Expression Of AHI-1 Research Articles

Loss of Ahi-1 Impairs the Self-Renewal Activity of Normal and BCR-ABL+ Stem Cells in Vitro and in Vivo

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have identified AHI-1 (Abelson helper integration site-1) oncogene as a potential therapeutic target in CML. It encodes a scaffold protein with a SH3 domain, multiple proline rich motifs and several WD40 repeats. Its expression is highly increased in BCR-ABL+ LSCs. Overexpression of Ahi-1 in primitive hematopoietic cells enhances cell proliferation in vitro and induces leukemia in vivo, and these effects can be enhanced by BCR-ABL. We have also demonstrated that AHI-1 interacts with multiple kinases, including BCR-ABL, β-catenin and JAK2 to mediate LSC activities and TKI resistance in vitro and in vivo . Interestingly, mouse Ahi-1 is highly expressed in hematopoietic stem cells (HSCs, CD45+EPCR+CD48-CD150+ (E-SLAM)) and progenitors (lin-Sca-1+c-kit+ (LSK)), but expressed at low levels in differentiated cells. In addition, Ahi-1 transcripts are found to be expressed in human embryonic stem cells (ES) and at all stages of mouse embryo development, with increasing expression just prior to birth, suggesting that Ahi-1 expression is developmentally regulated. To investigate the precise role of Ahi-1 in regulating normal and LSC self-renewal and differentiation, we have developed a new conditional Ahi-1 knockout mouse model. Ahi-1 deficient mice (AKO) were viable and fertile, but displayed around 30% reduction of bone marrow (BM) and thymus cellularity and altered lineage differentiation as compared to control wild-type mice (AWT). Interestingly, cell cycle analysis showed that loss of Ahi-1 reduced quiescent G0 cells by about 17% in HSC (lin-Sca-1+c-kit+CD48-) as compared to AWT cells. Ahi-1 deficient LSK cells did not exhibit differences in output of colony forming cells (CFCs) but did have dramatically reduced CFC secondary replating efficiency as compared to control cells. In vivo limited dilution assays (LDA), measured by bone marrow transplantation, demonstrated that Ahi-1 loss did not result in significant changes in the frequency of HSC numbers in primary transplanted mice (AWT vs AKO: 1/85000 vs 1/74000), but greatly reduced the reconstitution ability of these cells in serial transplantation assays, since no detectable donor cells were found in tertiary transplanted mice with AKO cells, while engrafted donor cells were still detected in tertiary transplanted mice with AWT cells. These results suggest that Ahi-1 plays a key role in regulating long-term repopulation activity of normal HSCs.The effects of Ahi-1 on LSC growth and maintenance were more profound when BCR-ABL was expressed in Ahi-1-deficient LSCs. Primitive Ahi-1-deficient BM cells transduced with BCR-ABL retroviruses (AKOBA) displayed reduced cell proliferation in the presence of growth factors, especially 14 days after in vtiro culture, compared to BCR-ABL-expressing BM cells from AWT mice (AWTBA). The FACS analysis for these cells after 7 days in culture showed reduced LSK numbers (30%) in AKOBA cells compared to AWTBA control cells. CFC assays revealed a 40% reduction in colonies in AKOBA as compared to AWTBA cells, but the effect was strikingly enhanced in the second and third replating assays (~70% reduction) in AKOBA cells. Most interestingly, we demonstrated that loss of Ahi-1 significantly enhanced survival of leukemic mice generated by crossing of Ahi-1 conditional KO mice with BCR-ABL-transgenic mice as compared to Ahi-1 wild-type/BCR-ABL control mice (65 days vs. 14 days, P=0.008). These results demonstrate that Ahi-1 plays a critical role in regulating HSC functionality and that its deletion perturbs self-renewal and leukemogenesis of BCR-ABL+ LSCs. DisclosuresNo relevant conflicts of interest to declare.

Identifying Causal Genes at the Multiple Sclerosis Associated Region 6q23 Using Capture Hi-C

BackgroundThe chromosomal region 6q23 has been found to be associated with multiple sclerosis (MS) predisposition through genome wide association studies (GWAS). There are four independent single nucleotide polymorphisms (SNPs) associated with MS in this region, which spans around 2.5 Mb. Most GWAS variants associated with complex traits, including these four MS associated SNPs, are non-coding and their function is currently unknown. However, GWAS variants have been found to be enriched in enhancers and there is evidence that they may be involved in transcriptional regulation of their distant target genes through long range chromatin looping.AimThe aim of this work is to identify causal disease genes in the 6q23 locus by studying long range chromatin interactions, using the recently developed Capture Hi-C method in human T and B-cell lines. Interactions involving four independent associations unique to MS, tagged by rs11154801, rs17066096, rs7769192 and rs67297943 were analysed using Capture Hi-C Analysis of Genomic Organisation (CHiCAGO).ResultsWe found that the pattern of chromatin looping interactions in the MS 6q23 associated region is complex. Interactions cluster in two regions, the first involving the rs11154801 region and a second containing the rs17066096, rs7769192 and rs67297943 SNPs. Firstly, SNPs located within the AHI1 gene, tagged by rs11154801, are correlated with expression of AHI1 and interact with its promoter. These SNPs also interact with other potential candidate genes such as SGK1 and BCLAF1. Secondly, the rs17066096, rs7769192 and rs67297943 SNPs interact with each other and with immune-related genes such as IL20RA, IL22RA2, IFNGR1 and TNFAIP3. Finally, the above-mentioned regions interact with each other and therefore, may co-regulate these target genes.ConclusionThese results suggest that the four 6q23 variants, independently associated with MS, are involved in the regulation of several genes, including immune genes. These findings could help understand mechanisms of disease and suggest potential novel therapeutic targets.

Alterations in the expression of a neurodevelopmental gene exert long-lasting effects on cognitive-emotional phenotypes and functional brain networks: translational evidence from the stress-resilient Ahi1 knockout mouse

Many psychiatric disorders are highly heritable and may represent the clinical outcome of early aberrations in the formation of neural networks. The placement of brain connectivity as an ‘intermediate phenotype’ renders it an attractive target for exploring its interaction with genomics and behavior. Given the complexity of genetic make up and phenotypic heterogeneity in humans, translational studies are indicated. Recently, we demonstrated that a mouse model with heterozygous knockout of the key neurodevelopmental gene Ahi1 displays a consistent stress-resilient phenotype. Extending these data, the current research describes our multi-faceted effort to link early variations in Ahi1 expression with long-term consequences for functional brain networks and cognitive-emotional phenotypes. By combining behavioral paradigms with graph-based analysis of whole-brain functional networks, and then cross-validating the data with robust neuroinformatic data sets, our research suggests that physiological variation in gene expression during neurodevelopment is eventually translated into a continuum of global network metrics that serve as intermediate phenotypes. Within this framework, we suggest that organization of functional brain networks may result, in part, from an adaptive trade-off between efficiency and resilience, ultimately culminating in a phenotypic diversity that encompasses dimensions such as emotional regulation and cognitive function.

The Joubert Syndrome-associated Missense Mutation (V443D) in the Abelson-helper Integration Site 1 (AHI1) Protein Alters Its Localization and Protein-Protein Interactions

Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.

Expression changes of hypothalamic Ahi1 in mice brain: implication in sensing insulin signaling

Growing evidence suggests that the brain, in particular the hypothalamus, directly senses hormones and nutrients to initiate feeding behavior and metabolic responses in the control of energy homeostasis. However, the molecular mechanisms underlying this important process have remained largely unknown. Our study provides the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a sensor of insulin signaling in the hypothalamus. We found that fasting increased the expression of hypothalamic Ahi1 which was accompanied by lower levels of circulating insulin compared with satiated mice, while re-feeding decreased the expression of hypothalamic Ahi1 which was accompanied by higher levels of circulating insulin. We also found the up-regulated expression of hypothalamic Ahi1 in high-fat induced obese mice, db/db mice, and streptozotocin induced diabetic mice. In addition, we demonstrated that insulin could decrease the expression of Ahi1 in neuroblastoma cell line N18TG2. Taken together, our results indicate that hypothalamic Ahi1 functions as a sensor of insulin signaling.

Hypothalamic Ahi1 Mediates Feeding Behavior through Interaction with 5-HT2C Receptor

It is indicated that there are important molecules interacting with brain nervous systems to regulate feeding and energy balance by influencing the signaling pathways of these systems, but relatively few of the critical players have been identified. In the present study, we provide the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a mediator of feeding behavior through interaction with serotonin receptor 2C (5-HT(2C)R), known for its critical role in feeding and appetite control. First, we demonstrated the co-localization and interaction between hypothalamic Ahi1 and 5-HT(2C)R. Ahi1 promoted the degradation of 5-HT(2C)R through the lysosomal pathway. Then, we investigated the effects of fasting on the expression of hypothalamic Ahi1 and 5-HT(2C)R. Fasting resulted in an increased Ahi1 expression and a concomitant decreased expression of 5-HT(2C)R. Knockdown of hypothalamic Ahi1 led to a concomitant increased expression of 5-HT(2C)R and a decrease of food intake and body weight. Last, we found that Ahi1 could regulate the expression of neuropeptide Y and proopiomelanocortin. Taken together, our results indicate that Ahi1 mediates feeding behavior by interacting with 5-HT(2C)R to modulate the serotonin signaling pathway.

AHI1 gene expression levels and BCR-ABL1T315I mutations in chronic myeloid leukemia patients

With the availability of molecular monitoring of BCR-ABL1 and the use oftyrosine kinase inhibitors, treatment in chronic myeloid leukemia (CML)is now molecularly focused. Eighty-three samples taken at different time pointsfrom 38 CML patients; were subjected to T315I mutation analysis and gene expressionanalysis of AHI1; a novel gene that is thought to have arole in both BCR-ABL1 mediated leukemic transformation and response to tyrosinekinase inhibitors. Only one patient (2·63%) harbouredthe T315I mutation. While no significant difference in AHI1expression was observed between newly diagnosed CML samples and non-CML controls;CML samples under imatinib therapy had levels significantly higher than bothnewly diagnosed samples and controls. In the first 6 months of imatinib therapy, AHI1expression was found to increase and then gradually decrease. There was nosignificant difference between imatinib responders and non-responders, whiledasatinib caused significantly lower AHI1 levels. It is proposedthat the change in AHI1 expression during CML therapy mightbe under the control of mechanisms independent from BCR-ABL1. AHI1mediated signalling could be better understood by analyzing AHI1gene expression levels in a greater number of patients and concurrently investigatingJAK/STAT and Src family kinases pathways.

Neuronal Abelson helper integration site-1 (Ahi1) deficiency in mice alters TrkB signaling with a depressive phenotype

Recent studies suggest that the human Abelson helper integration site-1 (AHI1) gene on chromosome 6 is associated with susceptibility to schizophrenia and autism, two common neuropsychological disorders with depression symptoms. Mouse Ahi1 protein is abundant in the hypothalamus and amygdala, which are important brain regions for controlling emotion. However, the neuronal function of Ahi1 remains unclear. With the Cre-loxP system, we created a mouse model that selectively reduces Ahi1 expression in neuronal cells. Mice with neuronal Ahi1 deficiency show reduced TrkB level in the brain and depressive phenotypes, which can be alleviated by antidepressant drugs or by overexpression of TrkB in the amygdala. Ahi1 deficiency promotes the degradation of endocytic TrkB and reduces TrkB signaling in neuronal cells. Our findings suggest that impaired endocytic sorting and increased degradation of TrkB can induce depression and that this impaired pathway may serve as a previously uncharacterized therapeutic target for depression.

Ahi1, whose human ortholog is mutated in Joubert syndrome, is required for Rab8a localization, ciliogenesis and vesicle trafficking

The primary non-motile cilium, a membrane-ensheathed, microtubule-bundled organelle, extends from virtually all cells and is important for development. Normal functioning of the cilium requires proper axoneme assembly, membrane biogenesis and ciliary protein localization, in tight coordination with the intraflagellar transport system and vesicular trafficking. Disruptions at any level can induce severe alterations in cell function, giving rise to a myriad of human genetic diseases known as ciliopathies. Here we show that the Abelson helper integration site 1 (Ahi1) gene, whose human ortholog is mutated in Joubert syndrome, regulates cilium formation via its interaction with Rab8a, a small GTPase critical for polarized membrane trafficking. We find that the Ahi1 protein localizes to a single centriole, the mother centriole, which becomes the basal body of the primary cilium. In order to determine whether Ahi1 functions in ciliogenesis, loss of function analysis of Ahi1 was performed in cell culture models of ciliogenesis. Knockdown of Ahi1 expression by shRNAi in cells or targeted deletion of Ahi1 (Ahi1 knockout mouse) leads to impairments in ciliogenesis. In Ahi1-knockdown cells, Rab8a is destabilized and does not properly localize to the basal body. Since Rab8a is implicated in vesicular trafficking, we next examined this process in Ahi1-knockdown cells. Defects in the trafficking of endocytic vesicles from the plasma membrane to the Golgi and back to the plasma membrane were observed in Ahi1-knockdown cells. Overall, our data indicate that the distribution and functioning of Rab8a is regulated by Ahi1, not only affecting cilium formation, but also vesicle transport.

Identification of tyrosine kinase, HCK, and tumor suppressor, BIN1, as potential mediators of AHI-1 oncogene in primary and transformed CTCL cells

AbstractAHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4+CD7− Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1–mediated transformation, microarray analysis was performed to identify differentially expressed genes in AHI-1–suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4+CD7− Sezary cells from Sezary syndrome patients. HCK and BIN1 emerged as new candidate cooperative genes, with differential protein expression, which correlates with observed transcript changes. Interestingly, changes in HCK phosphorylation and biologic response to its inhibitor, dasatinib, were observed in AHI-1–suppressed or –overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells, which also exhibit differential MYC protein expression. In addition, aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1–mediated leukemic transformation of human CTCL cells.

AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells

Chronic myeloid leukemia (CML) represents the first human malignancy successfully treated with a tyrosine kinase inhibitor (TKI; imatinib). However, early relapses and the emergence of imatinib-resistant disease are problematic. Evidence suggests that imatinib and other inhibitors may not effectively eradicate leukemic stem/progenitor cells, and that combination therapy directed to complimentary targets may improve treatment. Abelson helper integration site 1 (Ahi-1)/AHI-1 is a novel oncogene that is highly deregulated in CML stem/progenitor cells where levels of BCR-ABL transcripts are also elevated. Here, we demonstrate that overexpression of Ahi-1/AHI-1 in murine and human hematopoietic cells confer growth advantages in vitro and induce leukemia in vivo, enhancing effects of BCR-ABL. Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL–transduced lin−CD34+ human cord blood cells and primary CML stem/progenitor cells reduces their growth autonomy in vitro. Interestingly, coexpression of Ahi-1 in BCR-ABL–inducible cells reverses growth deficiencies exhibited by BCR-ABL down-regulation and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2–STAT5. Moreover, we identified an AHI-1–BCR-ABL–JAK2 interaction complex and found that modulation of AHI-1 expression regulates phosphorylation of BCR-ABL and JAK2–STAT5 in CML cells. Importantly, this complex mediates TKI response/resistance of CML stem/progenitor cells. These studies implicate AHI-1 as a potential therapeutic target downstream of BCR-ABL in CML.

Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.

Interaction of a Novel Signaling Protein (AHI-1) with BCR-ABL Modulates BCR-ABL Transforming Activity and Imatinib Response of Chronic Myeloid Leukemia Progenitor Cells.

Ahi-1 (Abelson helper integration site 1) is a novel gene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma as a candidate cooperate oncogene. The Ahi-1 protein has a SH3 domain, multiple SH3 binding sites and WD-repeat domains, suggesting novel signaling activities. We recently demonstrated that Ahi-1/AHI-1 transcript levels are highly deregulated in human leukemic cells, particularly in highly enriched populations of BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML) where levels of BCR-ABL transcripts are also elevated. Interestingly, overexpression of Ahi-1 alone in primitive murine hematopotic cells confers a proliferative advantage in vitro and induces a lethal leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that drives CML pathogenesis. To investigate whether deregulated expression of AHI-1 may directly contribute to BCR-ABL-mediated transformation and altered signaling pathways in human CML, 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, P< 0.01) and in single cell cultures (2-fold, P< 0.05) by comparison to cells transduced with a control vector. Importantly, lentiviral-mediated suppression of AHI-1 in BCR-ABL transduced primitive human cord blood (CB) cells (lin−CD34+) further showed a significant reduction of up to 10-fold (P< 0.01) in colony-forming cell (CFC) output and up to 5-fold (P< 0.01) in liquid suspension cultures as compared to BCR-ABL transduced cells alone. In addition, suppression of AHI-1 expression also resulted in a reduction in CFC output in lin−CD34+ stem/progenitor cells from CML patients (n=3). Strikingly, co-expression of Ahi-1 in BCR-ABL inducible BaF3 cells immediately reverses growth deficiencies exhibited by down-regulation of BCR-ABL in both semi-solid cultures (>50-fold) and in liquid suspension cultures in the absence of IL-3 (>30-fold), and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2/STAT5. Moreover, a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels has now been identified in CML cells; this interaction complex is also associated with a 120 kDa tyrosine phosphorylated protein. In addition, significant changes in imatinib (IM, an inhibitor of the BCR-ABL) response have been further demonstrated in Ahi-1 transduced BCR-ABL inducible BaF3 cells (P< 0.01) and in AHI-1 suppressed CML stem/progenitor cells (n=3). These findings provide strong evidence that Ahi-1/AHI-1 interacts with BCR-ABL to form an interaction complex and that this interaction complex further modulates altered BCR-ABL signaling and transforming activities of CML progenitor cells, including their response to IM.

Microarray Identification of Differentially Expressed Genes in AHI-1- Mediated Leukemic Transformation of Human Cutaneous T-Cell Lymphoma Cells.

Abelson helper integration site 1 (Ahi-1) is a recently identified oncogene which is often the target of provirus insertional mutagenesis in murine leukemias and lymphomas. Ahi-1/AHI-1 encodes a unique cell signaling protein with a SH3 domain, multiple SH3 binding sites, seven WD40 repeats and a number of potential tyrosine kinase phosphorylation sites. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102) where 40-fold up-regulation of AHI-1 transcripts is seen. Hut78 cells are derived from a patient with Sezary syndrome, a common leukemic variant of human CTCL. Interestingly, we have recently demonstrated that aberrant expression of AHI-1 at both RNA and protein levels is found in CD4+CD7− leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells reduces autocrine production of interleukin (IL)-2, IL-4 and tumor necrosis factor-alpha (TNFα) and normalizes their transforming activity both in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, Microarray analysis was performed using the Affymetrix Human Genome U133 plus 2.0 Arrays which contains over 47,000 transcripts (54,330 probes). Comparative analysis of six RNA samples from AHI-1/sh4 cells (knockdown of AHI-1) and five samples from Hut 78 and Hut 78 cells transduced with a control vector (RPG) demonstrated that 101 genes (119 probes) were statistically differentially expressed with fold changes > or < 2 at p-values ≤ 0.001, using a DNA-Chip Analyzer (dChip). With the same selection criteria, the Linear Model for Microarray Data (Limma) analysis initially listed 239 genes (283 probes). Once the p-values for these genes were adjusted, using the approach of Benjamini and Hochberg, the list was refined to 27 genes (33 probes). After evaluation of differentially expressed genes selected by both dChip and Limma analyses, 15 up-regulated genes (fold change range: 2.2–11) and 6 down-regulated genes (fold change range: 2.1–8.3) in AHI-1 suppressed Hut 78 cells as compared to control cells, were chosen for further analysis. The expression patterns of these 21 genes identified by Microarray analyses were confirmed by quantitative real-time RT-PCR (fold change range: 6.7 higher to 33.3 lower, p≤0.001). Significant down-regulation of AHI-1 itself (p≤0.001) was confirmed in all six AHI-1/sh4 RNA samples studied. Interestingly, functional grouping of differentially expressed genes in AHI-1/sh4 cells shows several are involved in signal transduction (BRDG1, HCK, and REPS2), cell cycle control (CCNG2, CDKN1C, and PDCD6), cell proliferation and differentiation (BIN1 and MLLT11) and mRNA stability (ELAVL1). Additionally, our observed deregulated expression of NKG7 has also recently been documented in both Sezary syndrome and mycosis fungoides patient samples. Further, candidate tumor suppressor genes (BIN1 and CDKN1C) were found to be highly elevated in AHI-1/sh4 cells where AHI-1 expression is stably inhibited. These findings indicate that we have identified several new differentially expressed genes that may play critical roles in alteration of T-cell signaling in AHI-1 mediated leukemic transformation of human CTCL cells.

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.

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.

Evidence for an oncogenic role of AHI-1 in Sezary syndrome, a leukemic variant of human cutaneous T-cell lymphomas

Ahi-1 (Abelson helper integration site 1) is a novel gene frequently activated by provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in human leukemia cells, particularly in cutaneous T-cell lymphoma cell lines where increases in AHI-1 transcripts of 40-fold are seen. To test directly whether deregulated expression of AHI-1 contributes to their transformed properties, knockdown of AHI-1 expression in Hut78 cells, a cell line derived from a patient with Sezary syndrome (SS), was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited expression of AHI-1 and its isoforms in transduced cells by 80% and also reduced autocrine production of interleukin (IL)-2, IL-4 and tumor necrosis factor-alpha (TNFalpha) by up to 85%. It further significantly reduced their growth factor independence in vitro and the ability to produce tumors in immunodeficient mice. Interestingly, aberrant expression of AHI-1, particularly truncated isoforms, was present in CD4+CD7- Sezary cells from some patients with SS. Elevated expression of IL-2 and TNFalpha was also found in these cells. These findings provide strong evidence of the oncogenic activity of AHI-1 in human leukemogenesis and demonstrate that its deregulation may contribute to the development of SS.

Stable Suppression of a Novel Oncogene, AHI-1, in Human Cutaneous T-Cell Leukemia Cells Normalizes Its Transforming Activity In Vitro and In Vivo and Aberrant Expression of AHI-1 Is Also Present in Leukemic Sezary Cells from Patients with Sezary Syndrome.

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.

Forced Overexpression of Ahi-1 Confers a Proliferative Advantage on Primitive Murine Hematopoietic Cells and Enhances the Effects of BCR-ABL.

Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.