Aberrant Ras Activation Associated with Functional Loss of Tnk1/Kos1 In Human B-Cell Lymphoma

Abstract 3159

Tnk1/Kos1 (Thirty-eight negative kinase/Kinase of embryonic stem cells) is the first demonstrated nonreceptor protein tyrosine kinase (PTK) to be identified as a tumor suppressor (Hoare et al.2008). The Tnk1/Kos1 knockout mice (∼80%) spontaneously develop B-cell lymphomas, specifically DLBCL, which express aberrantly high levels of Ras activity due to the loss of Tnk1/Kos1. Mechanistically the high Ras activity in the tumor tissue results from the stabilization of the RasGEF (Grb2-Sos1) complex since Ras is not found to be mutated in B-cell lymphomas. Importantly, in humans, the allelic loss of Tnk1 and/or reduced expression of Tnk1 transcripts occur in patients with DLBCL (Oncomine.org). In support of a tumorigenic mechanism, Immunohistochemical (IHC) analysis using a phospho-specific Tnk1 antibody that we developed confirms the functional loss of Tnk1 activity in DLBCL tissue. These data underscore the potential clinical relevance of Tnk1 in human hematological malignancies. Interestingly, the L540 cell line, derived from a patient with Hodgkin Lymphoma was recently reported to express a 60 Kda truncated fusion Tnk1-C17orf61 gene product (Gu et al. 2010). Although the Tnk fusion gene product is heavily tyrosine phosphorylated, we find that it possesses poor to no enzymatic activity toward the Tnk1/Kos1 substrate Grb2. This indicates that its PTK activity is possibly inhibited by steric hindrance as a result of hyper Y-phosphorylation. This is reminiscent of kinase-dead Tnk1/Kos1 and hence the truncated L540 Tnk fusion protein may function as an oncogene (Hoare et al. 2003). Interestingly, when L540 cells were exposed to 5 Aza cytidine (40μM) or Trichostatin A (100ng/ml), we find a reduction in the expression of the truncated L540 Tnk fusion protein with a concomitant increase in Ras activity (hyper Ras activation) and accumulation of cells in sub G1 phase, indicating Ras mediated induction of cell senescence and death, an event commonly observed in the over expression of several oncogenes (Serrano et al 1997; Ruggero et al, 2004). Our findings may therefore, point the way to designing a novel and effective therapy that may prevent the progression of B-cell lymphomas associated with abnormal expression of mutated Tnk1. Importantly, since Ras is infrequently mutated to oncogenic Ras in human lymphoid tumors (unlike myeloid leukemias), aberrant Ras activation in such lymphoid tumors may result from the loss of Tnk1 kinase activity due to the constitutive activation of Ras-GEF activity. These findings indicate the mechanism by which wt-Ras can be aberrantly activated to an “oncogenic” equivalent form with the loss of Tnk1 to induce and/or contribute to spontaneous tumorigenesis in hematological malignancies.