Quantitative Tyrosine Phosphoproteome Profiling of AXL Receptor Tyrosine Kinase Signaling Network

Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.

AXL receptor tyrosine kinase: a possible therapeutic target in acute promyelocytic leukemia

Background Acute promyelocytic leukemia (APL) is a subset of acute myeloid leukemia (AML) which is characterized by the fusion of promyelocytic leukemia PML and retinoic acid receptor- alpha (RAR-alpha) genes. All-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) have resulted in durable cytogenetic and molecular remissions in most APL patients and have altered the natural history of the disease. Most APL patients treated with ATRA and/or ATO are now anticipated to have a nearly normal life expectancy. Unfortunately, relapse and resistance to the current treatment occur in APL patients and the outcome remains dismal in these refractory patients. AXL receptor tyrosine kinase (AXL-RTK) has been shown to increase tumour burden, provide resistance to therapy and is critical to maintain cancer stem cells (CSCs) in chronic myeloid leukemia (CML) by stabilizing β-catenin in the Wnt/β-catenin signalling pathway. However, the role of AXL-RTK has not been explored in PML/RARα-positive APL. This study aimed to explore the role of AXL-RTK receptor in PML/RARα-positive APL. Methods and results By using biochemical and pharmacological approaches, here we report that targeting of AXL-RTK is related to the down-regulation of β-catenin target genes including c-myc (p < 0.001), AXIN2 (p < 0.001), and HIF1α (p < 0.01) and induction of apoptosis in PML/RARα-positive APL cell line. Resistance to all-trans retinoic acid (ATRA) was also overcomed by targeting AXL-RTK with R428 in APL (p < 0.05). Conclusion Our results provide clear evidence of the involvement of AXL-RTK in leukemogenic potential of PML/RARα-positive APL and suggest targeting of AXL-RTK in the treatment of therapy resistant APL patients.

509 Potent and selective inhibition Of AXL receptor tyrosine kinase for the treatment of cancer

BackgroundAXL receptor tyrosine kinase (AXL) is a transmembrane protein that is over-expressed in a variety of cancer and immune cells. AXL signaling has been implicated in creating an immunosuppressive tumor microenvironment (TME) through both tumor-intrinsic and immunomodulatory mechanisms1,2,3,4,5 promoting resistance to various therapies.6,7,8,9MethodsCompound inhibition potency against the kinase activity of AXL and other kinases was determined by detecting phosphorylated substrate using homogeneous time-resolved fluorescence (HTRF). Binding affinity of inhibitor to intracellular AXL kinase was determined by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRET assay. Recombinant Gas6, cancer cell lines, whole blood or isolated cells from healthy donors were used to determine the reduction in AXL-mediated signaling in-vitro. PK/PD and anti-tumor effects of selected AXL inhibitors were evaluated in murine models.ResultsAXL is highly expressed on a subset of immune cells, including DC’s, NK cells and M2 macrophages as well as fibroblasts, which contribute to a blunted anti-tumor response. Consistent with these observations, AXL is strongly associated with increased infiltration of macrophages, exhausted NK and T-cells, as well as significantly increased CD73 expression in multiple cancer types in TCGA. Additionally, AXL expression is strongly and significantly correlated with epithelial-mesenchymal transition (EMT), which further generates an immunosuppressive TME and promotes resistance to immune, targeted and chemotherapies. High expression of AXL is also strongly associated with poor survival in NSCLC, pancreatic, breast, head & neck, stomach, colorectal, ovarian & prostate adenocarcinomas, especially in the metastatic setting. AXL inhibitors that exhibit high potency in both biochemical (IC <5nM) and cell-based (IC <25nM) assays in addition to good selectivity against closely related kinases MER and TYRO3 (>90x and >25x fold selectivity, respectively) as well as other kinases involved in downstream signaling such as PI3K have been developed. Initial studies in animal models indicate a favorable pharmacokinetic profile and anti-tumor efficacy.ConclusionsAXL is a promising therapeutic target involving both immunomodulatory and tumor-intrinsic mechanisms. AXL inhibition reduces the immunosuppressive TME, enables activation of an anti-tumor immune response and renders tumors more susceptible to previously resistant therapies. Highly potent and selective AXL inhibitors have been designed, displaying biological profiles superior to those of less-selective molecules currently advancing through clinical development.ReferencesGjerdrum C, Tiron C, Hoiby T, et al. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. PNAS 2010; 107(3):1124–1129.Ying X, Chen J, Huang X, Huang P, Yan S. Effect of AXL on the epithelial-to-mesenchymal transition in non-small cell lung cancer. Exp Ther Med 2017; 14:785–790.Tsukita Y, Fujino N, Miyauchi E, et al. Axl Kinase drives immune checkpoint and chemokine signalling pathways in lung adenocarcinomas. Molecular Cancer 2019;18:24.Terry S, Abdou A, Engelsen AST, et al. AXL targeting overcomes human lung cancer cell resistance to NK- and CTL-Mediated Cytotoxicity. Cancer Immunol Res 2019;7(11):1789–1802.Scutera S, Fraone T, Musso T, et al. Survival and migration of human dendritic cells are regulated by and IFN-alpha-Inducible Axl/Gas6 Pathway. J Immunol 2009; 183:3004–3013.Wilson C, Ye X, Pham T, et al. AXL Inhibition sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res 2014;74(20):5878–5890.Ludwig KF, Du W, Sorrelle NB, et al. Small-Molecule Inhibition of Axl targets tumor immune suppression and enhances chemotherapy in pancreatic cancer. Cancer Res 2018; 78(1):246–255.Brand TM, Iida M, Stein AP, et al. AXL mediates resistance to cetuximab therapy. Cancer Res 2015;74(18):5152–5164.Guo Z, Li Y, Zhang D, Ma J. Axl Inhibition induces the antitumor response which can be further potentiated by PD-1 blockade in the mouse cancer models. Oncotarget 2017; 8(52):89761–89774