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 6

Deficiency of Rac1 and Rac2 in KITD814V-bearing primary HSC/Ps represses ligand-independent growth.

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Deficiency of Rac1 and Rac2 in KITD814V-bearing primary HSC/Ps represses...
(A) PB cells were collected from WT, Rac1flox/flox, Rac2–/–, and Rac2–/–/Cre:Rac1flox/flox mice following four consecutive i.p. injections of polyI:C given at 48-hour intervals. Red blood cells were lysed, and equal amounts of protein were subjected to Western blotting using an anti-Rac1 antibody. Levels of Rac1 in each lane are shown. (B) Representative flow cytometric dot plots of low-density BM cells derived from WT, Rac1–/–, Rac2–/–, and Rac1–/–/Rac2–/– mice transduced with WT KIT– or KITD814V-expressing retrovirus. EGFP expression on the x axis is reflective of the transduction efficiency in the indicated genotypes. (C) WT, Rac1–/–, Rac2–/–, and Rac1–/–/Rac2–/– primary HSC/Ps expressing WT KIT or KITD814V were subjected to a thymidine incorporation assay. *P < 0.05 for KITD814V in WT versus Rac2–/–, Rac1–/–, or Rac1–/–/Rac2–/– cells. Shown are the combined data from three independent experiments in replicates of four. (D) WT, Rac1–/–, and Rac1–/–/Rac2–/– primary HSC/Ps expressing KITD814V were grown in the absence of growth factors for 0, 24, or 48 hours prior to being analyzed by annexin V and 7-AAD staining. Apoptosis was determined as a percentage of both annexin V– and 7-AAD–positive staining. Bars denote the mean ± SD percentage of annexin V– and 7-AAD–negative (viable) cells. *P < 0.05 for WT, Rac1–/– versus Rac1–/–/Rac2–/–.