Changes of 5-hydroxymethylcytosine distribution during myeloid and lymphoid differentiation of CD34+ cells

<p>BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis.</p><p>RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis.</p><p>CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.</p>

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.