Involvement of the CEBP Gene Family in Four IGH@ Chromosomal Translocations in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL).

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are rare in BCP-ALL (comprising ~3% of cases) and involve different breakpoints from those found in lymphoma and myeloma. Cytogenetics, metaphase and interphase FISH, using a split-signal probe specific for IGH@, identified four translocations: t(14;19)(q32;q13) (n = 10), t(8;14)(q11;q32) (n = 8), t(14;14)(q11;q32) (n =3) and t(14;20)(q32;q13) (n=2), among a series of BCP-ALL patients. Sequential FISH mapping of these translocations showed that four different members of the CEBP gene family flanked these breakpoint regions. Long-distance inverse PCR with primers specific for the IGHJ6 gene segment allowed molecular cloning of many of these breakpoints and confirmed the involvement of the CEBPA (19q13, n= 4), CEBPD (8q11, n=6), CEBPE (14q11, n =2) genes. Breakpoints were located within the 3′ UTR of CEBPA and either 3′ UTR or the 5′ region of CEBPE, whereas breakpoints in 8q11 were ~30kb centromeric of CEBPD. Over-expression of the respective target genes was shown by QRT-PCR. Evidence that the IGH@ locus directly contributed to gene deregulation was demonstrated by lower expression of CEBPA in 9/17 derived BCP-ALL cell lines without t(14;19)(q32;q13). CEBP proteins have been implicated in the differentiation of myeloid progenitors and CEBPA mutations have been detected in a subset of sporadic and familial acute myeloid leukemia, suggesting that CEBPA may act as a tumor suppressor gene in these malignancies. However, no mutations within the 5′ region of CEBPA comparable to those seen in AML were detected in any cases with t(14;19)(q32;q13), although one case did exhibit deletion of six nucleotides resulting in the loss of 2 amino acids. Moreover, the deregulated expression of CEBPA and other CEBP genes in a subset of BCP-ALL indicates that they may act as dominant oncogenes in B-cell precursors. Constitutive expression of CEBPA and CEBPB in normal lymphoid progenitors has previously been shown to induce myeloid differentiation whilst CEBPE is involved in the terminal differentiation of neutrophils. We showed that expression of full-length CEBPE in the IL3-dependent B-cell precursor cell line Ba/F3 did not result in enhanced proliferation or protection from a variety of apoptotic stimuli, but instead resulted in expression of myeloid differentiation antigens. Most CEBP genes are intronless, different protein isoforms with differing functions being generated translationally. 2-D western blot analysis of nuclear extracts from the SEM BCP-ALL cell line showed selective expression of the 32kd CEBPA protein isoform only, an isoform that lacks the anti-mitotic activity of full-length CEBPA. These data implicate deregulation of the CEBP gene family via juxtaposition to the IGH@ in the pathogenesis of a subset of BCP-ALL. They demonstrate for the first time, the involvement of four members of the same gene family in a single subtype of hematological disease. The transformation of B-cell progenitors may occur through the selective expression of shorter CEBP protein isoforms that lack the ability to induce myeloid differentiation.