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AXL and resistance to quizartinib in the hematopoietic niche
ChIP (Figure 3I). These data indicated that STAT5 binds to the AXL gene and increases AXL promoter activity.
Taken together, these data indicated that STAT5 medi- ates stroma-dependent AXL upregulation.
Low O2 concentration enhances AXL expression
In addition to stromal cells and cytokines, the hematopoietic niche is also characterized by low O2 con- centrations, which have been linked to AXL expression in other cancer cells.24,31 We thus assessed the contribution of low O2 levels (1% O2) in this model. Interestingly, hypoxia enhanced AXL expression in both FLT3-ITD AML cell lines and primary FLT3-ITD AML blasts (Figure 4A and AML#17 in Figure 4B and Online Supplementary Table S2). Several Hypoxia-Response Elements (HRE) have been described within a 2.4 kb fragment of the AXL promoter.24 AXL transcript levels were thus assessed in hypoxic cul- ture for both AML cell lines and primary AML blasts. In all cells, hypoxia enhanced AXL RNA expression, thereby indicating that O2 levels affect AXL expression (Figure 4C and AML#18-20 in Figure 4D and Online Supplementary Table S2).
As AXL contributed to FLT3-ITD AML cell survival in normoxia (see Figure 2C), we then analyzed its involve-
ment in AML cell survival in hypoxia by using the AXL- TKI R428. In the absence of stroma, R428-induced apop- tosis was similar in hypoxia and normoxia. Conversely, in the presence of stroma, R428 triggered much stronger apoptosis under hypoxia (Online Supplementary Figure S4). Overall, these data indicate that both low O2 concentra- tions and STAT5-activating stromal cytokines, in addition to GAS6, regulate AXL activity, thus mediating stronger AXL-dependent protection of AML cells.
AXL mediates microenvironment-dependent resistance of FLT3-ITD acute myeloid leukemia cells to AC220 treatment in vivo
Having shown that AXL mediates microenvironment- dependent protection against quizartinib in vitro, we ana- lyzed the contribution of AXL in vivo. To better investigate the role of AXL on a long timescale, we generated stable AXL KD FLT3-ITD AML cells through lentivirus-mediated RNA interference (Online Supplementary Figure S5A). MV4- 11 shCtrl and MV4-11 shAXL cells were engineered to sta- bly express the firefly luciferase gene. They were xenografted by vein injection and leukemic cell engraft- ment was analyzed by bioluminescence imaging (BLI). BLI analysis did not evidence any significant difference in
AB
Figure 4. Low O2 concentration up-regulates AXL expression of FLT3-ITD acute myeloid leukemia (AML) cells. (A) MV4-11 cells were incubated for 48 hours (h) at 20% or 1% O2 with or without MS5 stromal cells as indicated; MOLM-14 cells were incubated for 48 h at 20% or 1% O2 without stroma. Immunoblot analysis of the indicated proteins was performed with β actin as a loading control. (B) Primary FLT3-ITD AML blasts (AML#17) were cultured with HS27a stromal cells for two days in normoxia (20%) and hypoxia (1%). Samples were lysed at day zero and two days after growth on stromal cells. Immunoblot analysis of the indicated proteins was performed with β actin as a loading control. (C) MV4-11 and MOLM-14 cells were incubated for 48 h at 20%, 3%, 1% O2. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of AXL mRNA was performed. Results are normalized to GUSB expression and expressed relative to 20% O2 cultured cells. (D) Primary AML blasts (n=3, AML#18-20) were incubated for 48 h at 20% and 3% O2. RT-qPCR analysis of AXL mRNA was performed as in (B). Graphs show the mean±Standard Error of Mean of results of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
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