In this issue honoring the contributions of Greg Lemke, the Earp and Graham lab teams discuss several threads in the discovery, action, signaling, and translational/clinical potential of MERTK, originally called c-mer, a member of the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases. The 30-year history of the TAM RTK family began slowly as all three members were orphan RTKs without known ligands and/or functions when discovered by three distinct alternate molecular cloning strategies in the pre-genome sequencing era. The pace of understanding their physiologic and pathophysiologic roles has accelerated over the last decade. The activation of ligands bridging externalized phosphatidylserine (PtdSer) has placed these RTKs in a myriad of processes including neurodevelopment, cancer, and autoimmunity. The field is ripe for further advancement and this article hopefully sets the stage for further understanding and therapeutic intervention. Our review will focus on progress made through the collaborations of the Earp and Graham labs over the past 30 years.

Oligodendrocyte death is common in aging and neurodegenerative disease. In these conditions, dying oligodendrocytes must be efficiently removed to allow remyelination and to prevent a feedforward degenerative cascade. Removal of this cellular debris is thought to primarily be carried out by resident microglia. To investigate the cellular dynamics underlying how microglia do this, we use a single-cell cortical demyelination model combined with longitudinal intravital imaging of dual-labeled transgenic mice. Following phagocytosis, single microglia clear the targeted oligodendrocyte and its myelin sheaths in one day via a precise, rapid, and stereotyped sequence. Deletion of the fractalkine receptor, CX3CR1, delays the microglial phagocytosis of the cell soma but has no effect on clearance of myelin sheaths. Unexpectedly, deletion of the phosphatidylserine receptor, MERTK, has no effect on oligodendrocyte or myelin sheath clearance. Thus, separate molecular signals are used to detect, engage, and clear distinct sub-compartments of dying oligodendrocytes to maintain tissue homeostasis.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.

Organ fibrosis is a devastating medical challenge that is collectively responsible for an estimated 45% of all deaths in developed countries and poses a substantial health and economic burden. The process of fibrosis has common characteristics that can occur in various organs, such as the liver, kidney, lung, and skin. Currently, there is a paucity of effective treatments available for fibrosis. Therefore, it is crucial to identify new approaches to find potential therapeutic targets. Genetic studies have shown great promise in advancing the drug development process. Mer tyrosine kinase (MERTK) was recently identified as a crucial regulator of fibrosis that specifically controls the activity of transforming growth factor beta (TGFβ). In this brief review, we provide an overview of the potential role of MERTK as a targeted and valuable approach for treating organ fibrosis.

UNASSIGNED: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition.
UNASSIGNED: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning.
UNASSIGNED: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation.
UNASSIGNED: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Greg Lemke\'s laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke\'s scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG\'s role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

Exposure to fine particulate matter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) is known to be associated with the polarization of pro-inflammatory macrophages and the development of various cardiovascular diseases. The pro-inflammatory polarization of resident cardiac macrophages (cMacs) enhances the cleavage of membrane-bound myeloid-epithelial-reproductive receptor tyrosine kinase (MerTK) and promotes the formation of soluble MerTK (solMER). This process influences the involvement of cMacs in cardiac repair, thus leading to an imbalance in cardiac homeostasis, myocardial injury, and reduced cardiac function. However, the relative impacts of PM2.5 and PAHs on human cMacs have yet to be elucidated. In this study, we aimed to investigate the effects of PM2.5 and PAH exposure on solMER in terms of myocardial injury and left ventricular (LV) systolic function in healthy children. A total of 258 children (aged three to six years) were recruited from Guiyu (an area exposed to e-waste) and Haojiang (a reference area). Mean daily PM2.5 concentration data were collected to calculate the individual chronic daily intake (CDI) of PM2.5. We determined concentrations of solMER and creatine kinase MB (CKMB) in plasma, and hydroxylated PAHs (OH-PAHs) in urine. LV systolic function was evaluated by stroke volume (SV). Higher CDI values and OH-PAH concentrations were detected in the exposed group. Plasma solMER and CKMB were higher in the exposed group and were associated with a reduced SV. Elevated CDI and 1-hydroxynaphthalene (1-OHNa) were associated with a higher solMER. Furthermore, increased solMER concentrations were associated with a lower SV and higher CKMB. CDI and 1-OHNa were positively associated with CKMB and mediated by solMER. In conclusion, exposure to PM2.5 and PAHs may lead to the pro-inflammatory polarization of cMacs and increase the risk of myocardial injury and systolic function impairment in children. Furthermore, the pro-inflammatory polarization of cMacs may mediate cardiotoxicity caused by PM2.5 and PAHs.

Adenosine A3 receptor (ADORA3) belongs to the adenosine receptor families and the role of ADORA3 in vascular dementia (VaD) is largely unexplored. The present study sought to determine the therapeutic role of ADORA3 antagonist in a mouse model of VaD.
The GSE122063 dataset was selected to screen the differential expression genes and pathways between VaD patients and controls. A mouse model of bilateral carotid artery stenosis (BCAS) was established. The cognitive functions were examined by the novel object recognition test, Y maze test, and fear of conditioning test. The white matter injury (WMI) was examined by 9.4 T MRI, western blot, and immunofluorescence staining. The mechanisms of ADORA3-regulated phagocytosis by microglia were examined using qPCR, western blot, dual immunofluorescence staining, and flow cytometry.
The expression of ADORA3 was elevated in brain tissues of VaD patients and ADORA3 was indicated as a key gene for VaD in the GSE122063. In BCAS mice, the expression of ADORA3 was predominantly elevated in microglia in the corpus callosum. ADORA3 antagonist promotes microglial phagocytosis to myelin debris by facilitating cAMP/PKA/p-CREB pathway and thereby ameliorates WMI and cognitive impairment in BCAS mice. The therapeutic effect of ADORA3 antagonist was partially reversed by the inhibition of the cAMP/PKA pathway.
ADORA3 antagonist alleviates chronic ischemic WMI by modulating myelin clearance of microglia, which may be a potential therapeutic target for the treatment of VaD.

BACKGROUND: HuoXueTongFu Formula (HXTF) is a traditional Chinese herbal formula that has been used as a supplement and alternative therapy for intraperitoneal adhesion (IA). However, its specific mechanism of action has not been fully understood.
OBJECTIVE: In surgery, IA presents an inevitable challenge, significantly impacting patients\' physical and mental well-being and increasing the financial burden. Our previous research has confirmed the preventive effects of HXTF on IA formation. However, the precise mechanism of its action still needs to be understood.
METHODS: In this study, the IA model was successfully established by using the Ischemic buttons and treated with HXTF for one week with or without Mer Tyrosine Kinase (MerTK) inhibitor. We evaluated the pharmacodynamic effect of HXTF on IA mice. The MerTK/phosphoinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-associated proteins were detected by Western blotting. Neutrophil extracellular traps (NETs) were detected by immunofluorescence. Macrophage phenotype was assessed by immunohistochemistry and flow cytometry. Inflammatory cytokines were detected by Real Time Quantitative PCR and Western blotting.
RESULTS: HXTF reduced inflammatory response and alleviated IA. HXTF significantly enhanced MerTK expression, increased the number of M2c macrophages, and decreased the formation of NETs. In addition, the MerTK/PI3K/AKT pathway was significantly activated by HXTF. However, after using MerTK inhibitors, the role of HXTF in inducing M2c macrophage through activation of the PI3K/AKT pathway was suppressed and there was no inhibitory effect on NETs formation and inflammatory responses, resulting in diminished inhibition of adhesion.
CONCLUSIONS: HXTF may improve IA by activating the MerTK/PI3K/AKT pathway to induce M2c polarization, which removes excess NETs and attenuates the inflammatory response.