Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

Reversibility of acute B-cell leukaemia induced by BCR-ABL1.

Cancer is thought to arise from multiple genetic events that establish irreversible malignancy. A different mechanism might be present in certain leukaemias initiated by a chromosomal translocation. We have taken a new approach to determine if ablation of the genetic abnormality is sufficient for reversion by generating a conditional transgenic model of BCR-ABL1 (also known as BCR-ABL)-induced leukaemia. This oncogene is the result of a reciprocal translocation and is associated with different forms of leukaemia. The most common form, p210 BCR-ABL1, is found in more than 90% of patients with chronic myelogenous leukaemia (CML) and in up to 15% of adult patients with de novoacute lymphoblastic leukaemia (ALL). Efforts to establish a useful transgenic model have been hampered by embryonic lethality when the oncogene is expressed during embryogenesis, by reduced penetrance or by extremely long latency periods. One model uses the 'knock-in' approach to induce leukaemia by p190 BCR-ABL1(ref. 10). Given the limitations of models with p210, we used a different experimental approach. Lethal leukaemia developed within an acceptable time frame in all animals, and complete remission was achieved by suppression of BCR-ABL1expression, even after multiple rounds of induction and reversion. Our results demonstrate that BCR-ABL1is required for both induction and maintenance of leukaemia.

Multiple control elements are required for expression of the human CD34 gene.

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.

CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors.

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBPalpha in human granulopoiesis, we studied its expression and function in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. We show that the expression of C/EBPalpha initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBPalpha alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colony-stimulating factor receptor and secondary granule protein genes. Moreover, induced expression of C/EBPalpha in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBPalpha serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes.