Deregulated Expression of a Novel Oncogene, Ahi-1, in Murine Hematopoietic Cells Confers a Proliferative Advantage In Vitro and Leukemogenic Activity In Vivo and Enhances the Effects of BCR-ABL.
Authors: Zhou Leon , Ringrose Ashley , Lin Ann E.-J. , Jiang Xiaoyan 
Published: October 2019

Abstract
Ahi-1 (Abelson helper integration site-1) is a novel gene that we recently identified based on its common activation in v-abl or myc-induced murine leukemias and lymphomas. It encodes a unique protein with known signaling features, including SH3 and WD40-repeat domains, but its function is largely unknown. We have recently demonstrated that Ahi-1/AHI-1 transcript levels are normally down-regulated during both early murine and human hematopoietic cell differentiation and are highly increased in human leukemic cells, particularly in highly enriched populations of primitive BCR-ABL+ leukemic stem cells (lin−CD34+CD38−) in patients with chronic myeloid leukemia (CML). To investigate the potential cooperative activity of Ahi-1 in BCR-ABL-mediated signal transduction and leukemogenesis, we transduced IL-3-dependent BaF3 cells with MSCV-Ahi-1-IRES-YFP and/or MSCV-BCR-ABL-IRES-GFP retroviruses and compared the biological behavior of these cells in vitro and in vivo. All Ahi-1-transduced clonal cell lines showed increased proliferative activity and reduced apoptosis in the absence of IL-3, compared to parental BaF3 cells or control GFP-transduced cells. Interestingly, overexpression of both Ahi-1 and BCR-ABL caused more enhanced perturbations when compared to cells transduced with either Ahi-1 or BCR-ABL alone. Strikingly, intravenous injection of NOD/SCID-β2microglobulin−/−mice with BaF3 cells induced to overexpress Ahi-1 alone induces a lethal leukemia within 70 days. These leukemogenic activities were further increased by introduction of co-transduced Ahi-1 and BCR-ABL cells, producing a shorter latency of 30 days. A disease latency of 40 days was revealed by introduction of BCR-ABL-transduced cells alone. Western blot analysis showed that both protein expression and the tyrosine kinase activity of p210BCR-ABL were highly increased in cells co-transduced with Ahi-1 and BCR-ABL compared to BaF3 cells transduced with BCR-ABL alone. Similarly, we also observed higher levels of Ahi-1 protein expression in the same dually transduced cells (Ahi-1+BCR-ABL+) than in those transduced with Ahi-1 alone. We further demonstrated a similarly perturbed proliferative activity, growth factor independence and colony-forming cell (CFC) output in long-term culture initiated cell (LTC-IC) assays of 5-fluorouracil (5-FU)-treated primitive murine BM cells (Lin−Sca1+ cells) transduced with Ahi-1 and BCR-ABL, either alone or in combination. To investigate directly the cooperating oncogenic role of AHI-1 in BCR-ABL-mediated malignant transformation of CML cells, knockdown of AHI-1 expression in K562 cells, a cell line that was derived from a patient with CML and that is characterized by highly increased expression of AHI-1, was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited endogenous AHI-1 expression in transduced cells by 70% as evaluated by Q-RT-PCR and Western blot analyses. It further caused a significant reduction in their growth factor independence in semi-solid cultures (up to 5-fold) and in single cell cultures (2-fold) by comparison to cells transduced with a control vector. Taken together, these findings provide strong evidence of the transforming potential of Ahi-1/AHI-1 in primitive hematopoietic cells. This effect is additive with those of BCR-ABL, suggesting that AHI-1 and BCR-ABL can play a cooperate role in the development of BCR-ABL-associated diseases like CML.