In normal haematopoiesis, multipotent hematopoietic stem cells (HSC) give rise to all blood cells. Somehow similar, although impaired, leukemia is composed of a heterogeneous population of cells, within which resides a small population of leukemia stem cells (LSC) that are responsible for the growth and propagation potential. HSC depend on an appropriate niche of cells and extra cellular substrates that control their self-renewal and progeny production. AFT024 is one of the most potent stromal cell lines able to sustain HSC expansion in vitro. Different genomics approaches were undertaken to identify genes differentially expressed by AFT024 over other stromal lineages unable to support HSC. SLIT is one of the identified genes, which is a secreted protein known to function through the Roundabout (ROBO) receptor as a chemorepellent in axon guidance and neuronal migration, as an inhibitor in leukocyte chemotaxis, and in inducing tumor angiogenesis.  Humans have three SLIT (SLIT-1, -2, -3) and ROBO genes (ROBO-1, -2, -4). Considering the similarities between HSC and leukemia stem cells, and the bone marrow (BM) angiogenic phenotype associated with AML, we sought to investigate whether SLIT/ROBO could be implicated in the crosstalk between AML cells and bone marrow stromal cells. In this study, we investigated the expression of SLIT and ROBO genes in AML and in its BM microenvironment. A multiplex RT-PCR assay was developed for specific amplification of all known human SLIT and ROBO transcripts. In general, AML do not express SLIT and ROBO with a few exceptions: (i) AML presenting with inv(16) express ROBO-4. Interestingly, we also found ROBO-4 expression in Acute Lymphoblastic Leukemia and AML associated with translocations involving the AML1 gene [t(12;21) or t(8;21)], which like inv(16) disrupt the CBF transcription factor; (ii) in 2 out of 3 patients with t(8;21), we found the expression of ROBO-1, corroborating recent microarray results from literature. SLIT and ROBO expression was also investigated in different BM stromal cell lineages and in endothelial cells. We found that BM stromal cells, BM mesenchimal cells and BM endothelial cells express ROBO-1 plus SLIT-2 and SLIT-3. Human umbilical endothelial cells (HUVEC) express ROBO-1 and ROBO-4, but only SLIT-2. ROBO and SLIT genes will be cloned and recombinant proteins produced in order to perform functional assays on in vitro co-cultured AML and BM stromal cells.

* Financial Support: FAPESP