In all, 1?g of RNA was reverse-transcribed with Transcriptor First-Strand cDNA synthesis kit (Roche)
In all, 1?g of RNA was reverse-transcribed with Transcriptor First-Strand cDNA synthesis kit (Roche). identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition TRi-1 targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and TRi-1 confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials. values were calculated using paired test. E GSEA plots showing downregulation of IL2-STAT5 components and MYC targets in KG1 and MOLM14 cell lines after R406 treatment. Data were derived from the publicly accessible dataset available from GEO at the accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE46302″,”term_id”:”46302″GSE46302. FDR: false discovery rates, ES: enrichment score, NES: normalized enrichment score. SYK signals through ERK1/2 to block differentiation of AML cells Since activated ERK1/2 phosphorylates CCAAT/enhancer-binding protein (C/EBP) on serine 21 and inhibits activity of this myeloid differentiation transcription factor23, we hypothesized that the aberrant activation of the MEK/ERK1/2 pathway through SYK might contribute to the differentiation blockade in AML cells. To test this hypothesis, we retrovirally transduced KG1 and MOLM14 cell lines with a constitutively active form of an upstream MEK1 kinase (MEK-DD)24,25 and assessed the differentiation status of cells incubated either with R406 or DMSO. Consistent with previous results, in cells expressing empty vector, R406 treatment markedly reduced the p-ERK1/2 level, enhanced superoxide production, increased CD14 surface level and expression of genes involved in myeloid maturation, and increased the number of cells with morphological signs of differentiation (Fig. ?(Fig.22 and Supplementary Fig. S2). In contrast, in MEK-DD-transduced cells, R406 only moderately reduced the level of p-ERK1/2, and MEK-DD cells treated with R406 did not exhibit features of differentiation (Fig. 2ACD and Supplementary Fig. S2). These data indicate that MEK/ERK1/2 pathway activation downstream of SYK plays an important role in differentiation arrest in AML cells. Mouse monoclonal to CD49d.K49 reacts with a-4 integrin chain, which is expressed as a heterodimer with either of b1 (CD29) or b7. The a4b1 integrin (VLA-4) is present on lymphocytes, monocytes, thymocytes, NK cells, dendritic cells, erythroblastic precursor but absent on normal red blood cells, platelets and neutrophils. The a4b1 integrin mediated binding to VCAM-1 (CD106) and the CS-1 region of fibronectin. CD49d is involved in multiple inflammatory responses through the regulation of lymphocyte migration and T cell activation; CD49d also is essential for the differentiation and traffic of hematopoietic stem cells Reduced MEK/ERK1/2 activity after R406 treatment is responsible for the induction of myeloid maturation. Open in a separate window Fig. 2 SYK signals through the MAPK/ERK1/2 pathway to block differentiation of leukemic cells.A KG1 and MOLM14 cells transduced with an empty vector or a vector containing constitutively active form of MEK1 kinase (MEK-DD) were treated with R406 (KG1: 4?M, MOLM14: 0.075?M) for 24?h; thereafter, the phosphorylation status of ERK1/2 was assessed by immunoblotting. B Transfected cells were incubated for 3 days with R406 (KG1 0.4?M, MOLM14 0.075?M), and NBT reduction was assessed. The graph shows a relative change in absorbance at 620?nm. The experiment was repeated TRi-1 twice. Bars indicate mean?+?/? SD from biological replicates (value was calculated using Students test. *value was calculated using Students test. *test. Since functionally defined LSCs in AML are characterized by a low rate of energy metabolism and low levels of reactive oxygen species, we further tested the R406 effects on sorted ROS-low AML cells10. For these experiments, we used TEX line, given its hierarchical organization similar to normal hematopoiesis and TRi-1 AML34. First, TEX cells were sorted to obtain subsets with low and high endogenous ROS levels (ROS-low and ROS-high cells). The stem-cell.