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The vascular niche in chronic myeloid leukemia
Indeed, specific knockdown of SCL/TAL1 in K562 cells led to upregulation of CD44 (P=0.0001) (Figure 3B and Online Supplementary Figure S5A) and, conversely, overexpression of SCL/TAL1 in K562 (P<0.0001) (Figure 3C and Online Supplementary Figure S5B, C) or in human CD34+ cells (P=0.0001) (Online Supplementary Figure S5D) led to decreased expression of CD44. We performed luciferase assays on K562 cells transfected with a control plasmid or a CD44 regulatory element with SCL/TAL1-binding sites and transduced with SCL/TAL1 shRNA- or SCL/TAL1- overexpressing lentivirus, in order to test activity of the CD44 regulatory element (Online Supplementary Figure S5E). This revealed decreased activity of the CD44 regula- tory element when SCL/TAL1 was overexpressed (P=0.02) (Figure 3D and Online Supplementary Figure S5B, C) and increased activity when SCL/TAL1 was knocked down (P=0.049) (Figure 3D and Online Supplementary Figure S5A). Having shown the influence of SCL/TAL1 on the activity of the CD44 regulatory element, we tested the depen- dence of this on BCR-ABL1 in a chromatin immunopreci- pitation assay using K562 cells treated with vehicle or ima- tinib. This revealed that imatinib treatment significantly increased the binding of SCL/TAL1 to the CD44 regulato- ry element (Figure 3E and Online Supplementary Figure S6A), but not to a control region (Online Supplementary Figure S6B). Furthermore, overexpression of SCL/TAL1 (Online Supplementary Figure S7A) in BCR-ABL1+ LIC in vivo significantly prolonged survival (P=0.013) (Figure 3F), led to decreased median fluorescence intensity of CD44 on BCR-ABL1+ CD11b+ myeloid cells (P=0.031) (Figure 3G) and a trend towards decreased disease clonality (Online Supplementary Figure S7B) in murine recipients in our CML model. This resembled what we had observed when using CD44-deficient BCR-ABL1+ donor BM,8 although the effect was not as pronounced. Overexpression of SCL/TAL1 in K562 cells (Online Supplementary Figure S5B) also decreased the median fluorescence intensity of CD44 (P=0.019) (Online Supplementary Figure S5C). Taken togeth- er, these data suggest that SCL/TAL1 negatively regulates the expression of CD44 and that overexpression of SCL/TAL1 on LIC leads to prolongation of survival in CML, similar to the effect of CD44 deficiency on CML- initiating cells.8
Binding of CD44 to E-selectin influences the cell cycle of BCR-ABL1+ cells
In order to test the nature of the interaction between CD44 and the BM endothelium directly, we performed in vivo imaging of the murine calvarium of live anesthetized mice as shown in Figure 1A, B and Online Supplementary Figure S1A-D. Indeed, in vivo imaging of CD44-overex- pressing BCR-ABL1+ or BCR-ABL1+ BaF3 cells injected into Tie2-GFP mice revealed an increased contact time with the endothelium by CD44-overexpressing BCR-ABL1+ compared to BCR-ABL1+ BaF3 cells (P<0.001) (Figure 4A, B). In recapitulation of our findings with human CML cells (Figure 1B) treatment of mice with GMI-1271 decreased the contact time of BCR-ABL1+ BaF3 cells with BM endothelium (P=0.05) (Online Supplementary Figure S7C). Testing a possible influence of CD44, a marker for cancer stem cells,27 and SCL/TAL125 on cell cycle progression via binding to E-selectin, we plated BaF3 cells coexpressing BCR-ABL1 and CD44 on E-selectin in the presence of the four drugs or their combination. This demonstrated that GMI-1271 (with or without imatinib) did not alter the cell
cycle of the non-adherent fraction, either when the cells were plated on E-selectin (Figure 4C and Online Supplementary Figure S8A), or on bovine serum albumin (Online Supplementary Figure S8B). In contrast, cell cycle analysis of the non-adherent fraction of BCR-ABL1+ BaF3 cells plated on E-selectin showed an increase of cells in the G2-S-M phase and a decrease in the G0 phase of the cell cycle in the presence of GMI-1271 (P=0.03 and P=0.01, respectively) (Figure 4D and, Online Supplementary Figure S8A) or GMI-1271 plus imatinib (P=0.02) (Figure 4D). No such differences were observed in the adherent fraction (Figure 4E and Online Supplementary Figure S8A) or when the BCR-ABL1+ BaF3 cells were plated on bovine serum albumin (Online Supplementary Figure S8C). BCR-ABL1+ BaF3 cells plated on E-selectin in the presence of GMI- 1271 also exhibited increased expression of the cell cycle promoter cyclin dependent kinase (CDK) 6 (P<0.0001) (Figure 4F), a possible trend towards increased nuclear CDK4 (Online Supplementary Figure S8D, E), which has been associated with cell cycle progression, and decreased expression of the cell cycle inhibitor p16 (or cyclin dependent kinase inhibitor 2A) (P<0.0001) (Figure 4G). Consistently, GFP+ (BCR-ABL1+) Lin- c-Kit+ cells from mice with CML treated with GMI-1271 or GMI-1271 and ima- tinib had increased proportions of cells in the G2-S-M phase of the cell cycle compared to those treated with vehicle (P=0.01) or imatinib alone (P<0.0001) (Figure 4H). GFP+ (BCR-ABL1+) Lin- cells also expressed higher levels of SCL/TAL1 (P=0.04) (Online Supplementary Figure S8F) and lower levels of p16 (P=0.002) (Online Supplementary Figure S8G) when derived from CML mice treated with GMI- 1271 compared to vehicle-treated animals. CD44 was sig- nificantly more highly expressed on most GFP+ (BCR- ABL1+) progenitor fractions or CD11b+ cells compared to GFP- (BCR-ABL1-) cells (Online Supplementary Figure S8H), in line with our previous results.8 Taken together, these data suggest that CD44 is involved in mediating contact with BM endothelium. Treatment with GMI-1271 leads to non-adhesion of BCR-ABL1+ cells to E-selectin, an increase in cell cycle and a concomitant increase of Scl/Tal1 in BCR- ABL1+ cells, which leads to downregulation of CD44. These data suggest a possible role for CD44 in cell cycle regulation of BCR-ABL1+ cells.
Expression of CD44 is synergistically influenced by SCL/TAL1 and BCR-ABL1
In order to test whether the expression of CD44 and/or SCL/TAL1 may be BCR-ABL1-dependent, we treated BaF3 cells transduced with empty vector or BCR-ABL1 with imatinib.23,24 This led to a significant decrease of the median fluorescence intensity of CD44, specifically on BCR-ABL1+ compared to empty vector-transduced BaF3 cells (P=0.05) (Figure 5A and Online Supplementary Figure S9A, B). Furthermore, in the non-adherent, but not the adherent fraction, of BCR-ABL1+ BaF3 cells plated on E- selectin the median fluorescence intensity of CD44 was synergistically reduced by combined treatment with GMI- 1271 and imatinib compared to imatinib alone (P=0.05) (Figure 5B). Consistently, in non-adherent, but not adhe- rent, BCR-ABL1+ BaF3 cells plated on E-selectin, expres- sion of SCL/TAL1 and pSCL/TAL1 was significantly high- er upon treatment with GMI-1271 or GMI-1271 and ima- tinib (Online Supplementary Figure S9C). These results sug- gest that the expression of CD44 is BCR-ABL1-dependent and that reduction of CD44 expression by GMI-1271 (via
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