MKK4 and MKK7 Interact with NPM-ALK and Contribute to Tumor Cell Proliferation and Survival in NPM-ALK+ Anaplastic Large Cell Lymphoma
G Rassidakis, P Georgoulis, V Atsaves, P Tsioli, E Drakos, LJ Medeiros, P Panayiotidis, E Patsouris. National and Kapodistrian University of Athens, Athens, Greece; The University of Texas M.D. Anderson Cancer Center, Houston, TX
Background: Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in overexpression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncoprotein. NPM-ALK is known to activate a number of oncogenic pathways including Ras, JAK/STAT, PI3K/AKT/mTOR, and others. In a previous study we showed that NPM-ALK activates JNK and promotes cell cycle progression through activation of c-Jun. (Leventaki et al, Blood 2007; 110:1621). In this study we hypothesized that NPM-ALK interacts with MKK4 and MKK7, members of the MAP kinase family, thus contributing to tumor cell proliferation.
Design: To investigate the activation (phosphorylation) status of MKK4 and MKK7 in NPM-ALK+ ALCL, Western blot analysis, immunofluorescence and immunohistochemistry were performed in primary and cultured cells. Co-immunoprecipitation followed by Western blot analysis were used to assess the physical interaction between NPM-ALK and MKK4/MKK7. Transfection experiments were performed in human embryonic kidney (HEK) cell line 293T and in 2 ALCL cell lines, Karpas 299 and SU-DHL1. Cell viability and proliferation of viable cells were assessed by trypan blue exclusion and MTS assays, respectively.
Results: Activated (phosphoryated) MKK4 and MKK7 were expressed at a variable level in NPM-ALK+ cell lines and tumors tested. Co-immunoprecipitation studies revealed that NPM-ALK physically interacts with both MKK4 and MKK7. Transient transfection of active NPM-ALK plasmid in HEK 293T resulted in MKK4 and MKK7 activation, which was not present after transfection with the inactive, kinase-dead, NPM-ALK plasmid. Silencing of MKK4 and MKK7 using specific siRNA's in NPM-ALK+ cell lines, Karpas 299 and SUDHL-1, resulted in a dramatic decrease in cell number and proliferation and, at a lesser degree, in a decrease (by 20-25%) in cell viability. Similar biologic effects were obtained after pharmacologic inhibition of ALK kinase activity in Karpas 299 and SUDHL-1 cells, which were associated with deactivation of MKK4 and MKK7.
Conclusions: Our data suggest that NPM-ALK is capable of activating MKK4 and MKK7, which substantially contribute to tumor cell proliferation in NPM-ALK+ lymphoma cells, thus providing novel potential targets for investigational therapy in patients with ALK+ ALCL.
Monday, March 22, 2010 11:45 AM
Platform Session: Section B, Monday Morning