Mutations of the E2F4 gene in hematological malignancies having microsatellite instability

We previously analyzed for MSI using 48 childhood ALL, 17 AML, and 22 ATL samples.11-13 MSI was present in 9 (19%) childhood ALL specimens, 5 (29%) AML specimens, and 10 (45%) ATL specimens. These 24 samples with MSI were used in this study. Informed consent was obtained from the patients, their parents, or both, as appropriate. Mutation analysis of the E2F4, TGF-βRII, BAX, IGFIIR, andhMSH3 genes were performed as reported previously.6-10 A trinucleotide (AGC) repeat spanning codons 306-321, which normally encodes 13 serine residues of theE2F4 gene, was studied.6 For the TGF-βRIIgene, the poly (A) sequence (nucleotides 709-718) was analyzed.7 For the BAX gene, the poly (G) sequence in the third coding exon was analyzed.8 A fragment containing the deoxyguanine repeat (nucleotides 4030-4140) was investigated in the IGFIIR gene.9 For the hMSH3gene, the track of 8 deoxyadenine in exon 7 was assessed.10 

Three (13%) samples contained mutations of the poly (AGC) tract within E2F4 (Table 1). One sample contained an E2F4 allele that was 6 codons (18 nucleotides) shorter than the normal sequence. The 2 other samples containedE2F4 alleles that were 3 codons (9 nucleotides) longer than the normal sequence (Figure 1). By leukemia subtype, 1 of 9 (11%) childhood ALL samples and 2 of 10 (20%) ATL samples contained E2F4 mutations. The ALL patient with anE2F4 mutation had a T-cell phenotype; 2 ATL patients withE2F4 mutations had acute-type ATL. All 3 samples withE2F4 mutations had MSI at 1 or 2 loci. None of the 5 AML samples had mutations within E2F4. No mutations were found within the repeat sequence of TGF-βRII, BAX, IGFIIR, andhMSH3 genes (data not shown).

The present study has found for the first time that mutations within the important cell cycle gene E2F4 were present in hematological malignancies with MSI. E2F is a family of transcription factors, the activity of which influences the progression through the G1-S transition of the cell cycle.14 E2F consensus binding sites have been identified in the promoters of several growth-regulatory genes, including c-myc, cyclin-dependent kinases, and cyclin D1.15-17 E2F-mediated transcriptional activation is repressed through a physical association between E2F and proteins of the retinoblastoma family (pRB, p107, p130). Five different E2Fs have been identified: E2F1, E2F2, and E2F3 interact preferentially with pRB18; E2F5 with p13019; and E2F4 associates with all 3 of these proteins, especially p107 and p130 in a cell cycle–dependent manner.14,18,20,21 The p107 and p130 proteins inhibit the ability of E2F4 to transactivate genes whose promoter contains E2F binding sites, whereas the capacity of E2F4 to bind DNA is retained.22 This regulation of E2F4 is important in the normal control of cell growth. For example, an E2F4 mutation that prevented the protein from binding to p107 resulted in transformation of NIH3T3 cells, allowing the cells to form tumors.14 The poly (AGC) tract of E2F4 lies between the “marked box” region and the binding region of the retinoblastoma family of proteins. This serine repeat domain may induce transactivation of target genes.6 Even though a functional effect of a mutation of this region has not been studied, changes in this region might potentiate transcriptions of growth-stimulatory E2F4 target genes.

In this study, mutations were found only at the trinucleotide repeat sequence within the E2F4 gene, and no mutations were found at the single nucleotide repeat within the TGF-βRII, BAX, IGFIIR,and hMSH3 genes. This finding is congruent with the previous findings that no mutations of the TGF-βRII andBAX genes were found in childhood ALL.23,24Moreover, Kaneko et al25 reported that mutations of theTGF-βRII gene were not found in MDS patients with MSI. Taken together, these findings suggest that the mechanisms underlying the occurrence of such trinucleotide replication errors could differ from those of the single nucleotide frameshifts in hematological malignancies.

We thank Naoko Hayase for excellent secretarial help.