Background: The Bcr-Abl kinase expressed in ALL drives malignant transformation of human pre-B cells and promote aberrant alternative splicing of genes involved in B-cell signaling and differentiation. The fact that Ikaros (Ik) transcription factor functions as a critical regulator of normal lymphocyte development and the observation of rapid development of leukaemia in mice expressing non-DNA binding isoforms, prompted our study to investigate whether normal Ik expression and function might be altered in Ph+ ALL. Patients and methods: Bone marrow and peripheral blood samples from 47 adult patients (median age 55 yrs, 18-76) with Ph+ ALL were collected after informed consent. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning, nucleotide sequencing and western blot analyses were performed to identify the specific Ik isoforms. Results: In 43/47 (90%) patients the Ik6 isoform, lacking all N-terminal zinc-fingers responsible for DNA-binding, was detected and in 23 of them (54%) it was the predominant isoform. Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patients expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In the patients who seemed to express by RT-PCR only full-length Ik isoforms cloning and subsequent sequencing revealed clones harboring different transcripts. We detected an in frame deletion of 10 aa (ΔKSSMPQKFLG) at the end of ex6 due to the selection of an alternative donor splice site. The consequences were impaired activation of transcription and impaired ability to form dimers. We also observed isoforms with a 60-bp insertion (TYGADDFRDFHAIIPKSFSR) immediately upstream of exon 4 at the exon 2/exon 4 junction either alone or together with the in-frame deletion. Also in this in case the aberration is due to the selection of alternative splice site which determines the skipping of exon 3 and the insertion of a sequence inside the intron 3-4 which starts with an alternative acceptor splice site. Aberrant Ik isoforms had a cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. Conclusions: Our data demonstrated that aberrant splicing of Ikaros represents a frequent feature in Ph+ ALL patients and that dominant negative Ik6 splice variant correlates with BCR-ABL mRNA levels, disease progression and relapse.
Iacobucci, I., Lonetti, A., Ottaviani, E., Cilloni, D., Messa, F., Paolini, S., et al. (2008). The Bcr-Abl kinase promotes aberrant expression of spliced oncogenic Ikaros isoforms in Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) patients treated with tyrosine kinase inhibitors.
The Bcr-Abl kinase promotes aberrant expression of spliced oncogenic Ikaros isoforms in Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) patients treated with tyrosine kinase inhibitors
LONETTI, ANNALISA;Papayannidis, Cristina;Soverini, Simona;
2008
Abstract
Background: The Bcr-Abl kinase expressed in ALL drives malignant transformation of human pre-B cells and promote aberrant alternative splicing of genes involved in B-cell signaling and differentiation. The fact that Ikaros (Ik) transcription factor functions as a critical regulator of normal lymphocyte development and the observation of rapid development of leukaemia in mice expressing non-DNA binding isoforms, prompted our study to investigate whether normal Ik expression and function might be altered in Ph+ ALL. Patients and methods: Bone marrow and peripheral blood samples from 47 adult patients (median age 55 yrs, 18-76) with Ph+ ALL were collected after informed consent. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning, nucleotide sequencing and western blot analyses were performed to identify the specific Ik isoforms. Results: In 43/47 (90%) patients the Ik6 isoform, lacking all N-terminal zinc-fingers responsible for DNA-binding, was detected and in 23 of them (54%) it was the predominant isoform. Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patients expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In the patients who seemed to express by RT-PCR only full-length Ik isoforms cloning and subsequent sequencing revealed clones harboring different transcripts. We detected an in frame deletion of 10 aa (ΔKSSMPQKFLG) at the end of ex6 due to the selection of an alternative donor splice site. The consequences were impaired activation of transcription and impaired ability to form dimers. We also observed isoforms with a 60-bp insertion (TYGADDFRDFHAIIPKSFSR) immediately upstream of exon 4 at the exon 2/exon 4 junction either alone or together with the in-frame deletion. Also in this in case the aberration is due to the selection of alternative splice site which determines the skipping of exon 3 and the insertion of a sequence inside the intron 3-4 which starts with an alternative acceptor splice site. Aberrant Ik isoforms had a cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. Conclusions: Our data demonstrated that aberrant splicing of Ikaros represents a frequent feature in Ph+ ALL patients and that dominant negative Ik6 splice variant correlates with BCR-ABL mRNA levels, disease progression and relapse.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
