Abstract
Abstract 912
Background:
Recent genome-wide analyses in B-precursor acute lymphoblastic leukemia (ALL) demonstrated that deletions of IKZF1, which encodes the transcription factor Ikaros, play an important role in the pathogenesis of BCR-ABL1-positive and BCR-ABL1-like acute leukemias. IKZF1 deletions have been associated with poor outcome in children with ALL but a full understanding of their biological implications and clinical significance has not yet been defined in adult patients.
Purpose and Methods:
In order to address this issue and to evaluate whether the cases harbouring IKZF1 alterations display a peculiar gene expression profile, a cohort of 144 adult de novo ALL patients (106 BCR-ABL1-postive and 38 B-progenitor ALL negative for known molecular rearrangements) were analyzed with the use of single-nucleotide–polymorphism (SNP) microarrays (Affymetrix 250K NspI and SNP 6.0), FISH for IKZF1 deletions and gene expression profiling (HGU133 Plus 2.0 gene chips, Affymetrix). Patients had a median age of 49 years (range 18-78) and were enrolled into institutional (n = 17) or GIMEMA AL Working Party (n = 121) clinical trials.
Results:
Deletions of IKZF1 were identified in 75% adult BCR-ABL1-positive and in 58% BCR-ABL1-negative ALL cases, suggesting that IKZF1 deletion is more frequent in the BCR-ABL1-positive ALL subtype (p= 0.04). FISH analysis using a pool of fosmid probes for IKZF1 and genomic quantitative PCR confirmed SNP results. Among 144 patients, the entire IKZF1 locus was deleted in 18 (13%) whereas in 84 (58%) patients only a subgroup of exons or the genomic region immediately upstream of IKZF1 was deleted. In particular, in 46 patients (32%) there was a deletion of the coding exons 4 through 7, which resulted in the expression of a dominant-negative isoform, Ik6, lacking the DNA binding domain. In 24 cases (17%) we identified the loss of exons 2 through 7, producing an Ikaros isoform lacking the translation start site. Using gene-set enrichment analysis to compare the gene-expression data from patients with IKZF1 deletion versus wild-type patients, we identified a peculiar signature irrespective of BCR-ABL1 rearrangement but dependent on IKZF1 genomic status. Indeed, it was characterized by the presence of two subgroups of genes, the expression of which was deregulated in a reciprocal fashion. One subgroup was enriched with up-regulated genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A), activation of signalling via JAK-STAT pathway (CISH, SOCS1, SOCS3, STAT3) and DNA damage (GADD45A, GADD45B, NFKBIA, the protoncogene REL). The second subgroup contained down-regulated genes, which are normally expressed during lymphocyte differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK) or are involved in DNA damage repair (MSH2, MSH6) supporting the hypothesis that B-ALL cells with IKZF1 deletions are prone to a block of B-cell differentiation and accumulation of DNA damage events. To investigate whether Ikaros transcription factor is directly involved in the regulation of putative target genes identified in gene expression analysis, cross-linking chromatin immunoprecipitation (ChIP) assay was performed in cell lines and primary ALL cells. We found that the promoters of IGGL1, CD79A, BLK, EBF1, BLC2, MSH2, BUB3, ETV6, YES1, CDKN1A (p21) and CDKN2C (p18) genes, were bound in vivo only by Ikaros full-length protein, but not by Ik6 mutant. These data strongly support a model in which Ikaros deleted isoforms loose the ability to regulate a large set of genes, many of which may play crucial roles in B-ALL development. We next investigated whether the IKZF1 deletions associated with a poor outcome in ALL patients. Univariate analysis showed that the IKZF1 deletion negatively influenced the cumulative incidence of relapse (p=0.02) and disease-free survival (p=0.04, Wilcoxon test) as confirmed by multivariate analysis.
Conclusion:
In conclusion, our findings shed light on a new subgroup of adult ALL including BCR-ABL1 positive and BCR-ABL1 negative patients and characterized by a unique signature dependent on Ikaros genomic status. Loss of normal Ikaros activity results in the activation of JAK-STAT pathway, DNA repair gene down-regulation and a block of B-cell differentiation.
Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.
Disclosures:
No relevant conflicts of interest to declare.
Homeostatic and Interferon-induced gene expression represent different states of promoter-associated transcription factor ISGF3