The Overexpression of Spliced Oncogenic Ikaros Isoforms in Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Patients Is a New Mechanism of Resistance to Tyrosine Kinase Inhibitors.

The zinc-finger transcription factor Ikaros (Ik) plays an important role in the control of differentiation and proliferation of all lymphoid lineages. By means of alternative splicing, different isoforms are encoded. Forced expression of short isoforms alters the differentiation capacities of haematopoietic progenitors arresting lineage commitment. We analyzed the differential expression pattern of Ik isoforms in Ph+ ALL patients (pts) in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 47 adult pts (median age 55 yrs, 18–76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (31 pts) were collected. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning and nucleotide sequencing were performed to identify the specific Ik isoforms. Amplification and genomic sequencing of the exon-intron splice junctions were performed in search of mutations. BCR-ABL transcript levels were monitored in each sample by real-time quantitative PCR. In 43/47 (90%) pts 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. In one patient we found a new Ik isoform in which exon 1 was directly juxtaposed to exon7, maintaining the frame. The expression of only the functional DNA-binding isoforms Ik1, Ik2 was detected in 4 (9%) pts. Western blot analysis showed that the Ik6 transcript is encoded in a smaller immunoreactive protein of approximately 37 to 40 kda which has a predominant cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon1/2 splice junction and of rs10262731 [A/G] in the exon 6/7 splice junction in 50% and 36% of pts, respectively. A variant of the rs11329346 [-/C], in which a A was substituted by a G in 16% of pts was also found. Other two different single nucleotide substitutions not recognized as SNP were observed in our samples. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.