SysTAMatic mouse model toolbox to disentangle signal transduction pathways

About This Grant

TYRO3, AXL and MERTK receptor tyrosine kinases (TAM RTKs) function in maintaining homeostasis of a number of tissues and organs by eliciting two complementary but non-identical principal effector functions in cells – phagocytosis of dead cells and anti-inflammatory signaling. Understanding of TAM RTK biology comes from three decades of mouse knockout studies that revealed phenotypes spanning increased susceptibility to endotoxic shock, lymphoproliferation and broad-spectrum autoimmunity, defective spermatogenesis and sterility, retinal degeneration and vision loss, and worsening synaptogenesis index and memory in an Alzheimer’s disease (AD) model. Despite the unequivocal strength of these studies, advances have somewhat stalled as the precise effector function of TAM RTK that underlies the phenotype in each of these scenarios – phagocytosis, anti-inflammatory signaling or both – remains undetermined. We propose to generate and validate a series of TAM RTK mutant mice that are not knocked out or devoid of all functions of TAM RTKs, but instead have a single, specific effector function knocked out. RTKs are phosphorylated on a series of tyrosines (Y), each of which recruits specific adaptors and engages a particular cascade. By substitution of a single Y residue by phenylalanine (F), a specific signaling axis can be ablated while maintaining intact other downstream signaling. Pioneering studies from Hanafusa and Birge labs, and more recently from Creixell, White and Meyer labs have demonstrated that TAM RTK pleiotropic signaling and function can also be disentangled, albeit using in vitro cell culture systems. Inspired by these studies we have established the proof-of-concept that TAM RTK signaling is amenable for in vivo disentanglement by generating Mertk Y867F/Y867F YàFIN motif-mutant mice to disable MERTK-dependent phagocytosis and Mertk Y680F/ Y680F immunoreceptor tyrosine-containing inhibitory motif (ITIM) mutant which has resulted in increased inflammation. We have employed CRISPR-Cas9 to introduce YàF substitutions in mice in defined motifs in MERTK and AXL, including YàF/VIN, ITIM and immunoreceptor tyrosine-based switch motif (ITSM). We will extend this approach to engineer the corresponding mutations in converved motifs in TYRO3. We will test the concept that TAM RTK-dependent phagocytosis can be segregated from TAM RTK-dependent negative regulation of inflammation by assaying primary cells for phagocytosis of dead cells and NF-kB activation both in vitro and in vivo. We anticipate that these T/A/M YàF mouse models will comprise an essential toolkit for the next decades, to be used by us and to be shared with colleagues, to untangle TAM RTK effector functions in a vast array of physiologic system and disease models including immune resolution and chronic inflammatory diseases, photoreceptor turnover and vision loss, macrophage function and cardiovascular diseases, microglial biology and AD, magnitude of the immune response and anti-tumor immunity. This in turn would improve our mechanistic understanding and precise therapeutic targeting of TAM RTKs to overcome the current bottleneck in translating TAM-based therapeutics.