Distinct Transforming and Leukemogenic Activities of Tel-Arg and Tel-Abl Oncogenes in Mice: Possible Negative Role for the Transforming Activity in C-Terminus of Arg
Authors: Okuda Keiko , Harakawa Nari , VanEtten Richard A , Patel Nihal , Domae Naochika , Sato Yuko , Hirai Hideyo 
Published: April 2020

Abstract
ARG (ABL2) is a member of ABL tyrosine kinase gene family that is highly homologous to c-ABL (ABL1) in overall domain structure (SH3-SH2-SH1) and amino acid sequence. ARG has recently been implicated in the pathogenesis of human acute leukemia though t(1;12) translocations that fuse a transcription factor gene, ETV6/TEL, to ARG (Iijima et al., Blood2000;95:2126). The resulting TEL-ARG fusion tyrosine kinase is similar in structure to the TEL-ABL fusion found in some acute leukemia and atypical CML patients, and, like TEL-ABL, can transform Ba/F3 cells and fibroblasts in vitro and activate a similar set of intracellular signaling pathways (Iijima et al., Oncogene2002;21:4374). To assess the leukemogenic activity of TEL-ARG, we co-expressed TEL-ARG with GFP in mouse bone marrow using a retroviral bone marrow transduction/transplantation strategy. Whereas TEL-ABL induces rapidly fatal myelolproliferative disease (MPD) in recipient mice (Million et al., Blood2000;96:664), recipients of TEL-ARG-transduced BM did not develop overt MPD, but succumbed instead to long-latency (30–45 weeks) T-cell acute lymphoblastic leukemia/lymphoma characterized by modest leukocytosis and a malignant pleural effusion composed of Thy-1+B220- tumor cells. To study the molecular basis of the marked difference in the leukemogenic activity of TEL-ARG and TEL-ABL, we produced TEL-ARG mutants that swapped the kinase domain or C-terminus of ARG with the corresponding domain in ABL. The mutants were introduced into Ba/F3 cells by retroviral transduction and transduced cells selected for equal expression of the fusion protein by flow sorting for populations with equivalent intensity of GFP fluorescence. All chimeric proteins were expressed and showed equivalent levels of auto-phosphorylation by western blot analysis of the sorted cells. However, the quantitative transforming activity of TEL-ARG in Ba/F3 cells, measured by the number of days required to achieve measurable cell growth following IL-3 deprivation, was significantly lower than for TEL-ABL (25 ± 4.3 days for TEL-ARG vs. 1 to 2 days for TEL-ABL). A TEL-ARG mutant containing the ABL kinase domain was similar to TEL-ARG in this assay, but replacing the ARG C-terminal domain with that of ABL increased Ba/F3 transformation to levels equivalent to TEL-ABL. To further dissect the functional domains that are responsible for this effect, a new series of mutants containing internal deletions in ARG C-terminus of TEL-ARG [aa.826–976 (Delta Box1), aa.977–1213 (Delta Box2), aa 1214–1316 (Delta Box3), aa.1317–1465 (Delta Box4)] were generated. Regarding to their function, only Box2 and Box4 were reported as F- or F,G- actin binding domain. From a preliminary study using the cell lines which express each of the mutants, Delta Box 1 clones obtained an accelerated proliferation which compared with that of Tel-Abl, suggesting that there is a disadvantage for cell proliferation by ARG c-terminus. These results indicate that distinct bio-phenotype associated with Abl family tyrosine kinase is the most likely regulated by their c-terminus and the c-terminus of Arg contains functional subdomain that impairs growth signal induced by Abl family tyrosine kinase.