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P.-Y. Dumas et al.
AXL contributes to stromal cell-dependent FLT3-ITD AML resistance to FLT3-TKI
To determine how STAT5 contributes to TKI-resistance of AML cells by stromal cells, we looked for STAT5 target genes in myeloid cells. We searched for STAT5 regulated genes by conducting a comparative large-scale genome expression analysis of STAT5 knocked-down (KD) and control cells using normal human primitive CD34+ CD38– HSPC. Among the top-ranked down-regulated transcripts upon STAT5 KD, we looked for mediators that might be involved in stromal protection. AXL was the only RTK down-regulated in these STAT5 KD cells (Online Supplementary Table S3) and was the only one reported in resistance to treatment in AML.21,23 To examine the role of AXL in cancer resistance, we investigated whether there was a link between STAT5-dependent AXL regulation, stroma-dependent STAT5 tyrosine phosphorylation main- tenance and stromal protection for AC220. We found that AXL mRNA and protein were both decreased in STAT5 KD HSPC (Online Supplementary Figure S2A). Like HSPC, STAT5 KD induced AXL loss in MV4-11 and MOLM-14 FLT3-ITD or UT7 FLT3 wt AML cell lines (Figure 2A and Online Supplementary Figure S2B). STAT5 is encoded by two highly similar genes, STAT5A and STAT5B, whose combined KD led to cell death. However, STAT5A- restricted KD did not affect AML cell survival; it allowed us to perform rescue experiments. Silencing of STAT5A followed by expression of either murine STAT5A or murine STAT5B enhanced AXL expression, thus providing evidence that both STAT5 regulated AXL expression (Online Supplementary Figure S2B). Finally, we found that stromal cells enhanced both STAT5 phosphorylation and AXL expression in MV4-11 and MOLM-14 cells (Figure 2B). These activities were both prevented when cells were incubated in the presence of the STAT5 inhibitor AC-4- 130 (Online Supplementary Figure S2C). Overall, these data indicated that stroma supports STAT5 activation of AML cells, which enhances AXL expression of AML cells.
We thus analyzed the role of AXL in AML cell survival with a well-characterized and clinically investigated AXL- TKI compound, R428.28 To determine whether AXL and FLT3-ITD pro-survival activity were connected, we first determined the minimal effective AC220 and R428 dose to induce apoptosis after 48 h of incubation (Online Supplementary Figure S2D). The minimal effective AC220 dose was 1-3 nM, depending on the FLT3-ITD AML cell line, whereas for R428 it was 0.3 μM for all. Using these doses, we observed that AXL and FLT3 co-inhibition trig- gered additive apoptosis (MOLM-13) and even a weak synergistic activity (MV4-11 and MOLM-14) as compared to single drug treatment (Figure 2C).
Next, we investigated whether such effects were observed in primary AML blasts. In all primary AML sam- ples tested, HS27a stroma induced upregulation of AXL expression (AML#1, #2 in Figure 2D and Online Supplementary Table S2). However, this increase did not provide extra AML cell survival when AXL activity was inhibited. Instead, AXL and FLT3 co-inhibition induced a significantly stronger apoptosis than single drug treat- ments on primary AML blasts cultivated ex vivo on stromal cells (AML #3 to #9 in Figure 2E and Online Supplementary Table S2).
AXL is activated through the binding of its ligand GAS6, for which autocrine and paracrine activities have been reported in various contexts, including AML. Although
both stromal and leukemic cells are known to secrete GAS6, stromal secretion was 10- to 20-fold more abun- dant than leukemia ones, as detected by species-specific ELISA (Online Supplementary Figure S2E).29 We analyzed the contribution of GAS6 to the protective activity of stro- mal cells toward AC220-induced AML cell apoptosis. Protection of FLT3-ITD AML cells by stromal cells was significantly reduced yet was not suppressed in the pres- ence of AXL-Fc, a well-known GAS6-neutralizing mole- cule22 (Figure 2F). Moreover, GAS6-KD OP9 stromal cells provided weaker protection than parental OP9 (Figure 2G and Online Supplementary Figure S2F), confirming the results observed with AXL-Fc.
Taken together, using an AXL inhibitor (R428) or a GAS6-neutralizing molecule (AXL-Fc) and genetically engineered stromal cells (OP9 shGAS6), these results con- firm that AXL activity contributes to the stromal protec- tion of FLT3-ITD AML.
STAT5-activating cytokines up-regulate AXL expression and activity
Since AXL expression is associated with STAT5 activa- tion, we wondered whether and how stroma activates STAT5. We first analyzed GAS6 activity but did not detect any STAT5 tyrosine phosphorylation upon GAS6 treat- ment in AML cells. We therefore wondered whether STAT5-activating cytokines, secreted by stromal cells, could trigger AXL overexpression. Therefore, we first used the UT7-mpl AML cell line (FLT3 wild type) which is highly sensitive to GM-CSF and TPO for its growth and survival.25 Both GM-CSF and TPO activated STAT5 and enhanced AXL expression within a few hours (Figure 3A and B). These cytokines are known to activate STAT5 by binding to their receptors through subsequent activation of JAK2 kinase. In the presence of the STAT5 inhibitor pimozide (pi) or the JAK2 inhibitor-I (ji), GM-CSF and TPO no longer up-regulated AXL expression, whereas inhibition of the PI3K/AKT pathway by LY294002 (ly) had no effect (Figure 3B). We further observed that AXL acti- vation was also induced by GM-CSF and TPO in AML cells, as assessed by Tyr779 AXL phosphorylation detec- tion and by global Tyr phosphorylation in AXL immuno- precipitates (Figure 3B and C). Similar results were obtained using primary CB CD34+ HSPC in the presence of interleukin (IL)-3 or TPO. However, this was not the case with the FLT3 ligand, which did not activate STAT5 (Online Supplementary Figure S3A). These results indicated that STAT5-activating cytokines can up-regulate both AXL expression and activation in normal HSPC and FLT3 wild-type AML.
FLT3-ITD AML cells are known to co-express cytokine receptors such as IL-3 and TPO receptors, whose activity remains ill-defined.29,30 We wondered whether cytokines could trigger AXL upregulation, specifically in FLT3-ITD AML cells. Despite the presence of FLT3-ITD inhibitor AC220, IL-3 sustained STAT5 phosphorylation (Figure 3D). This activation was correlated with an increase in AXL protein expression (Figure 3E). Similarly, in primary AML samples, an IL-3/GM-CSF/TPO cytokine cocktail increased AXL RNA expression (AML#10-12 and #15-16 in Figure 3F and Online Supplementary Table S2) associated with STAT5 activation (AML#10-14 in Figure 3G and Online Supplementary Table S2).
To unravel how STAT5 up-regulates AXL transcript lev- els, we focused on the AXL genomic sequence. STAT5
2020
haematologica | 2019; 104(10)