Ahi-1 (Abelson helper integration site 1) is a novel gene that is commonly activated by provirus insertional mutagenesis in v-abl and myc-induced murine leukemias and lymphomas. It encodes a unique protein with SH3 and WD40-repeat domains suggesting novel signaling activities. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and lymphomas and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemia cell lines, particularly in the cutaneous T-cell leukemia cell lines, Hut 78 and Hut 102, where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether the deregulated expression of AHI-1 in leukemic cells contributes to their transformed properties, knockdown of AHI-1 expression in Hut 78 cells, a cell line derived from peripheral blood of a patient with Sezary syndrome, was performed using retroviral-mediated RNA interference (RNAi). In a screen of 9 constructs that produce specific short hairpin AHI-1 transcripts, one was found to specifically inhibit AHI-1 expression in transduced Hut 78 cells by 80%, as evaluated by quantitative real-time RT-PCR, Northern and Western blot analyses. Retroviral-mediated suppression of AHI-1 also reduced the autocrine production of IL-2, IL-4 and TNFalpha in Hut 78 cells by up to 85% and caused a significant reduction in their growth factor independence in semi-solid cultures (up to 10-fold) and in single cell cultures (4-fold) by comparison to cells transduced with a control vector. Interestingly, although addition of IL-4, TNFalpha or a combination of 3 growth factors restored colony formation from the shRNA-transduced Hut 78 cells in semi-solid cultures, this was not achieved if only IL-2 was added, even though AHI-1 expression was inhibited. The ability of Hut 78 cells to produce tumors in NOD/SCID-β2microglobulin−/− mice within 3 weeks was also lost when AHI-1 expression was suppressed. Microarray analysis on RNA from Hut 78 cells with the suppression of AHI-1, using the Affymetrix Human Genome U133 plus 2.0 Arrays, identified differentially expressed molecules critical in T-cell activation, signal transduction, as well as cell proliferation and differentiation. Q-RT-PCR analysis revealed that the transcript levels of AHI-1 and its isoforms were significantly increased in CD4+CD7− Sezary cells, in which more than 85% of these cells are leukemic cells, in 5 of 6 blood samples obtained from patients with Sezary syndrome as compared to T cells similarly isolated from 8 healthy individuals. Elevated AHI-1 transcript levels were not found in 3 patient samples containing less than 35% leukemic Sezary cells. Taken together, these findings provide strong evidence of the oncogenic activity of AHI-1 in human T-cell leukemic cells and its deregulation can contribute to the development of human cutaneous T-cell lymphomas, including Sezary syndrome.