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Molecular abnormalities involved in the multistep leukemogenesis of adult T-cell leukemia (ATL) remain to be clarified. Based on our integrated database, we focused on the expression patterns and levels of Ikaros family genes, Ikaros, Helios, and Aiolos, in ATL patients and HTLV-1 carriers. The results revealed profound deregulation of Helios expression, a pivotal regulator in the control of T-cell differentiation and activation. The majority of ATL samples (32/37 cases) showed abnormal splicing of Helios expression, and four cases did not express Helios. In addition, novel genomic loss in Helios locus was observed in 17/168 cases. We identified four ATL-specific short Helios isoforms and revealed their dominant-negative function. Ectopic expression of ATL-type Helios isoform as well as knockdown of normal Helios or Ikaros promoted T-cell growth. Global mRNA profiling and pathway analysis showed activation of several signaling pathways important for lymphocyte proliferation and survival. These data provide new insights into the molecular involvement of Helios function in the leukemogenesis and phenotype of ATL cells, indicating that Helios deregulation is one of the novel molecular hallmarks of ATL.
Adult T-cell leukemia (ATL) is a highly aggressive malignancy of mature CD4+ T cells and is caused by HTLV-1. After HTLV-1 infection, ATL is thought to develop following a multitude of events, including both genetic and epigenetic changes in the cells. Although many aspects of HTLV-1 biology have been elucidated, the detailed molecular mechanism of ATL leukemogenesis remains largely unknown.[1, 2] Therefore, to precisely define the comprehensive abnormalities associated with ATL leukemogenesis, we previously carried out global mRNA and miRNA profiling of ATL cells derived from a large number of patients.[3, 4] In this study, we focused on Ikaros family genes, especially Helios, on the basis of our integrated profiling of expression and gene copy number in ATL cells, which revealed the deregulated expression of this family of genes and genomic loss of Helios locus.
Ikaros family genes are specifically expressed in the hematopoietic system and play a vital role in regulation of lymphoid development and differentiation.[5-11] In addition, they are known to function as tumor suppressors during leukemog-enesis according to several genetic studies carried out in mouse models.[12-15] Recently, many studies reported the deregulated splicing of Ikaros and the deletion of Ikaros locus in several human leukemias.[16-23] These abnormalities are associated with poor prognoses.[24-27] Helios is mainly expressed in the T-cell lineage.[10, 11] Genomic changes and abnormal expression of Helios are also observed in some patients with T-cell malignancies.[18, 28-31] However, in contrast to Ikaros, the substantial impact of aberrant Helios expression remains to be elucidated because of the absence of functional information, including the target genes of Helios.
In this study, we carried out a detailed expression analysis of Ikaros family genes in a large panel of clinical samples from ATL patients and HTLV-1 carriers and consequently identified a novel molecular characteristic, that is, abnormal splicing of Helios and loss of expression, which seems to be a significant key factor in leukemogenesis affecting the regulation of T-cell proliferation.
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- Materials and Methods
- Disclosure Statement
- Supporting Information
In the present study, on the basis of the integrated analysis of ATL cells using our biomaterial bank in Japan, we revealed a novel molecular characteristic of ATL cells, which is a profound abnormality in the expression of Helios. The abnormal alternative splicing and, in some cases, loss of Helios expression appear to be a part of the basis for advantageous cell growth and survival in ATL cells. We also showed the tumor-suppressive function and target genes, as well as pathways of Helios, in mature human T cells.
Characterization of Ikaros family members revealed profound abnormalities in Helios expression in ATL cells: (i) biased and increased expression of alternatively spliced variants; (ii) suppression of Hel-1 expression; (iii) lack of Helios expression in some cases; and (iv) frequent genomic defects of the Helios locus. Our results also revealed that alternatively spliced Helios variants are expressed in PBMCs of HTLV-1 carriers, suggesting that the abnormal splicing of Helios may occur in HTLV-1-infected cells at the carrier state until progression to leukemia development. However, the genomic deletions appear to be one of the important genetic events during the latter stages of leukemia development, as they were observed only in aggressive subtypes of ATL.
The structural characteristics of the ATL-type Helios variants involve a selective lack of one or more zinc fingers in the N-terminal domain. The results of this study indicated that these variant proteins lost DNA binding activity, whereas the capacity of dimerization was preserved. Therefore, these variant proteins hindered transcriptional activities of Ikaros family proteins, showing dominant-negative effects. In addition, a part of ATL-type Helios isoform, which lacks exon 6, is linked to abnormal localization of wild-type Helios and Ikaros. We confirmed that Helios isoforms lacking exon 6 were overexpressed in primary ATL cells (Fig. S5). Interestingly, Hel-2 has reduced transcriptional suppressive activity compared with Hel-1, although it can bind to the target sequence as well as Hel-1. This is similar to a previous report, which noted that the activity of mouse Ik-2 protein for the reporter gene was remarkably lower than that of Ik-1, whereas the binding affinities of Ik-1 and Ik-2 were similar. The exon 3 skip occurred more frequently in ATL cells, compared to PBMCs from normal volunteers (Fig. S6). These results collectively indicate that all abnormalities of Helios expression, including loss of or decreased Hel-1 expression and upregulated Hel-2 and ATL-type Helios, result in abrogation of Ikaros family functions in ATL cells.
We also confirmed that Hes1, a target gene of the Notch pathway, is one of the targets of Helios as well as Ikaros.[34, 35] A recent study reported that activated Notch signaling may be important to ATL pathogenesis and that Hes1 is upregulated in ATL cells. Thus, we examined expression levels of Hes1 mRNA by quantitative RT-PCR and confirmed the upregulation in our ATL samples (Fig. S7). Hes1 has been reported to directly promote cell proliferation through the transcriptional repression of p27kip1. Taken together, our results suggest a possibility that abnormalities in Helios expression are one of the causes of Hes1 activation, which may be one of the genetic events involved in ATL leukemogenesis.
Our results show that the Hel-5 variant may have an oncogenic role, whereas the wild-type Helios, Hel-1, shows tumor suppressor-like activity. These findings are consistent with previous findings in mice. Furthermore, our description of expression profiles of stable cells followed by pathway analyses showed activation of several important pathways in lymphocytes for the regulation of proliferation, survival, and others. In particular, we discovered novel molecular cross-talk between the Ikaros family and the S1P pathway. The S1P–S1PR1 axis is known to play important roles in regulation of the immune system, apoptosis, cell cycle, and migration of lymphocytes.[40-42] Recently, activation of the S1P pathway in various diseases, including leukemia, has been reported, and the therapeutic potential of S1PR1 inhibitors was suggested. Studies of functional roles of S1P pathway activation in ATL cells are now underway in our laboratory.
In conclusion, our present study revealed a novel aspect of molecular abnormalities in ATL cells: a profound deregulation in Helios expression, which appears to play an important role in T-cell proliferation. Our experimental approaches also imply that, in addition to genetic and epigenetic abnormalities, ATL shows abnormal splicing, which has been observed in various human diseases including cancers.[43-45]