Mechanism of resistance to tyrosine kinase inhibitors in
philadelphia-positive acute lymphblastic leukaemia (all): from
genetic alterations to impaired RNA editing
Creators: name: Iacobucci, Ilaria <1980>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: MED/15 Malattie del sangue

description: The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it
represents the single most significant adverse prognostic marker. Despite imatinib has led to
significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases
resistance developed quickly and disease progressed. Some mechanisms of resistance have been
widely described but the full knowledge of contributing factors, driving both the disease and
resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia
in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a
zinc finger transcription factor required for normal hemopoietic differentiation and proliferation,
particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several
proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding
isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer
partners to bind DNA.
The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed 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 46 adult patients (median age 55 yrs, 18-76) with
Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were
collected. We set up a fast, high-throughput method based on capillary electrophoresis technology
to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non
DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display
abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6
failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA
binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and
at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and
Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a
60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the
end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the
insertion and deletion were due to the selection of alternative splice donor and acceptor sites. 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. In order to mechanistically
demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase
inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line
(SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6
strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a
previously unknown link between specific molecular defects that involve the Ikaros gene and the
resistance to TKIs in Ph+ ALL patients. 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 exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice
junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of
patients was also found. Other two different single nucleotide substitutions not recognized as SNP
were observed. Some mutations were predicted by computational analyses (RESCUE approach) to
alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional
regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients
treated with TKIs. 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.