6929

TCF3

E2A|E47|ITF1|TCF-3|VDIR|bHLHb21;transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47);TCF3;transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)

VDR interacting repressor|class B basic helix-loop-helix protein 21|helix-loop-helix protein HE47|immunoglobulin transcription factor 1|kappa-E2-binding factor|negative vitamin D response element-binding protein|transcription factor E2-alpha|transcription

19p13.3

protein-coding

The chimeric transcription factor E2a-Pbx1 is expressed as a result of the 1;19 chromosomal translocation in some 5% of cases of pediatric acute lymphoblastic leukemia. We investigated the biological and transcriptional consequences of forced expression of E2a-Pbx1 in the interleukin-3 (IL-3) dependent, bone marrow-derived cell line Ba/F3. We show that forced expression of E2a-Pbx1 induces apoptosis in Ba/F3 cells without apparent effects on cell cycle progression. This pro-apoptotic effect is enhanced on cytokine deprivation. Furthermore, using cDNA representational difference analysis (RDA), we show thatthese cellular effects are associated with marked induction of the gene NDRG1, which was previously identified as a target of transcriptional repression by N-myc and induction by the tumor suppressor protein p53. We identify a portion of the NDRG1 promoter capable of mediating transcriptional induction by E2a-Pbx1 and show that NDRG1 is also induced on simple IL-3 deprivation of BaF3 cells. Although we show that E2a-Pbx1 induction of NDRG1 is not impaired as a result oftargeting p53 using HPV E6, and therefore does not appear to be p53-dependent, our results overall are consistent with the notion that induction of NDRG1 by E2a-Pbx1 may represent part of an apoptotic or cytostatic cellular response to oncogene activation.

The t(1;19) translocation that characterizes 25% of pediatric pre-B cell acute lymphoblastic leukemias (pre-B ALL) produces a chimeric gene, joining 5' sequences that encode a transcriptional activator domain of E2A with 3' sequences that, in part, encode a homeo box domain of a new gene called pbx1. Two E2A-pbx1transcripts have been cloned. They encode the putative fusion proteins, p85(E2A-Pbx1) and p77(E2A-Pbx1), which differ in Pbx1 sequences alone, containing unique carboxyl termini whose sequences diverge after the Pbx1 homeo box. In this study, an antiserum to Pbx1 was used to investigate the identity and abundance of E2A-Pbx1 fusion proteins in both the pre-B ALL cell line, 697, and in cryopreserved leukemic bone marrow cells, obtained from six children with t(1;19)-positive pre-B ALL. Five species of E2A-Pbx1 proteins were identified inall cells containing t(1;19), two of which were indistinguishable from in vitro-translated p85(E2A-Pbx1) and p77(E2A-Pbx1). To assess the biological properties of p85(E2A-Pbx1) and p77(E2A-Pbx1) in fibroblasts, the cDNAs encodingthese proteins were cloned into retroviral vectors, and each was introduced intoNIH-3T3 cells. Both p85(E2A-Pbx1) and p77(E2A-Pbx1) are localized in the nucleus, and expression of either resulted in malignant conversion of NIH-3T3 cells as assayed by tumor formation in nude mice. When scored by focus formation, density-independent growth, and growth in agar assays, p77(E2A-Pbx1) was a much more potent transforming protein than was p85(E2A-Pbx1). Because subtle mutations in p85(E2A-Pbx1) converted its transforming activity into that of p77(E2A-Pbx1),we suggest that a sequence within the unique carboxyl terminus of p85(E2A-Pbx1) serves to negatively regulate its biochemical activity.

Approximately 25-30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. Thechimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colony-stimulating factor receptor (G-CSFr) is specifically upregulated in pre-B cells expressing E2A-Pbx1. G-CSFr is also expressed in celllines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2A-Pbx1.

The p16INK4A (p16) and p15INK4B (p15) tumor suppressor genes are inactivated by homozygous gene deletion and p15 promoter hypermethylation in a significant proportion of childhood acute lymphoblastic leukemias (ALLs). However, little isknown about the potential association between p16/p15 gene alterations and specific genetic abnormalities implicated in leukemogenesis. The t(1;19)(q23;p13) and t(17;19)(q21-22;p13) are non-random translocations observed in childhood ALLthat create distinct E2A fusion proteins: E2A-PBX1 and E2A-HLF, respectively. Previously, a negative association was found between the t(1;19) and homozygous p16/p15 deletions. In this study we determined p16 and p15 gene status in additional t(1;19)+ ALLs and compared this incidence to that observed in t(17;19)+ ALLs. No homozygous p16 or p15 deletions were observed among 13 t(1;19)+ ALLs analyzed. In contrast, homozygous deletions of both p16 and p15 were present in two of four t(17;19)+ ALLs. None of 10 t(1;19)+ ALLs contained p15 promoter hypermethylation. In contrast, one of the two t(17;19)+ ALLs that lacked p15/p16 homozygous deletions showed probable hemizygous p15 hypermethylation. We conclude that homozygous p16 and/or p15 deletions and p15 hypermethylation rarely accompany E2A-PBX1 fusion, but occur in concert with E2A-HLF fusion in a subset of t(17;19)+ ALLs. These findings suggest that there may be different modes of cooperative leukemogenesis in ALLs associated with different E2A fusion proteins.