Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane association and depletes Notch1 mRNA. GGDPS inhibition increases phosphorylation of histone H2A.X, and inhibitors of ataxia telangiectasia-mutated kinase (ATM) mitigate GGDPS inhibitor-induced apoptosis. GGDPS inhibition also influences c-abl activity downstream of caspases, and inhibitors of these enzymes prevent GGDPS inhibitor-induced apoptosis. Surprisingly, induction of apoptosis by GGDPS inhibition is reduced by co-treatment with γ-secretase inhibitors. While inhibitors of γ-secretase deplete one specific form of the Notch1 intracellular domain (NICD), they also increase Notch1 mRNA expression and increase alternate forms of Notch1 protein expression in cells treated with a GGDPS inhibitor. Furthermore, inhibitors of γ-secretase and ATM increase Notch1 mRNA stability independent of GGDPS inhibition. These results provide a model by which T-ALL cells use Notch1 to avoid DNA-damage-induced apoptosis, and can be overcome by inhibition of GGDPS through effects on Notch1 expression and its subsequent response.
The properly programmed “stemness” of tissue specific stem cells, including adipose tissue stem cells (ASCs), is the core feature for their function of tissue regeneration. Although ASCs harbor great potential for clinical application, it is poorly understood how ASCs respond to obesity, an affliction now affect over one third of the national population and health risks of cardiovascular diseases. We recently identified a set of cell surface markers to enrich ASCs from murine visceral adipose tissue. We observed that adipose tissue stromal cells bearing CD45 - CD31 - Sca-1 + CD49f - CD34 + were highly enriched for cells with sphere-initiation and multilineage differentiation capacity. Interestingly, the frequency of ASCs was markedly reduced in obese visceral stroma and their capability to derive multilineage progeny were severely impaired, compared to those in lean mice. Given the heterogeneity of isolated ASCs, we performed single cell sequencing analysis to understand mechanisms underlying the impaired “stemness” of obese ASCs. Freshly isolated ASCs from obese or age matched lean mice were subjected to single cell library generation (10X Genomics), followed by high-throughput sequencing. An average of 5500 UMI (unique molecular index) were detected in each cell and a total of 18,835 genes were detected in each sample (mapped to mm10). Interestingly, ASCs isolated from lean or obese adipose tissue displayed distinct pattern after K-mean clustering. Further, functional analysis revealed that critical networks in ASCs are significantly altered under obese conditions, including suppressed adipogenic program initiation and impaired impaired antioxidative defense. Further, multiple signaling networks triggered by both innate and adaptive immune responses displayed high activation Z-score in obese ASCs than that in lean ASCs. Overall, our results suggested that obesity severely impaired ASCs “stemness”, which may be attributed to multiple factors in obese adipose tissue, including the proinflammatory microenvironment. Further analyses of these profiles will provide critical information on how obesity alters the “stemness” program in ASCs and how to “revive” obese ASCs tissue regenerative ability.
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