The key role of stem cell factor/KIT signaling in the proliferation of blast cells from Down syndrome-related leukemia.

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome.

Mutations of the GATA1 gene on chromosome X have been found in almost all cases of transient myeloproliferative disorder and acute megakaryoblastic leukemia (AMKL) accompanying Down syndrome (DS). Although most GATA1 mutations lead to the expression of GATA1s lacking the N-terminal activation domain, we recently found two novel GATA1 proteins with defects in another N-terminal region. It has been suggested that loss of the N-terminal portion of GATA1 might interfere with physiological interactions with the critical megakaryocytic transcription factor RUNX1, and this would imply that GATA1s is not able to interact properly with RUNX1. However, the interaction domain of GATA1 remains controversial. In this study, we show that GATA1 binds to RUNX1 through its zinc-finger domains, and that the C-finger is indispensable for synergy with RUNX1. All of the patient-specific GATA1 mutants interacted efficiently with RUNX1 and retained their ability to act synergistically with RUNX1 on the megakaryocytic GP1balpha promoter, whereas the levels of transcriptional activities were diverse among the mutants. Thus, our data indicate that physical interaction and synergy between GATA1 and RUNX1 are retained in DS-AMKL, although it is still possible that increased RUNX1 activity plays a role in the development of leukemia in DS.

Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform.

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Cloning and expression of human B cell-specific transcription factor BACH2 mapped to chromosome 6q15.

The transcription factor Bach2, a member of the BTB-basic region leucine zipper (bZip) factor family, binds to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and the related Maf-recognition element (MARE) by forming homodimers or heterodimers with Maf-related transcription factors. Bach2 regulates transcription by binding to these elements. To understand the function in hematopoiesis, we isolated a cDNA clone for human Bach2 (BACH2) encoding a protein of 841 amino acid residues with a deduced amino acid sequence having 89.5% identity to mouse homolog. Among human hematopoietic cell lines, BACH2 is expressed abundantly only in some B-lymphocytic cell lines. RT-PCR analysis of hematopoietic cells revealed that BACH2 mRNA is expressed in primary B-cells. Enforced expression of BACH2 in a human Burkitt cell line, RAJI that does not express endogenous BACH2, resulted in marked reduction of clonogenic activity, indicating that BACH2 possesses an inhibitory effect on cell proliferation. By fluorescent in situ hybridization, the BACH2 gene was localized to chromosome 6q15. Because deletion of the long arm of chromosome 6 (6q) is one of the commonest chromosomal alterations in human B-cell lymphoma, we examined for the loss of heterozygosity (LOH) of the BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, five (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.