Year of publication
- 2012 (3) (remove)
- Novel role of Ras-GTPase Activating Protein SH3 Domain-Binding Protein G3BP in adhesion and migration of 32D myeloid progenitor cells (2012)
- Rho GTPases are involved in homing and mobilization of hematopoietic stem and progenitor cells due to their impact on cytoskeleton remodeling. We have previously shown that inhibition of Rho, Rac and Cdc42 clearly impairs adhesion of normal and leukemic hematopoietic progenitor cells (HPC) to fibronectin and migration in a three-dimensional stromal cell model. Here, we identified the Ras GTPase-Activating Protein SH3 Domain-Binding Protein (G3BP) as a target gene of Rho GTPases and analysed its role in regulating HPC motility. Overexpression of G3BP significantly enhanced adhesion of murine 32D HPC to fibronectin and human umbilical vein endothelial cells, increased the proportion of adherent cells in a flow chamber assay and promoted cell migration in a transwell assay and a three-dimensional stromal cell model suggesting a strong impact on the cytoskeleton. Immunofluorescent staining of G3BP-overexpressing fibroblasts revealed a Rho-like phenotype characterized by formation of actin stress fibers in contrast to the Rac-like phenotype of control fibroblasts. This is the first report implicating a role for G3BP in Rho GTPase-mediated signalling towards adhesion and migration of HPC. Our results may be of clinical importance, since G3BP was found overexpressed in human cancers.
- Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I (2012)
- Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.
- Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism (2012)
- ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.