The forkhead box (FOX) transcription factor FOXM1 is ubiquitously expressed in proliferating cells. FOXM1 expression peaks at the G2/M phase of the cell cycle and its functional deficiency in mice leads to defects in mitosis. To investigate the role of FOXM1 in the cell cycle, we used synchronized hTERT-BJ1 fibroblasts to examine the cell cycle-dependent regulation of FOXM1 function. We observed that FOXM1 is localized mainly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before entry into the G2/M phase and is associated with phosphorylation of FOXM1. Consistent with the dependency of FOXM1 function on mitogenic signals, nuclear translocation of FOXM1 requires activity of the Raf/MEK/MAPK signaling pathway and is enhanced by the MAPK activator aurintricarboxylic acid. This activating effect was suppressed by the MEK1/2 inhibitor U0126. In transient reporter assays, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter. RT-PCR analysis confirmed that different cell lines and tissues predominantly express the FOXM1c transcript. Mutations of two ERK1/2 target sequences within FOXM1c completely abolish the MEK1 enhancing effect, suggesting a direct link between Raf/MEK/MAPK signaling and FOXM1 function. Importantly, inhibition of Raf/MEK/MAPK signaling by U0126 led to suppression of FOXM1 target gene expression and delayed progression through G2/M, verifying the functional relevance of FOXM1 activation by MEK1. In summary, we provide the first evidence that Raf/MEK/MAPK signaling exerts its G2/M regulatory effect via FOXM1c.

The Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether

The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.

Primary resistance to tyrosine kinase inhibitors (TKI) is a significant barrier to optimal outcomes in chronic myeloid leukemia, but little is known about the factors contributing to response heterogeneity. Using scRNA-sequencing, we identified eight statistically significant features in pretreatment bone marrow mononuclear cells which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis transformation). Employing machine-learning, we also identified LSC and NK gene expression profiles predicting imatinib response with >80% accuracy, including zero false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor (ERP) expansion in vivo (p<0.05), and a marked 2-10-fold (6.3-fold in Group A vs 1.09-fold in Group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared to myeloid progenitors (p<0.001). These LSC features were lost with progressive resistance, and in patients who transformed, MYC- and IRF1-driven inflammatory regulons became evident. Patients with MMR also exhibited a 56-fold expansion (p<0.01) of a normally rare subset of hyperfunctional adaptive-like NK cells (CD57+NKG2C+) which diminished with progressive resistance, while patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance (through HLA-E binding on target cells). Finally, we developed a parsimonious set of antibodies to validate our scRNA-seq findings. This panel will be useful in prospective studies of primary resistance, and assessing the contribution of predetermined versus acquired factors in TKI response heterogeneity.

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34(+) CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells.