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Background. AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer and stromal cells and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL by its ligand, growth arrest specific protein 6 (Gas6), or ligand-independent dimerization facilitates AXL phosphorylation, initiates signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective novel AXL inhibitor. Materials and Methods. The potency and specificity of the novel Arcus inhibitor against AXL and other kinases was determined using a panel of HTRF KinEASE-TK assays. Intracellular target engagement was evaluated by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRETTM intracellular kinase assay. To further assess the inhibitory effect of the novel Arcus inhibitor on AXL kinase activity, AXL autophosphorylation induced SH2 domain translocation was measured in cells using a PathHunterÒ U2OS AXL Functional assay system. Inhibition of AXL phosphorylation in cancer cells was evaluated by Western blot and levels of soluble and surface AXL were assessed by ELISA and flow cytometry, respectively. Pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were evaluated in murine models. Results. A novel, potent, reversible, and highly selective AXL kinase inhibitor has been generated by Arcus. The novel inhibitor exhibits single-digit nanomolar potency in both biochemical and cell-based assays, retains significant activity in 100% human serum and does not show significant inhibition of the major CYP450 isoforms nor the hERG potassium channel. The novel Arcus molecule inhibits AXL phosphorylation mediated by both ligand-dependent Gas6 stimulation as well as ligand-independent autophosphorylation. AXL phosphorylation and subsequent signaling leads to receptor internalization, thereby decreasing both surface AXL expression and soluble AXL levels. AXL activity is inhibited in a concentration dependent manner significantly increasing both surface AXL expression and soluble AXL levels. More importantly, significant anti-tumor efficacy is observed in combination with targeted therapies in several in vivo models. Furthermore, AXL inhibition significantly reduces tumor growth after relapse to single-agent targeted therapy. Conclusions. A novel selective inhibitor of AXL tyrosine kinase activity has been developed that demonstrates single-digit nanomolar potency and inhibition of both ligand-dependent and ligand-independent AXL phosphorylation. Significant anti-tumor activity is observed in combination with targeted therapy and upon acquired resistance in xenograft models. Selective AXL inhibition is a promising therapeutic strategy to overcome resistance to chemotherapy, targeted therapy, and/or immunotherapy. Citation Format: Susan L. Paprcka, Subhasree Sridhar, Irene M. Luu, Salema Jafri, Dillon H. Miles, Suan Liu, Ruben Flores, Shiwei Qu, Manjunath Lamani, Sriivas Paladugu, Cesar Meleza, James Wu, Hema Singh, Yu Chen, Sean Cho, Akshata Udyavar, Angelo Kaplan, Enzo Stargnaro, Xiaoning Zhao, Lixia Jin, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters, Ester Fernandez-Salas. Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P253.