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Dormant HSCs represent a reservoir for hematopoiesis and are ready to be rapidly activated when required

Dormant HSCs represent a reservoir for hematopoiesis and are ready to be rapidly activated when required.16 We identified a dual role for CDK6 in HSCsin addition to its function as a cell-cycle kinase, CDK6 downregulates by directly binding to its promoter. do harbor LSCs. knock-down in BCR-ABLp210+ Cefpiramide sodium LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses in HSCs and LSCs. Introduction A cyclin-dependent kinase (CDK) is usually a critical regulator of cell-cycle progression, becoming activated upon binding to cyclins. Cefpiramide sodium Progression through the G1 phase of the cell cycle is usually mediated by activation of the CDK4/6-cyclinD complex and subsequent phosphorylation of the retinoblastoma protein, which triggers E2F-dependent transcription.1,2 CDK4 and CDK6 show 71% amino acid homology and have been considered to fulfill largely redundant functions because only Cefpiramide sodium the simultaneous deletion of both genes leads to embryonic lethality resulting from hematopoietic defects.3,4 deficiency is characterized by subtle defects in the hematopoietic system, such as defects in thymocyte development and a reduction in erythrocyte numbers.4,5 CDK6 has been shown to have a kinase-independent function in myeloid cells, where it interacts with RUNX1 to block RUNX1-dependent transcription.6 We recently discovered a key role for CDK6 in lymphoma formation: CDK6 transcriptionally regulates and by interacting with signal transducer and activator of transcription (STAT) and AP-1 transcription factors.7 A subsequent report described CDK6 as a transcriptional coregulator of nuclear factor B p65.8 CDK6 appears to have a key role in hematopoietic tumors, where it is frequently upregulated.5,7 CDK6 has also been shown to be critical in acute myeloid leukemia (AML) and acute lymphoblastic leukemia driven by mixed lineage leukemia fusion proteins.9,10 There is considerable interest in targeting CDK4/6 in cancer therapy, and the Food and Drug Administration nominated CDK4/6 inhibitors as the breakthrough therapeutic advance in 2013. All hematopoietic cells arise from hematopoietic stem cells (HSCs), which possess the ability to self-renew and to differentiate into all blood cell lineages.11 The existence of a deeply dormant HSC (BCR-ABLp210+ LSCs fail to repopulate upon transplantation. These results identify CDK6 as a crucial player in the activation of HSC and LSCs. Methods Mouse strains All mice were maintained under pathogen-free conditions at the University of Veterinary Medicine, Vienna, Austria. (from M. Malumbres4) HMOX1 mice were kept on a C57Bl/6J background. (and cells to individual LSK populations and mature lymphoid (CD19+, CD3+) and myeloid lineages (Gr1+ Mac1+). Transcriptional profiling Total RNA was extracted from the FACS fraction A cells (Lin?Sca1+c-Kit+CD150+CD48?) using the RNeasy Micro Kit Cefpiramide sodium (Qiagen). The RNA samples were quality controlled using the Laboratory-Chip technique (Agilent Bioanalyzer) and subsequently preamplified according to the TransPlex Whole Transcriptome Amplification WTA2 protocol (Sigma-Aldrich). Samples were then fluorescently labeled by in vitro transcription using the Two-Color Microarray-Based Gene Expression Analysis kit (Agilent) and hybridized onto Mouse Gene Expression G3 60K arrays (Agilent) made up of 56,000 60-mer probes. Images were acquired and quantified by confocal scanner and software (Agilent G2505C and Feature Extraction). Expression levels were processed using standard methods of normalization and significance analysis as described previously.23 A multiple testing correction with false discovery rate adjustment by the Benjamini-Hochberg method was performed. Gene ontology and pathways were analyzed using Ontologizer,24 JASPAR,25 and GeneMANIA databases.26 Heatmaps were generated using Caleydo software.27 Statistical analysis Data are reported as mean values standard deviation and were analyzed by GraphPad. Differences were assessed for statistical significance by Student test or 1-way analysis of variance. Kaplan-Meier plots were analyzed by the log-rank test. Statistical significance is as follows: *< .05, **< .01, ***< .001, ****< .0001. Homing assay Competitive setting. and BM cells were seeded on GP+E86 retroviral producer cells (pMSCV-IRES-GFP or pMSCV-IRES-dsRed) in Dulbeccos altered Eagle medium made up of 25 ng/mL IL-3, 50 ng/mL IL-6, 50 ng/ml stem cell factor (SCF), and 7 g/mL polybrene. After 48 hours incubation, equal numbers (100?000 cells/mouse) of dsRed+ LSKs and GFP+ LSKs were injected intravenously into lethally irradiated (9 Gy) animals together with 3 106 LSKCdepleted BM carrier cells. After 18 hours, mice were euthanized and BMs were analyzed for the presence of dsRed+ and GFP+ LSKs. Noncompetitive setting. and BM was sorted by FACS, and 1 106 cells (made up of comparable numbers of LSKs) were injected into lethally irradiated mice. After 18 hours, mice were euthanized and the BM was analyzed for the presence of LSKs by FACS. Results mice. Distinct ratios of and BM cells were transplanted into lethally irradiated mice (Physique 1A). Sixteen weeks later, the contribution of and cells to the individual hematopoietic cell populations were analyzed (supplemental Physique 1A, available on the Web site). Even when only a small proportion of donor cells were Gr1+Mac1+ or CD19+ and only low numbers of CD3+ cells (which.