Pak and Rac GTPases promote oncogenic KIT–induced neoplasms
Holly Martin, … , Suranganie Dharmawardhane, Reuben Kapur
Holly Martin, … , Suranganie Dharmawardhane, Reuben Kapur
Published September 16, 2013
Citation Information: J Clin Invest. 2013;123(10):4449-4463. https://doi.org/10.1172/JCI67509.
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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 1

Constitutive activation of GEF Vav1 and Rac GTPase in KITD814V-expressing cells.

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Constitutive activation of GEF Vav1 and Rac GTPase in KITD814V-expressin...
(A) 32D cells expressing either WT KIT or KITD814V were serum and growth factor starved for 8 hours, and equal amounts of lysates were subjected to IP using an anti-Vav1 antibody. The position of tyrosine phosphorylated Vav1 is indicated at the right of the blot. Right panel indicates the Vav1 protein whole-cell lysate loading control. (B) Proliferation as assessed by thymidine incorporation in KITD814V-expressing WT and Vav1-deficient primary BM cells in the absence of growth factors. Bars represent the mean thymidine incorporation (cpm) in primary BM cells expressing the indicated receptors. Similar results were observed in three independent experiments. *P < 0.05 for KITD814V versus Vav1–/– KITD814V. (C) Cell lysates derived in B were analyzed for Rac-GTP levels by incubating with agarose beads conjugated to the Pak binding domain and by subjecting the IPs to Western blot analysis using an anti-Rac1, anti-Rac2, or anti–pan Rac antibody. Rac-GTP position is indicated at the right of the blot. Bottom panel shows total Rac protein in each lane.