valeria Visconte, Ramon V. Tiu, Cornelis P. Vlaar, Suranganie Dharmawardhane, Reuben Kapur
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Research Article Oncology

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

Abstract

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21–activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Authors

Holly Martin, Raghuveer Singh Mali, Peilin Ma, Anindya Chatterjee, Baskar Ramdas, Emily Sims, Veerendra Munugalavadla, Joydeep Ghosh, Ray R. Mattingly, Valeria Visconte, Ramon V. Tiu, Cornelis P. Vlaar, Suranganie Dharmawardhane, Reuben Kapur

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Figure 8

KITD814V induces the activation of Pak in a Vav1- and Rac-dependent manner.

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KITD814V induces the activation of Pak in a Vav1- and Rac-dependent mann...
KITD814V-expressing WT or Rac1/Rac2 (A) or Vav1-deficient primary HSC/Ps (B) from two independent animals were starved and lysed and immunoblotted for active Pak. (C) KITD814V-bearing 32D cells expressing RacN17 were starved and lysed, and equal amounts of lysates were subjected to immunoblotting using a phospho-Pak antibody and total Pak. (D) Cells bearing WT KIT or KITD814V were starved and treated with the indicated concentrations of NSC23766 or EHop-016 and subjected to Rac activation assay. Shown are the levels of phosphorylated and total Pak. (E) 32D cells were transduced with KITD814V with or without dominant-negative PakK299R and starved; lysates were subjected to Western blot analysis using phospho-KIT, phospho-Pak, total KIT, total Pak, and total ERK antibodies. (F) Cells in E were starved for 6 hours before being plated in quadruplicate in the presence or absence of growth factors (IL-3, 5 ng/ml) for 48 hours. Cells were pulsed with [3H] thymidine for 6 hours and harvested. Bars denote the mean thymidine incorporation (cpm ± SD) from one of four independent experiments performed in quadruplicate. *P < 0.05 for KITD814V versus KITD814V plus PakK299R.