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 4

A novel Rac inhibitor, EHop-016, is a potent inhibitor of KITD814V-induced growth in SM and AML patient–derived cells.

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A novel Rac inhibitor, EHop-016, is a potent inhibitor of KITD814V-induc...
(A) Human SM patient–derived HMC1.2 cells bearing KITD816V and (B) human AML patient–derived Kasumi-1 cells bearing KITD816V were cultured in the presence of EHop-016 and assessed for proliferation by measuring [3H] thymidine incorporation. Bars represent the mean ± SD from four independent experiments performed in replicates of four; *P < 0.05. (C) Individual SM patient–derived cells positive or negative for KITD816V expression were cultured in the presence of the indicated concentrations of EHop-016. Bars represent the mean ± SD performed in replicates of three. *P < 0.05; 0 μM versus the indicated concentration. (D) 32D 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 active Rac1, Rac2, total Rac1, and Rac2 in each lane. (E) 32D cells expressing either WT KIT or KITD814V were starved and incubated with vehicle, 5 μM or 10 μM EHop-016; lysates were immunoprecipitated with anti-Rac1 antibody and immunoblotted with an anti-Vav1 or anti-Rac1 antibody, respectively. Lower panels indicate the Vav1 and Rac1 protein whole-cell lysate loading control.