Immuno-oncological Efficacy of RXDX-106, a Novel TAM (TYRO3, AXL, MER) Family Small-Molecule Kinase Inhibitor.

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models.

TAM receptors regulate multiple features of microglial physiology.

Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S.

The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.

The TAM receptors Tyro3, Axl and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and downregulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection.

A ligand for ALK.

The receptor tyrosine kinase ALK (anaplastic lymphoma kinase), as its name suggests, is aberrantly activated, mutated, or abundant in multiple cancers. Although widely studied in the context of cancer, the ligand that normally binds to and activates ALK in vertebrates has remained elusive. In this issue of Science Signaling, Murray and colleagues identify heparin as an ALK ligand.

Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl.

Diversification of TAM receptor tyrosine kinase function.

The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression.

Enveloped viruses disable innate immune responses in dendritic cells by direct activation of TAM receptors.

Upon activation by the ligands Gas6 and Protein S, Tyro3/Axl/Mer (TAM) receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection.

Genetic dissection of TAM receptor-ligand interaction in retinal pigment epithelial cell phagocytosis.

Although TAM receptor tyrosine kinases play key roles in immune regulation, cancer metastasis, and viral infection, the relative importance of the two TAM ligands-Gas6 and Protein S-has yet to be resolved in any setting in vivo. We have now performed a genetic dissection of ligand function in the retina, where the TAM receptor Mer is required for the circadian phagocytosis of photoreceptor outer segments by retinal pigment epithelial cells. This process is severely attenuated in Mer mutant mice, which leads to photoreceptor death.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells.

The precise sequence of FGF receptor autophosphorylation is kinetically driven and is disrupted by oncogenic mutations.

Autophosphorylation of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) is mediated by a sequential and precisely ordered three-stage autophosphorylation reaction. First-stage autophosphorylation of an activation loop tyrosine leads to 50- to 100-fold stimulation of kinase activity and is followed by second-stage phosphorylation of three additional tyrosine residues, which are binding sites for signaling molecules. Finally, third-stage phosphorylation of a second activation loop tyrosine leads to an additional 10-fold stimulation of FGFR1 catalytic activity.

Structural basis for reduced FGFR2 activity in LADD syndrome: Implications for FGFR autoinhibition and activation.

Mutations in fibroblast growth factor receptor 2 (FGFR2) and its ligand, FGF10, are known to cause lacrimo-auriculo-dento-digital (LADD) syndrome. Multiple gain-of-function mutations in FGF receptors have been implicated in a variety of severe skeletal disorders and in many cancers. We aimed to elucidate the mechanism by which a missense mutation in the tyrosine kinase domain of FGFR2, described in the sporadic case of LADD syndrome, leads to reduced tyrosine kinase activity.

Lacrimo-auriculo-dento-digital syndrome is caused by reduced activity of the fibroblast growth factor 10 (FGF10)-FGF receptor 2 signaling pathway.

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations.

Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction.

Tyrosine phosphorylation of cellular proteins induced by extracellular cues serves as a critical mediator in the control of a great variety of cellular processes. Here, we describe an integrated experimental approach including rapid quench methodology and ESI-LC-MS/MS as well as time-resolved ESI-MS to demonstrate that tyrosine autophosphorylation of the catalytic tyrosine kinase domain of FGF-receptor-1 (FGFR1) is mediated by a sequential and precisely ordered reaction. We also demonstrate that the rate of catalysis of two FGFR substrates is enhanced by 50- to 100-fold after autophosphorylation of Y653 in the activation loop, whereas autophosphorylation of the second site in the activation loop (Y654) results in 500- to 1,000-fold increase in the rate of substrate phosphorylation.

Mutations in different components of FGF signaling in LADD syndrome.

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.