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AXL and resistance to quizartinib in the hematopoietic niche
the hematopoietic niche, thus confirming our in vitro and ex vivo data.
Next, we wondered whether the bone marrow niche protection of FLT3-ITD AML cells is specific to the hematopoietic niche conditions or if it would be similar in other microenvironments. We thus evaluated the impact of quizartinib on subcutaneous xenograft tumor growth using control (MV4-11 shCtrl) and AXL KD (MV4-11 shAXL) AML cells. Subcutaneous implantation of these cells into immunodeficient NSG mice resulted in solid tumors without any difference between MV4-11 shCtrl and MV4-11 shAXL cells (Online Supplementary Figure S5C). After 21 days, when tumors reached 300-500 mm3, half of the mouse cohort received AC220 by daily oral gavage (5 mg/kg/day). This resulted in complete regres- sion of the tumors after 14 days. We did not observe any difference between MV4-11 shCtrl and MV4-11 shAXL in terms of response to AC220 treatment regarding both the kinetic and intensity of tumor regression (Online Supplementary Figure S5C). The daily administration of AC220 was ceased after full tumor regression, allowing tumor regrowth 12 days after AC220 withdrawal. Again, a similar progression was observed post-relapse between MV4-11 shCtrl and MV4-11 shAXL-injected cohorts both in time to relapse and in tumor volumes, thus confirming the specific role of the hematopoietic niche (Online Supplementary Figure S5C). Similar tumor weights at sacri- fice confirmed these observations (Online Supplementary Figure S5D).
The absence of any difference in subcutaneous tumor growth between MV4-11 shCtrl and MV4-11 shAXL- injected animals shows that the instrumental role of AXL in protecting AML cells against FLT3-targeted therapy in vivo is specifically mediated by the hematopoietic niche.
Discussion
These findings show that the bone marrow hematopoi- etic niche provides specific protection for FLT3-ITD AML cells against quizartinib by multiple signaling pathways converging to AXL upregulation and activation. In addi- tion to the established role of the AXL canonical ligand GAS6, the bone marrow niche enhances AXL expression and the activity of AML cells through both STAT5-activat- ing soluble factors and local hypoxic environment.
The microenvironment was already known to promote AML cell resistance via several mechanisms such as CXCL12-CXCR4 signaling,32 adhesion molecules such as CD44 and selectins,33 vasculature by VEGF,34 and angiopoi- etins/TIE2 signaling35 and well-reviewed by Brenner et al.36 More recently, FLT3-ligand and FGF1/2 were also identi- fied as protective molecules against FLT3-TKI in vitro.37 Hypoxia, a key factor of the microenvironment, was also reported to drive pro-tumoral signaling in AML via a HIF1α/MIF/IL8 pathway that is also thought to play a role in chronic lymphocytic leukemia survival.38 Stroma has also been shown to sustain AML cell resistance to cytara- bine via activation of the AXL receptor upon AML cell- dependent education of stroma to secrete GAS6 in vitro.21 We now provide evidence that several microenvironment messages converge to enhance AXL expression and activa- tion, which sustain pro-survival signals to selectively pro- tect FLT3-ITD AML cells from quizartinib treatment in situ. Beyond demonstrating the value of AXL as a thera-
peutic target for AML burden, our data suggest a more appealing approach in which prevention of AXL expres- sion will be key to alleviating protection against treatment of the leukemic initiating cells provided by their hematopoietic niche.
The role of cytokines and growth factors inside the hematopoietic niche with regard to AML cell survival has long been debated. TPO receptor expression level has been shown in approximately 50% of AML patients.30 In addition, primary human AML engrafts with higher effi- cacy in mice in which human versions of CSF1, CSF2, IL3 and TPO genes are knocked-in into murine loci, suggest- ing the central role of these cytokines.39 The IL-3 receptor (CD123), whose expression carries a poor prognosis,40 is thought to be a marker for AML-initiating cells41 and to be closely related with FLT3-ITD mutation.42 Our data now demonstrate a new survival mechanism provided by STAT5/hypoxia in the upregulation of AXL.
AXL expression is often up-regulated in solid tumors. Its activation has been mostly observed under stress condi- tions associated with metastatic disease or in situations where tumor cells are under severe nutrient deprivation. Knowledge about the extracellular messages that regulate AXL expression is limited. However, interferon-α has been reported to enhance AXL expression in monocytes through a STAT1-dependent pathway, but the mechanism by which this is achieved has not yet been investigated.43 Our results show for the first time that the AXL gene exhibits a conserved SRE that binds cytokine-activated STAT5 which up-regulates AXL gene expression through enhanced recruitment of RNAPolII. All the cytokines stud- ied (IL-3, GM-CSF and TPO) activated both STAT5A and STAT5B, but we observed that STAT5A-selective knock- down triggered massive AXL downregulation, whatever the levels of STAT5B expression and activation (Dumas et al., 2019, personal communications). These observations sug- gest that STAT5A plays a dominant role in AXL regula- tion, an observation that fits with major regulation by STAT5A of other genes like HIF2α or PHD3 that promote HSPC and leukemia cell maintenance, as we and others have observed.44 Whether other STAT5-activating factors also enhance AXL expression and contribute to tumorige- nesis and resistance of other myeloid leukemia, non- myeloid leukemia and/or solid tumors awaits further investigation.
The current findings show that STAT5-activating factors not only enhance AXL expression but also trigger AXL activation. Indeed, TK receptors such as EGFR or VEGFR, which activate Src kinases,45 and FLT3, were shown to activate AXL.22 AXL also functions as a docking site for non-receptor kinases such as Syk and Lyn,46 which could be activated by hematopoietic niche signals. Whether other kinases mediate AXL activation remains to be stud- ied.
AXL expression is known to be modulated by O2 con- centration in various solid tumors.24,31 Our present data extend these observations to AML. Low O2 concentra- tions are a well-known BM niche hallmark.47 Hypoxia was shown to down-regulate FLT3 and FLT3-ITD signaling in AML cells.48 Under such conditions, the PI3K/AKT path- way was shown to sustain AML cell survival, rescuing FLT3 activity,49 while AXL is known to activate PI3K/AKT signaling in AML cells. We and others have previously shown that cytokine-activated STAT5 enhances HIF2α expression directly44 but also indirectly by inhibiting one
haematologica | 2019; 104(10)
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