Development of an in vitro model for the simultaneous study of the efficacy and hematotoxicity of antileukemic compounds.

Hematopoietic system displays a wide spectrum of cell populations hierarchically organized in the bone marrow. Homeostasis in this system requires equilibrium between the self-renewal of the stem cells and their capacity of differentiation. Any failure on this equilibrium could lead to fatal consequences, such as the development of leukemia. Due to its rapid rate of renewal, hematopoietic tissue is a major target for antitumoral compounds and often becomes a dose limiting factor in the development of antineoplastics. Our aim was to develop an in vitro model for predicting the efficacy of antitumoral compounds on leukemic cells and their toxic effects on the healthy hematopoietic cells. The mouse myelomonocytic leukemia WEHI-3b was transduced with a lentiviral vector for expressing the green fluorescence protein. Mixed semisolid clonogenic cultures of transduced WEHI-3b and murine bone marrow cells were exposed to five pharmaceuticals: daunorubicin (positive control), atropine sulphate (negative control) and three in different stages of clinical development (trabectedin, Zalypsis(®) and PM01183). Colonies of leukemic cells were distinguishable from healthy CFU-GM under fluorescence microscope. The sensitivity of leukemic cells to daunorubicin, trabectedin, Zalypsis(®) and PM01183 was higher compared to healthy cells. The effect of a non-antitumoral compound, atropine sulphate, was the same on both populations. Our results show that this in vitro model is a valuable tool for studying the effect of antitumoral compounds in both tumoral and normal hematopoietic cells under the same toxic microenvironment and could safe time and facilitate the reduction of the number of animals used in preclinical development of pharmaceuticals.

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.