Abstract
Abstract 3909
Poster Board III-845
Myeloproliferative disorder (MPD) and acute myeloid leukemia (AML) are heterogeneous hematologic malignancies which share the common characteristics of myeloid cell overproduction and aberrant differentiation. With the exception of chronic myelogenous leukemia (CML), therapeutic intervention for these diseases is largely ineffective. There are increasing examples of mutations in tyrosine kinases (TKs) that contribute to MPD as well as AML. Key examples are the well characterized BCR-ABL transgene causing constitutively activated Abl tyrosine kinase function in CML. FLT3 internal tandem duplications (ITDs) induce ligand-independent autophosphorylation in AML, and activating KIT mutations seen in AML and in over 90% cases of systemic mastocytosis (SM). Although hyperactivation of several signaling molecules downstream from these tyrosine kinases have been reported; little is known about the identity of signaling molecules that are shared among these mutations. Rho kinases or Rho-associated coiled coil-containing protein kinases (ROCK1 and ROCK2) are protein serine/threonine kinases that control a wide variety of cellular functions, including cell growth, survival and motility. Rho kinases have been shown to be hyperactive in several cancers. Importantly, in preclinical models, ROCK inhibitors have demonstrated significant efficacy in repressing aspects of tumorigenesis. However, the role of ROCK kinases in MPDs/Leukemogenesis has not been investigated. We show that expression of wild-type (WT) KIT receptor or vector alone in 32D murine myeloid cells or WT FLT3 receptor in BaF3 murine lymphoid cells does not result in ligand independent activation of ROCK kinases as evident by lack of phosphorylation of its substrate MYPT1. In contrast, expression of oncogenic KITD816V, FLT3N51, and BCR-ABL in these cells results in constitutive activation of Rho kinases, which is completely inhibited in the presence of Rho kinase inhibitor dimethyl-fasudil. Importantly, treatment of oncogene bearing cells with dimethy-fasudil did not affect the activation of PKA, PKC, AKT, STAT3 or ERK MAPkinase. Furthermore, treatment of oncogenic KITD816V, FLT3N51 and BCR-ABL bearing cells with additional Rho kinase inhibitors (Y27632 and fasudil) also resulted in a dose dependent repression in ligand independent growth in a thymidine incorporation assay. In contrast, WT KIT, FLT3, or vector alone containing cells demonstrated little to no repression of growth in the presence of cytokines and Rho kinase inhibitors. A human mast cell line (HMC1.2) derived from a patient with mastocytosis bearing the activation loop mutation D816V in the KIT receptor, also showed a dose dependent suppression in ligand independent growth upon treatment with Rho kinase inhibitors. Similar inhibition of growth was observed when primary hematopoietic stem and progenitor cells (HSC/Ps) bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL mutations were treated with Rho kinase inhibitors. In an effort to identify the mechanism behind reduced growth, we examined opoptosis in oncogene bearing cells treated with Rho kinase inhibitors. We found increased apoptosis in these cells compared to WT KIT receptor, FLT3 receptor, or vector alone expressing cells. Apoptosis was observed as early as two hours after Rho kinase inhibitor treatment in oncogene bearing cells. To determine the mechanism of such rapid apoptosis in oncogene bearing cells, we examined the activation of several known downstream substrates of ROCK kinases. We found that cells bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL receptors demonstrated enhanced and constitutive phosphorylation of myosin light chain (MLC). Since inhibition of MLC phosphorylation in breast cancer lines has been associated with apoptosis, we next measured the activation of MLC in the presence of ROCK inhibitors. We found that MLC was dephosphorylated within minutes of treating oncogene bearing cells with Rho kinase inhibitors but not in wildtype receptor bearing cells. Taken together, these results suggest that dephosphorylation of a major downstream Rho kinase substrate (i.e. MLC) plays an essential role in inducing cell death in oncogene bearing cells but not in normal cells. These studies indicate Rho kinases as possible therapeutic targets for treating diseases such as AML and MPD.
Disclosures:
No relevant conflicts of interest to declare.
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