Summary We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth and loss of actin polymerization in oncogene bearing cells leading to significantly prolonged life span of leukemic mice. In summary, we describe a pathway involving PI3K/Rho/ROCK/MLC which may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.
Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KITinduced growth and survival in vitro and myeloproliferative disease (MPD) in vivo.Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85␣, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85␣, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD. (Blood. 2012; 120(13):2669-2678) IntroductionGain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). [1][2][3][4] An activating KIT receptor mutation of aspartic acid to valine at codon 814 in mice (KITD814V) or codon 816 in humans (KITD816V) results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous signaling. 5,6 Consequently, cell lines and primary BM cells that express the oncogenic KITD814V demonstrate ligand-independent proliferation in vitro and myeloproliferative disease (MPD) in vivo. [5][6][7][8][9] However, the intracellular signals that contribute to KITD814V-induced MPD are not known. Although activating mutations of KIT involving the juxtamembrane domain found in GIST are highly sensitive to inhibition by imatinib mesylate (ie, Gleevec), KIT mutations within tyrosine kinase domain found in SM and AML, including KITD816V, are relatively resistant to imatinib treatment. [10][11][12] Thus, it is vital to identify novel drug targets for diseases involving KITD816V mutation.Emerging data suggest an essential role for SHP2 in MPD. SHP2 is a protein tyrosine phosphatase that is encoded by PTPN11 gene and has been implicated in diverse signaling pathways induced by a number of stimuli, including growth factors, cytokines, extracellular matrix, and even cellular stress. [13][14][15] Given that activating mutations in SHP2 have been found in leukemias and solid tumors, 16,17 efforts are ongoing to define the potential efficacy of SHP2 phosphatase inhibition in diseases bearing SHP2 hyperactivation, either because of activating SHP2 mutations or those in which SHP2 collaborates with other oncogenes. Using genetic approaches, including primary BM cells derived from SHP2 Ϫ/Ϫ and Gab2 Ϫ/Ϫ mice and a novel SHP2 inhibitor, II-B08, identified from a focused library of indole-based salicylic acid derivatives, 18 we demonstrate that SHP2 is essential for KITD814V-induced MPD. We further demonstrate that SHP2 constitutively binds to p85␣ and Gab2 in KITD814V-bearing cells, which can be disrupted by II-B08 resulting in impaired ligand-independent growth in vitro a...
Oncogenic activation loop KIT mutations are observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants, the activation loop mutants are insensitive to imatinib mesylate. Furthermore, as prior studies primarily used heterologous cell lines, the molecular mechanism(s) underlying oncogenic KITinduced transformation in primary cells is poorly understood. We demonstrate that expression of KITD814V in primary hematopoietic stem/progenitor cells (HSC/Ps) and mast cell progenitors (MCps) induces constitutive KIT autophosphorylation, supports ligand-independent hyperproliferation, and promotes promiscuous cooperation with multiple cytokines. Genetic disruption of p85␣, the regulatory subunit of class IA lipid kinase phosphoinositol-3-kinase (PI3K), but not of p85, or genetic disruption of the hematopoietic cellspecific Rho GTPase, Rac2, normalizes KITD814V-induced ligand-independent hyperproliferation. Additionally, deficiency of p85␣ or Rac2 corrects the promiscuous hyperproliferation observed in response to multiple cytokines in both IntroductionStem cell factor (SCF) is a unique cytokine with important functional roles in melanocytes, germ cells, interstitial cells of Cajal, mast cells, and hematopoietic stem cells. 1 Consistent with the importance of SCF signaling within these defined tissues, activating mutations of KIT, which encodes the receptor for SCF, have been described in germ cell tumors, gastrointestinal stromal tumors (GISTs), sinonasal lymphomas, acute myeloid leukemia (AML), and systemic mastocytosis (SM). [2][3][4][5][6] SM is characterized by clonal expansion of myelomastocytic progenitors with tissue accumulation of malignant mast cells and commonly bears the KIT activation loop mutant KITD816V. 7 Although this disease may assume an indolent course, it can also take an aggressive direction (aggressive systemic mastocytosis or mast cell leukemia) or can be associated with a non-mast-cell hematologic malignancy (associated clonal hematologic non-mast-cell lineage disease). Oncogenic KIT mutations are also observed in core binding factor-acute myeloid leukemia (CBF-AML), leukemias that bear either the t(8;21) or inv(16) cytogenetic abnormality, generating the fusion genes AML1-ETO or CBF-MYH11 and disrupting CBF␣ (AML1) or CBF, respectively. 8 Studies examining both adult and pediatric AML have indicated that the presence of the KITD816V mutant in CBF-AML carrying t(8;21) worsens the prognosis based on several clinical indices. [9][10][11][12] Oncogenic KIT is constitutively phosphorylated, suggesting that signals emanating from this receptor are not regulated by ligand stimulation, 13,14 and, consistently, cell lines expressing oncogenic KIT demonstrate ligand-independent proliferation. 13,15,16 KIT contains an extracellular portion containing 5 immunoglobulinlike repeats, a transmembrane domain, a juxtamembrane domain, and a cytoplasmic tyrosine kinase domain that is split by an insert sequence. Activating KIT mutations within the juxtame...
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