Abstract 4869: Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK
Authors: Holland Sacha J. , Owyang Alex M. , Yi Sothy , Young Chi , Braselmann Sylvia , Frances Roy , Bagos Arthur , Tai Ernest , Siu Stacey , Park Gary , Lau David , Duan Matt , Kolluri Rao , Bhamidipati Somasekhar , Darwish Ihab , Duncton Matthew , Singh Rajinder , Masuda Esteban , Payan Donald G. 
Published: July 2016

Abstract
Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome.
Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK.
Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry.
Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 &lt;100nM. Compounds were not overtly cytotoxic or antiproliferative and, importantly, do not block the activity of TLR and T cell immune effector pathways. Compounds also phenocopied the inhibition of efferocytosis observed using a MerTK blocking antibody. In vivo activity of these novel MerTK inhibitors is under investigation in PD and tumor models.
Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response.
Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.