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TWIST1 regulates normal hematopoiesis and leukemia
CSF secretion in the BM supernatant of Twist1-deleted mice, we then determined VEGF mRNA expression in BM cells and its protein concentration in BM supernatant. As expected, the results revealed a marked increase of VEGF production in Twist1-deleted mice compared with control mice (Online Supplementary Figure S8A,B). These observa- tions provide a possible explanation for the EC alterations, i.e., Twist1 deficiency in the BM microenvironment leads to increased production of G-CSF, which in turn induces the secretion of VEGF, exerting a promotive effect on the pro- liferation of EC. This effect overrides the direct inhibitory role of Twist1 deletion on EC, and results in increased num- bers of EC. As VEGF can be produced by various cell types,
the specific mechanism needs further investigation.
Our understanding of niche contributions to AML has increased tremendously over the past decade. However, most studies have focused on how the leukemic cells actively shape their microenvironment to reinforce disease progression. There are a limited number of reports show- ing that certain niche alterations can act as a driver of AML initiation or progression, without having been educated by leukemic cells.40,44,45 Our present study demonstrates that environmental deletion of Twist1, a conserved transcrip- tional factor gene, results in diverse cellular and factor alter- ations common to the microenvironmental dysregulation exhibited by AML. These alterations appear to be predis-
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Figure 6. Notch signaling is activated in leukemic stem cells and inhibition of Notch signaling partially rescues MLL-AF9-induced acute myeloid leukemia progres- sion in Twist1-deficient mice. Expression of Jagged-2 in RNA-sequencing analysis of stromal cells (CD45-Ter119-) from chimeric control (Ctrl) and knockout (KO) mice. (B-D) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Notch ligands (Dll1, Dll3, Dll4, Jagged-1 and Jagged-2) in freshly sorted mesenchymal stem cells (MSC) (B), osteolineage cells (OLC) (C) and endothelial cells (EC) (D) from chimeric Ctrl and KO mice. (E) qRT-PCR analysis of Notch receptors (Notch1-4) in freshly sorted GFP+c-Kit+Gr-1- from chimeric Ctrl and KO mice. (F) Western blot showed a significant increase of cleaved Notch1 expression in leukemic stem cells (LSC) from chimeric KO mice compared to Ctrl mice. (G) qRT-PCR analysis of downstream genes (Dtx, Hes1, Hes5, Hey1 and Hey2) regulated by the Notch pathway in freshly sorted GFP+c-Kit+Gr-1- from chimeric Ctrl and KO mice. (B-E, G) Data represent the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (Student t test). (H-J) GFP+ leukemic cells were transplanted into chimeric Ctrl and KO recipient mice. Five days later, the mice were treated daily with vehicle (dimethylsulfoxide, DMSO) or g-secretase inhibitor (DBZ) (2 mmol per kg body weight) for 10 days. The counts of white blood cells (WBC) (H), leukemic cells in peripheral blood (PB) (I), and L-GMP (IL-7R-Lin-GFP+c-KithiCD34+CD16/32hi) cells in bone marrow (BM) (J) are shown (n=4, two independent exper- iments). Column plots show the mean ± standard deviation. *P<0.05; **P<0.01, Student t test). (K) Survival curve of chimeric Ctrl and KO recipients treated with DMSO or DBZ (n=7-8, log-rank test).
haematologica | 2018; 103(12)
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