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X. Liu et al.
(Online Supplementary Figure S3A,B). We next assessed HSPC mobilization after infusion of exogenous G-CSF and found that Twist1 deletion significantly increased exoge- nous G-CSF-induced mobilization of total cells and colony-forming cells to the blood and spleen, and SLAM LSK to the spleen (Online Supplementary Figure S3C-H).
Taken together, these results demonstrate a functional role of TWIST1 in HSC homing, retention, baseline mobi- lization and stress mobilization in response to G-CSF.
Twist1 deficiency impairs hematopoietic stem cell qui- escence and self-renewal, and induces enhanced early myeloid lineage differentiation
Quiescence, self-renewal and committed differentiation are important properties of HSC, which could be con- trolled by stromal cells, extracellular matrix, cytokines and chemokines.32,33 Our study revealed that Twist1 deletion altered multiple stromal cells and the level of expression of HSC supportive factors, so we next investigated whether these HSC features were consequently changed. Immunophenotypic analysis demonstrated that Twist1 deletion resulted in a significant decrease in the number of long-term HSC (CD34-Flt3-LSK) in the BM (Online Supplementary Figure S4A). Ki67 staining revealed a signifi- cant decrease in the percentage of HSC (CD34-LSK) in G0 phase in Twist1-deleted mice, together with an increase in G1 phase (Figure 4A). Bromodeoxyuridine labeling further identified a higher frequency of proliferating cells in CD34- LSK cells from Twist1-deleted mice compared to control mice (Figure 4B). These data suggest that TWIST1 in the microenvironment plays an important role in maintaining HSC quiescence, and loss of Twist1 drives aberrant prolifer- ation of HSC.
To clarify the role of TWIST1 in HSC self-renewal, we conducted serial transplantation assays (Figure 4C). Three hundred long-term HSC (CD34-Flt3-LSK, CD45.1) from Twist1-deleted or control chimeric mice were transplanted into lethally irradiated recipients (CD45.2), together with 2×105 CD45.2+ support BM cells. Then secondary trans- plantation assays were performed 16 weeks later. Three hundred long-term HSC (CD45.1+) from the primary recip- ients were transplanted into lethally irradiated secondary recipients, together with 2×105 CD45.2+ support BM cells. Donor cells from Twist1-deleted mice showed lower engraftment capacity than control cells out to 16 weeks in both primary and secondary transplantation (Figure 4D). From the above, it can be concluded that Twist1 deficiency in niche cells impairs HSC self-renewal capacity.
We then performed FACS analysis to evaluate the dif- ferentiation capacity of HSC, and found increases in the numbers of common myeloid progenitors (CD34+CD16/32-Lin-Sca-1-c-Kit+, 1.5-fold, P=0.047) and granulocyte/macrophage progenitors (GMP: CD34+CD16/32+Lin-Sca-1-c-Kit+, 1.3-fold, P=0.022) in the BM of Twist1-deleted mice in comparison with those in controls, accompanied by decreases in the numbers of megakaryocyte/erythroid progenitors (MEP, CD34- CD16/32-Lin-Sca-1-c-Kit+, 1.5-fold, P=0.028) and common lymphoid progenitors (Lin−Sca-1lowc-KitlowIL7R+, 2.5-fold, P=0.046) (Figure 4E,F). In accordance with the change of progenitors, the proportion of mature myeloid cells (Mac1+, 1.2-fold, P=0.007; Gr-1+, 1.2-fold, P=0.008; Mac- 1+Gr-1+, 1.2-fold, P=0.011) was also significantly increased, accompanied by reduced proportions of B lymphoid cells (B220+, 1.4-fold, P=0.004) and erythrocytes (Ter119+, 1.3-
fold, P=0.008) in the BM of Twist1-deleted mice (Figure 4G,H). These results suggest that Twist1 deletion in niche cells promotes HSC differentiation into the myeloid line- age. We observed that apart from the increase of HSPC in the spleen of Twist1-deleted mice, the numbers of mature myeloid cells and erythrocytes were also increased, while the number of lymphoid cells remained unchanged (Online Supplementary Figure S4B-D), indicating the occurrence of extramedullary hematopoiesis in the spleen of Twist1- deleted mice.
Taken together, these data suggest that TWIST1 in the BM microenvironment plays a critical role in HSC mainte- nance, and Twist1 deletion impairs all the fundamental fea- tures of HSC.
Twist1 deficiency promotes progression
of MLL-AF9-induced acute myeloid leukemia
It has been reported that the MLL-AF9 AML model exhibited multiple alterations in the niche compartments, including decreased frequencies of MSC and osteoblasts, an increased number of vascular EC, and downregulated expression of Vcam1, Cxcl12, Angpt1, and Scf, together with upregulation of Opn.34 Intriguingly, these pheno- types are quite similar to the niche alterations in our Twist1-deleted mice. Considering the emerging impor- tance of the BM niche for leukemia maintenance and pro- gression, we were tempted to speculate that the altered niche in Twist1-deleted mice may play a role in the devel- opment of MLL-AF9 AML. To validate this hypothesis, we transduced WT BM c-Kit+ cells with retrovirus expressing MLL-AF9, and injected these cells into lethally irradiated WT recipient mice, in which the disease was rapidly induced with massive BM and spleen infiltration of GFP+ leukemic cells. We next injected 5×105 GFP+ spleen cells from these mice into Twist1-deleted and con- trol chimeric recipient mice (Figure 5A). Notably, the overall survival of Twist1-deleted recipients was signifi- cantly shorter than that of control recipients (Figure 5B), and Twist1-deleted recipient mice exhibited a greater infiltration of total cells and leukemic cells in the BM, peripheral blood and spleen than that of control mice (Online Supplementary Figure S5A-G).
Previous studies using MLL-AF9 AML models have established that LSC are enriched in the leukemic GMP populations (IL-7R-Lin-GFP+c-KithiCD34+CD16/32hi)34,35 or c- Kit+Gr-1−.36 We found that the infiltration of leukemic GMP cells was significantly increased in the BM and periphery of Twist1-deleted recipient mice compared to control mice (Figure 5C,D), and so was that of GFP+c-Kit+Gr-1- cells (Online Supplementary Figure S6A-C). Additionally, cell cycle analysis showed that the proportion of LSC (GFP+c- Kit+Gr-1-) in the G0 phase was decreased and that in the G1 or S/G2/M phases was increased in both BM and spleen from Twist1-deleted mice, but rates of LSC apoptosis in BM and spleen did not differ between Twist1-deleted and control mice (Online Supplementary Figure S6D-G). We then performed secondary transplants using LSC (Figure 5A), injecting 5000 GFP+c-Kit+Gr-1- cells isolated from Twist1- deleted and control mice into WT recipient mice. We found that the survival of the secondary recipients of LSC grafts from leukemic Twist1-deleted donors was signifi- cantly reduced compared to that of recipients of control LSC grafts (Figure 5E).
Taken together, these results demonstrate that the altered niche in Twist1-deleted mice accelerates the pro-
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haematologica | 2018; 103(12)