Tyro3, Axl, MerTK Research Articles

TYRO3 and EPHA2 Expression Are Dysregulated in Breast Cancer.

Receptor tyrosine kinases (RTKs) are involved in cell growth, motility, and differentiation. Deregulation of RTKs signaling is associated with tumor development and therapy resistance. Potential RTKs like TAM (TYRO3, AXL, MERTK), RON, EPH, and MET have been evaluated in many cancers like lung, prostate, and colorectal, but little is known in breast tumors. In this study, 51 luminal breast cancer tissue and 8 triple negative breast cancer (TNBC) subtypes were evaluated by qPCR for the expression of TAM, RON, EPHA2, and MET genes. Statistical analysis was performed to determine the correlation to clinical data. TYRO3 is related to tumor subtype and stage, patient's age, smoking habits, and obesity. MET expression is correlated to EPHA2 and TAM gene expression. EPHA2 expression is also related to aging and smoking habits. The expression levels of the TAM and EPHA2 genes seem to play an important role in breast cancer, being also influenced by the patient's lifestyle.

Targeting MERTK on tumour cells and macrophages: a potential intervention for sporadic and NF2-related meningioma and schwannoma tumours

Meningioma and schwannoma are common tumours of the nervous system. They occur sporadically or as part of the hereditary NF2-related schwannomatosis syndrome. There is an unmet need for new effective drug treatments for both tumour types. In this paper, we demonstrate overexpression/activation of TAM (TYRO3/AXL/MERTK) receptors (TAMs) and overexpression/release of ligand GAS6 in patient-derived meningioma tumour cells and tissue. For the first time, we reveal the formation of MERTK/TYRO3 heterocomplexes in meningioma and schwannoma tissue. We demonstrate the dependence of AXL and TYRO3 expression on MERTK in both tumour types, as well as interdependency of MERTK and AXL expression in meningioma. We show that MERTK and AXL contribute to increased proliferation and survival of meningioma and schwannoma cells, which we inhibited in vitro using the MERTK/FLT3 inhibitor UNC2025 and the AXL inhibitor BGB324. UNC2025 was effective in both tumour types with superior efficacy over BGB324. Finally, we found that TAMs are expressed by tumour-associated macrophages in meningioma and schwannoma tumours and that UNC2025 strongly depleted macrophages in both tumour types.

Co-targeting JAK1/STAT6/GAS6/TAM signaling improves chemotherapy efficacy in Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.

Phosphatidylserine and Tyro3-Axl-Mertk Receptor Tyrosine Kinase level detection in plasma and on plasma-derived extracellular vesicle surface in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.

Abstract 6341: Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE

Abstract Background: Myeloid epithelial reproductive tyrosine kinase (MERTK), a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family, is overexpressed in cancer and associated with reduced apoptosis and chemoresistance, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) (Inspirna, Inc), selectively binds to MERTK with high affinity on tumor cells, resulting in internalization and degradation of the receptor. It then induces cytotoxicity through the release of the payload, MMAE (monomethyl auristatin E), which disrupts mitosis. We hypothesize that targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: First, to determine the clinical relevance of MERTK, using the Oregon Health and Science University (OHSU) acute myeloid leukemia (AML) dataset, we divided patients into low and high MERTK mRNA expression based on mean expression and compared their overall survival. To identify the AML subtypes with the highest MERTK expression, we analyzed samples from 810 AML patients at MD Anderson using reverse phase protein arrays (RPPA), as well as 8 AML cell lines and the peripheral blood of 9 AML patients using flow cytometry. Then, the cell lines with the highest MERTK expression were treated with varying doses of RGX-019-MMAE or isotype control for 120 hours and assessed for viability with CellTiter-Glo 2.0. Likewise, we administered RGX-019-MMAE to primary cells from AML patients and determined the anti-leukemic effect. Finally, we investigated the synergistic effect of RGX-019-MMAE with venetoclax, a BCL2 inhibitor, or cytarabine, a chemotherapy agent, in vitro. Results: In the AML OHSU dataset, patients with high MERTK mRNA expression had significantly worse overall survival (p=0.02). RPPA revealed that patients with PTPN11 mutation, t (9;11), and monocytic AML subtypes had significantly higher MERTK protein expression. MERTK protein expression varied in AML cell lines and was highest in Kasumi-1 and OCI-AML3. Treatment of these two cell lines with RGX-019-MMAE resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p<0.01). Similarly, in primary AML cells with high MERTK expression, RGX-019-MMAE treatment induced significant cell death compared to the control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated with RGX-019-MMAE combined with venetoclax or cytarabine showed a synergistic cytotoxic effect in a dose-dependent manner. Conclusion: High MERTK expression in monocytic AML suggested that MERTK is a promising therapeutic target in this subtype. RGX-019-MMAE significantly induced AML cell death and produced a synergistic anti-leukemic effect in combination with chemotherapeutic agents and targeted therapy in vitro, suggesting its potential for targeting patients with high-risk AML. Citation Format: Anudishi Tyagi, Maryam Siddiqui, Isabel Kurth, Shugaku Takeda, Amanda Eckstrom, Jenny Borgman, Abhishek Maiti, Venkata Lokesh Battula. Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6341.

Targeting Myeloid Epithelial Tyrosine Kinase (MERTK) Receptor in Acute Myeloid Leukemia Using a Novel Antibody Drug Conjugate, Rgx-019-MMAE

Background: Myeloid epithelial reproductive tyrosine kinase receptor (MERTK) is a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family. It is known to be overexpressed in various solid and hematological cancers. Overexpression of MERTK is associated with reduced apoptosis, increased metastasis, and drug resistance in tumor cells, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) developed by Inspirna, selectively binds to MERTK on tumor cells, which results in internalization and degradation of the receptor, and induces cytotoxicity through the release of the payload, MMAE, which disrupts the mitosis process of actively dividing cells. Several monoclonal antibodies targeting MERTK have shown promising results against various solid cancers, however, the effect of targeting MERTK in AML progression via RGX-019-MMAE is not known. Here, we hypothesize MERTK is overexpressed in distinct subsets of AML and associated with leukemia progression, therefore, targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: To determine the clinical/prognostic relevance of MERTK, we analyzed a gene expression dataset (OHSU) and segregated the patients into low and high MERTK mRNA expression groups based on their mean expression value. To identify the specific AML subset with the highest MERTK expression, MERTK protein expression was analyzed in 818 patients with AML by performing Reverse Phase Protein Arrays (RPPA). MERTK protein expression was also measured by flow cytometry in OCI-AML3, OCI-AML2, Kasumi-1, ThP-1, Molm-13, Molm-14, MV4-11, and U937 leukemic cell lines and in the peripheral blood or bone marrow of five AML patients. Further, we treated Kasumi-1 and OCI-AML3 AML cell lines with varying doses of RGX-019-MMAE or isotype control for 72 and 120 hours and measured its effect on AML cell viability using CellTiterGlo 2.0 assay. Likewise, we treated primary cells from 5 different AML patients and determined the anti-leukemic effect of RGX-019-MMAE in primary cells. Finally, we investigated the synergistic effect of RGX-019-MMAE with Venetoclax (BCL2 inhibitor) or 5-Azacytidine in vitro. Results: Analysis of AML OHSU dataset showed that patients with high MERTK mRNA expression had significantly worse overall survival than those having low MERTK mRNA expression (p=0.02). RPPA analysis revealed significantly higher MERTK protein expression in monocytic AML subtypes, such as M4 and M5, especially in those with PTPN11 mutation or t (9;11) translocation. Moreover, in the RPPA we noted that high expression of MERTK protein levels is significantly correlated with inferior survival. This finding suggests that MERTK could serve as a therapeutic target in AML, especially in patients with monocytic subtypes of leukemia. In addition, we observed varying degrees of MERTK protein expression in AML cell lines, with Kasumi-1 and OCI-AML3 expressing the highest amount of MERTK protein. Next, treatment of Kasumi-1 and OCI-AML3 cell lines with varying doses of anti-MERTK ADC, RGX-019-MMAE, resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p<0.01), suggesting that RGX-019-MMAE inhibits AML cell proliferation. Further, we validated this finding in primary AML cells with high MERTK expression. As expected, we observed that treatment with RGX-019-MMAE induced significantly greater cell death (˜80% apoptosis) in leukemic cells than the isotype control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated in combination with RGX-019-MMAE and either Venetoclax or 5-Azacytidine showed a synergistic effect with increased cytotoxicity in a dose-dependent manner. These findings suggest that RGX-019-MMAE targets MERTK-expressing leukemic cells, sensitizes cells to chemotherapies, and promotes overall cytotoxicity. Conclusion: RGX-019-MMAE significantly induced cell death in AML cell lines and primary AML patient samples. RGX-019-MMAE, combined with chemotherapeutic agents and targeted therapy, produces a synergistic anti-leukemic effect. Our data indicates that MERTK is a potential therapeutic target in AML patients with monocytic subtypes of leukemia.

All-trans retinoic acid and dexamethasone regulate phagocytosis-related gene expression and enhance dead cell uptake in C2C12 myoblast cells

Extensive mechanical stress frequently causes micro-traumas in skeletal muscle, followed by a regeneration period. The effective removal of dead myofibers is a prerequisite for proper regeneration, and several cell types, including professional phagocytes, were reported to be active in this process. Myoblasts express several molecules of the phagocytic machinery, such as BAI1, stabilin-2, and TAM (Tyro3, Axl, Mertk) tyrosine kinase receptors, but these molecules were reported to serve primarily cell fusion and survival, and their role in the phagocytosis was not investigated. Therefore, we aimed to investigate the in vitro phagocytic capacity of the C2C12 mouse myoblast cell line. RNA sequencing data were analyzed to determine the level and changes of phagocytosis-related gene expression during the differentiation process of C2C12 cells. To study the phagocytic capacity of myoblasts and the effect of dexamethasone, all-trans retinoic acid, hemin, and TAM kinase inhibitor treatments on phagocytosis, C2C12 cells were fed dead thymocytes, and their phagocytic capacity was determined by flow cytometry. The effect of dexamethasone and all-trans retinoic acid on phagocytosis-related gene expression was determined by quantitative PCR. Both undifferentiated and differentiated cells engulfed dead cells being the undifferentiated cells more effective. In line with this, we observed that the expression of several phagocytosis-related genes was downregulated during the differentiation process. The phagocytosis could be increased by dexamethasone and all-trans retinoic acid and decreased by hemin and TAM kinase inhibitor treatments. Our results indicate that myoblasts not only express phagocytic machinery genes but are capable of efficient dead cell clearance as well, and this is regulated similarly, as reported in professional phagocytes.

Abstract 4991: Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC)

Abstract MERTK is a transmembrane receptor that belongs to the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases. The role of MERTK in cancer progression and resistance to therapies are reported in many cancer types, including non-small cell lung cancer (NSCLC). In canonical signaling, MERTK is localized at the cell membrane and mediates intracellular signal transduction pathways. Non-canonical signaling of receptor tyrosine kinases (RTKs) has been reported, including signaling which occurs when RTKs translocate to the nucleus. Our group previously reported nuclear localization of MERTK in leukemia cells and we now extend these findings to non-small cell lung cancer. We found exogenous MERTK localized to the nucleus in cells transfected with a plasmid expressing wild-type MERTK. Additionally, endogenous nuclear MERTK was phosphorylated, suggesting that MERTK may have a functional role in the nucleus. MERTK kinase inhibitor MRX-2843 modulates its nuclear translocation. Introducing point mutations to MERTK autophosphorylation sites diminished nuclear translocation in comparison to cells transfected with wild-type MERTK. Our analysis of proteins that may interact with MERTK to regulate nuclear localization and function demonstrated co-immunoprecipitation of nuclear MERTK and STAT1. Current studies are underway to further explore nuclear MERTK functions and how these roles may be modulated upon interaction with STAT1. Citation Format: K.M. Tanim, Deborah DeRyckere, Douglas K. Graham. Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4991.

First-in-human phase 1/1b study to evaluate sitravatinib in patients with advanced solid tumors

Sitravatinib (MGCD516), a spectrum-selective receptor tyrosine kinase inhibitor targeting TAM (TYRO3, AXL, MERTK) and split kinase family receptors, has demonstrated preclinical anti-tumor activity and modulation of tumor microenvironment. This first-in-human phase 1/1b study included sitravatinib dose exploration and anti-tumor activity evaluation in selected patients with advanced solid tumors. Primary objectives included assessment of safety, pharmacokinetics and clinical activity of sitravatinib. Secondary objectives included identifying doses for further investigation and exploring molecular markers for patient selection. In phase 1, 32 patients received 10–200 mg, while phase 1b dose expansion comprised 161 patients (150 mg n = 99, 120 mg n = 62). Maximum tolerated dose was determined as 150 mg daily. Dose-limiting toxicity was reported in 4/28 evaluable phase 1 patients (three at 200 mg, one at 80 mg). In phase 1b, 120 mg was defined as the recommended dose due to tolerability. Treatment-related adverse events (TRAEs) were experienced by 174/193 patients (90.2%); grade ≥ 3 TRAEs in 103 patients (53.4%). Most common TRAEs were diarrhea, fatigue, hypertension and nausea; TRAEs led to treatment discontinuation in 26 patients (13.5%) and death in one patient. Sitravatinib was steadily absorbed and declined from plasma with a terminal elimination half-life of 42.1–51.5 h following oral administration. Overall objective response rate was 11.8% in phase 1b, 13.2% in patients with non-small cell lung cancer (NSCLC) and 4.2% in patients with NSCLC with prior checkpoint inhibitor experience. Sitravatinib demonstrated manageable safety and modest clinical activity in solid tumors. NCT02219711 (first posted August 14, 2014).

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.

Dissecting the Role of AXL in Cancer Immune Escape and Resistance to Immune Checkpoint Inhibition.

The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.

43P MRTX-500: Phase II trial of sitravatinib (sitra) + nivolumab (nivo) in patients (pts) with non-squamous (NSQ) non-small cell lung cancer (NSCLC) progressing on or after prior checkpoint inhibitor (CPI) therapy

Therapy with CPI has improved OS in a subset of pts with NSCLC. Mechanisms of CPI resistance, however, have been described, including an immunosuppressive tumor microenvironment (TME), which may recruit immunosuppressive myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and M2-polarized macrophages in the TME. Sitra, a spectrum-selective TKI targeting TAM (Tyro3/Axl/MerTK) receptors and VEGFR2, reduces the number of MDSCs and Tregs and increases the M1/M2-polarized macrophage ratio. It is hypothesized to overcome an immunosuppressive TME and augment antitumor immune responses. MRTX-500 (NCT02954991) is a phase II study evaluating sitra (120 mg QD) + nivo (Q2W or Q4W) in pts with NSQ NSCLC who have progressed on or after treatment, with a CPI-based regimen (anti-PD1/PD-L1) and/or platinum doublet chemotherapy. The primary endpoint is ORR per RECIST 1.1. Secondary endpoints include OS, PFS, and safety. We report updated efficacy data for pts with NSCLC with PCB (prior clinical benefit; CR, PR, or SD ≥12 weeks) from a CPI who were treated with sitra + nivo as either 2L or 3L therapy. As of 17 October 2020, 68 pts with PCB (57% female; median age, 66 years; ECOG PS 0/1/2, 27%/66%/7%) were treated. Median follow-up was 28 months, median OS was 15 months (95% CI 9.3, 21.1),1- and 2-year OS rates were 56% and 32%, respectively. Median PFS was 6 months, and ORR was 16% (11/68), including 2 CRs. Median duration of response was 13 months. In all CPI-experienced pts evaluable for safety (n=124), treatment related adverse events (TRAEs) occurred in 91% of pts, with Gr 3/4 TRAEs occurring in 60% of pts. The most common (≥10%) Gr 3/4 TRAEs were hypertension and diarrhea. There were no Gr 5 TRAEs. Discontinuation rates for sitra and nivo due to any AE were 30% and 27%, respectively. Sitra + nivo demonstrated antitumor activity and encouraging OS compared to historical controls and no new safety signals were observed in pts with NSQ NSCLC who progressed on prior CPI. This combination is being evaluated in the phase III SAPPHIRE study. Previously presented at ESMO 2021, FPN (Final Publication Number): 1191O, Ticiana Leal et al. - Reused with permission.

Computational Interspecies Translation Between Alzheimer's Disease Mouse Models and Human Subjects Identifies Innate Immune Complement, TYROBP, and TAM Receptor Agonist Signatures, Distinct From Influences of Aging.

Mouse models are vital for preclinical research on Alzheimer’s disease (AD) pathobiology. Many traditional models are driven by autosomal dominant mutations identified from early onset AD genetics whereas late onset and sporadic forms of the disease are predominant among human patients. Alongside ongoing experimental efforts to improve fidelity of mouse model representation of late onset AD, a computational framework termed Translatable Components Regression (TransComp-R) offers a complementary approach to leverage human and mouse datasets concurrently to enhance translation capabilities. We employ TransComp-R to integratively analyze transcriptomic data from human postmortem and traditional amyloid mouse model hippocampi to identify pathway-level signatures present in human patient samples yet predictive of mouse model disease status. This method allows concomitant evaluation of datasets across different species beyond observational seeking of direct commonalities between the species. Additional linear modeling focuses on decoupling disease signatures from effects of aging. Our results elucidated mouse-to-human translatable signatures associated with disease: excitatory synapses, inflammatory cytokine signaling, and complement cascade- and TYROBP-based innate immune activity; these signatures all find validation in previous literature. Additionally, we identified agonists of the Tyro3 / Axl / MerTK (TAM) receptor family as significant contributors to the cross-species innate immune signature; the mechanistic roles of the TAM receptor family in AD merit further dedicated study. We have demonstrated that TransComp-R can enhance translational understanding of relationships between AD mouse model data and human data, thus aiding generation of biological hypotheses concerning AD progression and holding promise for improved preclinical evaluation of therapies.

Therapeutic Targeting of the Gas6/Axl Signaling Pathway in Cancer.

Many signaling pathways are dysregulated in cancer cells and the host tumor microenvironment. Aberrant receptor tyrosine kinase (RTK) pathways promote cancer development, progression, and metastasis. Hence, numerous therapeutic interventions targeting RTKs have been actively pursued. Axl is an RTK that belongs to the Tyro3, Axl, MerTK (TAM) subfamily. Axl binds to a high affinity ligand growth arrest specific 6 (Gas6) that belongs to the vitamin K-dependent family of proteins. The Gas6/Axl signaling pathway has been implicated to promote progression, metastasis, immune evasion, and therapeutic resistance in many cancer types. Therapeutic agents targeting Gas6 and Axl have been developed, and promising results have been observed in both preclinical and clinical settings when such agents are used alone or in combination therapy. This review examines the current state of therapeutics targeting the Gas6/Axl pathway in cancer and discusses Gas6- and Axl-targeting agents that have been evaluated preclinically and clinically.

1191O MRTX-500: Phase II trial of sitravatinib (sitra) + nivolumab (nivo) in patients (pts) with non-squamous (NSQ) non-small cell lung cancer (NSCLC) progressing on or after prior checkpoint inhibitor (CPI) therapy

Therapy with CPI has improved OS across many tumor types, including in a subset of pts with NSCLC. Mechanisms of CPI resistance, however, have been described, including an immunosuppressive TME, which may include recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and M2-polarized macrophages within the TME. Sitra, a spectrum-selective TKI targeting TAM (Tyro3/Axl/MerTK) receptors and VEGFR2, reduces the number of MDSCs and Tregs while increasing the ratio of M1/M2-polarized macrophages, and thus is hypothesized to overcome an immunosuppressive TME and augment antitumor immune responses. MRTX-500 (NCT02954991) is a phase II study evaluating sitra (120 mg QD) + nivo (Q2W or Q4W) in pts with NSQ NSCLC who have progressed on or after treatment, with a CPI-based regimen (anti-PD1/PD-L1) and/or platinum doublet chemotherapy. The primary endpoint is ORR per RECIST 1.1. Secondary endpoints include OS, PFS, and safety. We report updated efficacy data for pts with NSCLC with PCB (prior clinical benefit; CR, PR, or SD ≥12 weeks) from a CPI who were treated with sitra + nivo as either 2L or 3L therapy. As of 17 October 2020, 68 pts with PCB (57% female; median age, 66 years; ECOG PS 0/1/2, 27%/66%/7%) were treated. Median follow-up was 28 months, median OS was 15 months (95% CI 9.3, 21.1),1- and 2-year OS rates were 56% and 32%, respectively. Median PFS was 6 months, and ORR was 16% (11/68), including 2 CRs. Median duration of response was 13 months. In all CPI-experienced pts evaluable for safety (n=124), treatment related adverse events (TRAEs) occurred in 91% of pts, with Gr 3/4 TRAEs occurring in 60% of pts. The most common (≥10%) Gr 3/4 TRAEs were hypertension and diarrhea. There were no Gr 5 TRAEs. Discontinuation rates for sitra and nivo due to any AE were 30% and 27%, respectively. Sitra + nivo demonstrated antitumor activity and encouraging OS compared to historical controls and no new safety signals were observed in pts with NSQ NSCLC who progressed on prior CPI. This combination is being evaluated in the phase III SAPPHIRE study.

AXL Is a Key Factor for Cell Plasticity and Promotes Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA. IMPLICATIONS: These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

Growth Arrest-Specific Gene 6 Administration Ameliorates Sepsis-Induced Organ Damage in Mice and Reduces ROS Formation In Vitro.

Sepsis is a widespread life-threatening disease, with a high mortality rate due to inflammation-induced multiorgan failure (MOF). Thus, new effective modulators of the immune response are urgently needed to ameliorate the outcome of septic patients. As growth arrest-specific gene 6 (Gas6)/Tyro3, Axl, MerTK (TAM) receptors signaling has shown immunomodulatory activity in sepsis, here we sought to determine whether Gas6 protein injection could mitigate MOF in a cecal slurry mouse model of sepsis. Mice, divided into different groups according to treatment—i.e., placebo (B), ampicillin (BA), Gas6 alone (BG), and ampicillin plus Gas6 (BAG)—were assessed for vitality, histopathology and cytokine expression profile as well as inducible nitric oxide synthase (iNOS), ALT and LDH levels. BAG-treated mice displayed milder kidney and lung damage and reduced levels of cytokine expression and iNOS in the lungs compared to BA-treated mice. Notably, BAG-treated mice showed lower LDH levels compared to controls. Lastly, BAG-treated cells of dendritic, endothelial or monocytic origin displayed reduced ROS formation and increased cell viability, with a marked upregulation of mitochondrial activity. Altogether, our findings indicate that combined treatment with Gas6 and antibiotics ameliorates sepsis-induced organ damage and reduces systemic LDH levels in mice, suggesting that Gas6 intravenous injection may be a viable therapeutic option in sepsis.

MERTK Inhibition: Potential as a Treatment Strategy in EGFR Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer.

Epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) are currently the most effective treatment for non-small cell lung cancer (NSCLC) patients, who carry primary EGFR mutations. However, the patients eventually develop drug resistance to EGFR-TKIs after approximately one year. In addition to the acquisition of the EGFR T790M mutation, the activation of alternative receptor-mediated signaling pathways is a common mechanism for conferring the insensitivity of EGFR-TKI in NSCLC. Upregulation of the Mer receptor tyrosine kinase (MERTK), which is a member of the Tyro3-Axl-MERTK (TAM) family, is associated with a poor prognosis of many cancers. The binding of specific ligands, such as Gas6 and PROS1, to MERTK activates phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) cascades, which are the signaling pathways shared by EGFR. Therefore, the inhibition of MERTK can be considered a new therapeutic strategy for overcoming the resistance of NSCLC to EGFR-targeted agents. Although several small molecules and monoclonal antibodies targeting the TAM family are being developed and have been described to enhance the chemosensitivity and converse the resistance of EGFR-TKI, few have specifically been developed as MERTK inhibitors. The further development and investigation of biomarkers which can accurately predict MERTK activity and the response to MERTK inhibitors and MERTK-specific drugs are vitally important for obtaining appropriate patient stratification and increased benefits in clinical applications.

Single cell transcriptomics of mouse kidney transplants reveals a myeloid cell pathway for transplant rejection.

Myeloid cells are increasingly recognized as major players in transplant rejection. Here, we used a murine kidney transplantation model and single cell transcriptomics to dissect the contribution of myeloid cell subsets and their potential signaling pathways to kidney transplant rejection. Using a variety of bioinformatic techniques, including machine learning, we demonstrate that kidney allograft–infiltrating myeloid cells followed a trajectory of differentiation from monocytes to proinflammatory macrophages, and they exhibited distinct interactions with kidney allograft parenchymal cells. While this process correlated with a unique pattern of myeloid cell transcripts, a top gene identified was Axl, a member of the receptor tyrosine kinase family Tyro3/Axl/Mertk (TAM). Using kidney transplant recipients with Axl gene deficiency, we further demonstrate that Axl augmented intragraft differentiation of proinflammatory macrophages, likely via its effect on the transcription factor Cebpb. This, in turn, promoted intragraft recruitment, differentiation, and proliferation of donor-specific T cells, and it enhanced early allograft inflammation evidenced by histology. We conclude that myeloid cell Axl expression identified by single cell transcriptomics of kidney allografts in our study plays a major role in promoting intragraft myeloid cell and T cell differentiation, and it presents a potentially novel therapeutic target for controlling kidney allograft rejection and improving kidney allograft survival.

Targeting TAM to Tame Pancreatic Cancer.

Pancreatic cancer is expected to become the second leading cause of cancer-related death within the next few years. Current therapeutic strategies have limited effectiveness and therefore there is an urgency to develop novel effective therapies. The receptor tyrosine kinase subfamily TAM (Tyro3, Axl, MerTK) is directly implicated in the pathogenesis of the metastatic, chemoresistant, and immunosuppressive phenotype in pancreatic cancer. TAM inhibitors are promising investigational therapies for pancreatic cancer due to their potential to target multiple aspects of pancreatic cancer biology. Specifically, recent mechanistic investigations and therapeutic combinations in the preclinical setting suggest that TAM inhibition with chemotherapy, targeted therapy, and immunotherapy should be evaluated clinically.