Prognostic Significance and Target Potential of Axl in Acute Myeloid Leukemia

Abstract 940

Novel targets with potential to improve treatment of AML are urgently needed. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and circumstantial evidence in literature links Axl expression to AML pathobiology. However, a systematic study of the role of TAMRs and of their common ligand growth arrest-specific oncogene 6 (Gas6) in AML is missing until now. Therefore, we set out to first study the prognostic impact of Gas6 and TAMRs in a uniform cohort of cytogenetically normal AML patients (n=112) entered into the multicenter treatment trial AML SHG0199. By quantitative RT-PCR we determined pre-treatment expression levels of Gas6 and TAMR and subsequently correlated gene expression data with clinical data. We found expression of Axl, Tyro3, Mer and Gas6 in 57%, 79%, 85% and 90% of patients, respectively. Upon correlation of expression data with survival data of patients who expressed the respective gene, only Axl expression level represented an independent prognostic factor after correcting for age, sex, ECOG performance status, bone marrow blast count and white blood cell count at presentation. Patients expressing Axl above the median experienced a significantly shorter OS than patients expressing Axl below the median (28 months vs. not reached; p=0.037; median follow up 5.45 years). Axl expression levels were independent of patient age, sex and FAB type. Based on prognostic significance of Axl expression, we focused on evaluating the potential of Axl as therapeutic target in AML. First we analyzed a panel of different AML cell lines including HL-60, MV4-11, OCI-AML-5, KG-1, THP-1, U937 and Kasumi-1 for target expression. All cell lines except for Kasumi-1 showed expression of Axl and Gas6 was expressed in MV4-11, OCI-AML-5 and U937. For further studies we focused on MV4-11 and OCI-AML-5 cells. To investigate effects of Axl inhibition on leukemia pathobiology, we utilized R428, a well-characterized small molecule inhibitor of Axl. Application of R428 monotherapy inhibited proliferation of MV4-11 and OCI-AML-5 cells in a dose-dependent manner with an IC50 of 1.74 uM and 1.35 uM, respectively. Furthermore, R428 dose-dependently induced apoptosis in both cell lines. Because of the worse outcome of AML patients expressing high levels of Axl, we hypothesized that Axl might be implicated in resistance to chemotherapy. Indeed, treatment of MV4-11 and OCI-AML-5 cells with doxorubicin or cytarabine upregulated their Axl expression. Based on these data we conducted matrix experiments of R428 in combination with both drugs. Consistent with our hypothesis, R428 significantly enhanced chemosensitivity of MV4-11 and OCI-AML-5 cells. To investigate whether Gas6 is necessary for the pro-leukemic actions of Axl, we applied soluble Axl, which neutralizes Gas6, to MV4-11 and OCI-AML-5 cells, alone and in combination with chemotherapy and/or R428. In absence of Gas6 both the effect of R428 monotherapy and the additive effect of concomitant chemotherapy and Axl-inhibition were abrogated, hence the therapeutic effect of R428 specifically depends on disrupting Gas6-Axl interaction. Next, we analyzed different Axl-downstream signaling pathways in order to elucidate via which pathway R428 acts in MV4-11 and OCI-AML-5 cells. We could show that R428 dose-dependently inhibits Axl phosphorylation. Furthermore, R428 inhibits phosphorylation of Akt, which is increased in response to chemotherapy. Thus, the chemosensitizing effect of R428 is at least partly mediated by inhibiting activation of Akt. Encouraged by those data we investigated therapeutic potential of R428 in vivo by using the MV4-11 xenograft model. Mice bearing established tumors with a mean size of 294 mm³ (n=17) were randomized and subsequently treated with 75 mg/kg R428 or vehicle control twice daily by oral gavage. Treatment with R428 completely inhibited MV4-11 growth when compared to control treatment (end stage tumor size 1185 mm³ + 253 mm³vs. 184 mm³ + 46 mm³, respectively; n=8/9; p=0.0009). In summary, our data indicate that Axl represents a novel prognostic marker in CN AML. Therapeutic targeting of Axl by R428 exerts anti-leukemic activity in vitro and in vivo, thus treatment with R428 might open up novel therapeutic avenues in AML, potentially useful alone or in combination with chemotherapy.