TAM Family Of Receptor Tyrosine Kinases Research Articles

Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway.

Hepatic ischemia/reperfusion (I/R) injury is a major cause of complications in clinical liver surgery. AXL receptor tyrosine kinase (AXL) is a member of the TAM receptor tyrosine kinase family (TYRO3, AXL, and MERTK). Our previous study has shown that AXL expression was markedly upregulated in liver transplantation patients. However, the underlying mechanism of AXL in hepatic I/R injury remains unclear. A mouse liver warm I/R model and a primary hepatocyte hypoxia/reoxygenation model were established to investigate the role of AXL activation and ferroptosis in hepatic I/R injury by pretreating with recombinant mouse growth arrest-specific protein 6 (AXL activator) or R428 (AXL inhibitor). Moreover, we used LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor) to evaluate the relationship between the PI3K/AKT (the Ser and Thr kinase AKT) pathway and ferroptosis in hepatic I/R injury. Hepatic I/R injury decreased phosphorylation AXL expression and enhanced ferroptosis in liver transplantation patients and hepatic I/R-subjected mice. AXL activation attenuated lipid peroxidation and ferroptosis in hepatic I/R injury in vivo and in vitro. Inhibition of AXL activation exacerbated liver pathological damage and liver dysfunction, as well as iron accumulation and lipid peroxidation in hepatic I/R injury. Mechanistically, activated growth arrest-specific protein 6/AXL and its downstream PI3K/AKT signaling pathway inhibited ferroptosis during hepatic I/R injury. AXL activation protects against hepatic I/R injury by preventing ferroptosis through the PI3K/AKT pathway. This study is the first investigation on the AXL receptor and ferroptosis, and activating AXL to mitigate ferroptosis may be an innovative therapeutic strategy to combat hepatic I/R injury.

Abstract 6206: Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor. Despite high initial responses to frontline chemo-immunotherapy, therapeutic resistance develops rapidly. There are limited treatment options in the relapsed setting, where the prognosis remains dismal. SCLC tumors experience continuous and high levels of replication stress (RS) due to ubiquitous loss of key cell cycle checkpoints, RB1 and TP53. Frequent amplification and high expression of the transcription factor cMYC further contribute to increased RS. Thus, high levels of RS expose a potential SCLC vulnerability and provide a therapeutic opportunity. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its role in driving epithelial to mesenchymal transition (EMT). More recently, a novel role for AXL in DNA damage repair and tolerance has emerged. Therefore, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We first investigated the transcriptomic expression profile of AXL in SCLC clinical cohorts. AXL-high tumors were seen in a subset of treatment-naïve SCLC tumors, frequently among, but not limited to, the inflamed SCLC subtype. AXL expression was also seen in many relapsed SCLC tumors. As expected, tumors with high AXL expression also expressed several mesenchymal genes and higher EMT scores. Interestingly, among the treatment-naïve SCLC tumors, AXL expression was inversely correlated with a RS signature (rho=-0.54, p<0.001). Next, we tested the effects of AXL inhibition in SCLC in vitro and in vivo models. In a panel of 30 SCLC cell lines, bemcentinib, a selective AXL inhibitor in clinical trials for various advanced solid tumors, exhibited a range of antiproliferative activity, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM). Bemcentinib also significantly delayed tumor growth in in vivo SCLC models. Biomarkers associated with sensitivity to bemcentinib in SCLC cell lines included markers of RS (cMYC, replication stress score) and DNA damage response (phospho CHK1S345, phospho CHK2T68). Bemcentinib also induced RS, indicated by the activation of ATR/CHK1-mediated RS response pathway, and DNA damage, and the combination with an ATR inhibitor (ceralasertib) showed a greater than additive effect. In a syngeneic model of SCLC, the combination of bemcentinib, ceralasertib and an anti-PDL1 antibody induced significant tumor regression. Together, these promising findings demonstrate that AXL inhibition may be an effective strategy to target the RS vulnerability common in SCLC. Citation Format: Kavya Ramkumar, C. Allison Stewart, Azusa Tanimoto, Qi Wang, Yuanxin Xi, Benjamin B. Morris, Runsheng Wang, Li Shen, Robert J. Cardnell, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer [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 6206.

Abstract 1189: MERTK is a target in the microenviroenment of osteolytic bone metastasis

Abstract Metastatic bone disease often induces osteolytic lesions caused by an imbalance of osteoblastic bone formation and osteoclastic bone resorption. We could previously show that TAM family receptor tyrosine kinase MERTK exerts an oncogenic function in multiple myeloma, NSCLC and breast cancer bone metastasis, providing a rationale for using MERTK inhibitors in bone metastasis therapy. Furthermore, we reported a thus far unrecognized role of MERTK as a novel negative regulator of osteoblast function and showed that MERTK represents an osteoanabolic target in the tumor microenvironment of bone metastasis by targeting osteoblasts. It is described that MERTK exerts a prominent role in phagocytosis of macrophages. Nevertheless, the role of MERTK in osteoclasts is unknown. To assess a potential role of MERTK in physiological bone remodeling by osteoclasts in vivo, we performed microcomputed tomography (μCT) of tibia of LysM-cre+;Mertkflox/flox mice and observed increased bone volume. Histomorphometry revealed reduced osteoclast number in LysM-cre+;Mertkflox/flox mice, indicating that increased bone volume induced by loss of MERTK in the myeloid lineage is mediated by decreased osteoclast formation in vivo. Interestingly, analysis of TRAP+ mononuclear cells in the metaphyseal bone marrow showed decreased numbers and increased distance to closest bone surface, suggesting dysfunctional osteoclast precursor cell differentiation and migration in LysM-cre+;Mertkflox/flox mice. In vitro assays demonstrated that loss of Mertk results in decreased osteoclast precursor cell migration towards its ligand PROS1 and decreased osteoclast differentiation through a RHOA dependent mechanism. To elucidate if MERTK represents a target in the tumor microenvironment of osteolytic bone metastasis we utilized syngeneic EO771 breast cancer bone metastasis model and injected luciferase transduced EO771 cells into Mertkflox/flox and LysM-cre+;Mertkflox/flox C57BL/6J mice. Metastatic spread was monitored by bioluminescence imaging. Analysis of bone metastasis by μCT showed higher bone volume in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, osteolysis zones in the cortical bone were markedly decreased. Histomorphometry of TRAP/Hematoxylin staining indicated decreased osteoclast numbers in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, intratumoral TRAP+ mononuclear cells in breast cancer bone metastases were decreased, indicating that osteolytic bone metastasis recruit osteoclast precursors from its microenvironment in the bone marrow via MERTK to induce osteolysis. In summary, tumor-induced activation of MERTK suppresses and transforms osteoblasts, which counteracts bone formation and recruits osteoclast precursors to form osteoclasts and mediate osteolysis. Hence, MERTK is a novel target in the tumor microenvironment to inhibit osteolytic bone metastasis. Citation Format: Janik Engelmann, Jennifer Zarrer, Kristoffer Riecken, Jonas Waizenegger, Maria Elena Vargas-Delgado, Lara Meier, Carsten Bokemeyer, Klaus Pantel, Emily Alberto, Sourav Ghosh, Carla Rothlin, Eric Hesse, Hanna Taipaleenmäki, Isabel Ben-Batalla, Sonja Loges. MERTK is a target in the microenviroenment of osteolytic bone metastasis [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 1189.

TYRO3 promotes chemoresistance via increased LC3 expression in pancreatic cancer

Pancreatic cancer (PC) is an aggressive malignancy with few treatment options, and improved treatment strategies are urgently required. TYRO3, a member of the TAM receptor tyrosine kinase family, is a known oncogene; however, the relationship between TYRO3 expression and PC chemoresistance remains to be elucidated. We performed gain- and loss-of-function experiments on TYRO3 to examine whether it is involved in chemoresistance in PC cells. TYRO3 knockdown decreased cell viability and enhanced apoptosis following treatment of PC cells with gemcitabine and 5-fluorouracil (5-FU). In contrast, no such effects were observed in TYRO3-overexpressing PC cells. It is known that autophagy is associated with cancer chemoresistance. We then examined effects of TYRO3 on autophagy in PC cells. TYRO3 overexpression increased LC3 mRNA levels and induced LC3 puncta in PC cells. Inhibition of autophagy by chloroquine mitigated cell resistance to gemcitabine and 5-FU. In a xenograft mouse model, TYRO3 silencing significantly increased sensitivity of the cells to gemcitabine and 5-FU. To further investigate the involvement of autophagy in patients with PC, we immunohistochemically analyzed LC3 expression in the tissues of patients who underwent pancreatectomy and compared it with disease prognosis and TYRO3 expression. LC3 expression was negatively and positively correlated with prognosis and TYRO3 expression, respectively. Furthermore, LC3- and TYRO3-positive patients had a significantly worse prognosis among patients with PC who received chemotherapy after recurrence. These results indicated that the TYRO3-autophagy signaling pathway confers PC resistance to gemcitabine and 5-FU, and could be a novel therapeutic target to resolve PC chemoresistance.

The role of macrophage TAM receptor family in the acute-to-chronic progression of liver disease: From friend to foe?

Hepatic macrophages, the key cellular components of the liver, emerge as essential players in liver inflammation, tissue repair and subsequent fibrosis, as well as tumorigenesis. Recently, the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl and MerTK, was found to be a pivotal modulator of macrophages. Activation of macrophage TAM receptor signalling promotes the efferocytosis of apoptotic cells and skews the polarization of macrophages. After briefly reviewing the mechanisms of TAM receptor signalling in macrophage polarization, we focus on their role in liver diseases from acute injury to chronic inflammation, fibrosis and then to tumorigenesis. Notably, macrophage TAM receptor signalling seems to be a two-edged sword for liver diseases. On one hand, the activation of TAM receptor signalling inhibits inflammation and facilitates tissue repair during acute liver injury. On the other hand, continuous activation of the signalling contributes to the process of chronic inflammation into fibrosis and tumorigenesis by evoking hepatic stellate cells and inhibiting anti-tumour immunity. Therefore, targeting macrophage TAM receptors and clarifying its downstream pathways will be exciting prospects for the precaution and treatment of liver diseases, particularly at different stages or statuses.

Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation.

Bladder cancer is a common cancer; it is the tenth most common cancer in the world. Around one fourth of all diagnosed patients have muscle-invasive bladder cancer (MIBC), characterized by advanced tumors and which remains a lethal disease. The standard treatment for MIBC is the bladder removal by surgery. However, bladder-preserving alternatives are emerging by combining chemotherapy, radiotherapy and minimal surgery, aiming to increase the patient’s quality of life. The aim of the study was to improve these treatments by investigating a novel approach where in addition to radiotherapy, a receptor, TYRO3, a member of TAM receptor tyrosine kinase family known to be highly expressed on the bladder cancer cells and involved in the control of cell survival is targeted. For this, we evaluated the influence of TYRO3 expression levels on a colony or cell survival assays, DNA damage, γH2AX foci formation, gene expression profiling and cell cycle regulation, after radiation on different bladder cell models. We found that TYRO3 expression impacts the radiation response via the cell cycle dysregulation with noeffets on the DNA repair. Therefore, targeting TYRO3 is a promising sensitization marker that could be clinically employed in future treatments.

Abstract 2602: Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive lung tumor of neuroendocrine origin with a dismal 5-year survival rate. Despite high response rates to initial therapy, rapid development of therapeutic resistance limits overall survival. There are also limited treatment options, particularly in the relapsed setting. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its roles in driving epithelial to mesenchymal transition and DNA damage repair. AXL has also been implicated in immune escape. Based on these findings, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We screened BGB324 (bemcentinib), a selective small-molecule AXL inhibitor in clinical trials for various advanced solid tumors, in a panel of 60 human SCLC cell lines using cell viability assays. The SCLC cell lines showed a range of sensitivities, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM) with NeuroD1-driven SCLC cell lines being highly sensitive to BGB324 (ANOVA p<0.05). BGB324 also showed potent inhibition of tumor growth in an in vivo SCLC model. BGB324 further combined synergistically with an ATR inhibitor (AZD3738) and induces significant DNA damage. In a syngeneic model of SCLC, the combination of BGB324, AZD6738 and an anti-PDL1 antibody combination also induced significant tumor regression. Together, these promising findings show that AXL inhibition may be effective in SCLC and support further investigation of AXL and ATR inhibitor combinations with immune checkpoint blockade. Citation Format: Kavya Ramkumar, Azusa Tanimoto, C. Allison Stewart, Qi Wang, Li Shen, Robert J. Cardnell, B. Leticia Rodriguez, Don L. Gibbons, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2602.

Abstract 240: MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism

Abstract The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In previous studies, inhibition of MERTK decreased PD-1 checkpoint proteins in the leukemia microenvironment and prolonged survival in a syngeneic BCR-ABL+/Arf-/- B-cell acute leukemia model, implicating MERTK as a promising immune-oncology target in leukemia. Strikingly, Mertk-/- mice were largely protected from leukemia. In our current studies, Tyro3-/- almost completely prevented development of leukemia, comparable to Mertk-/-, while Axl-/- mice died with similar timing to wild type (WT) mice (20-40 days). These data demonstrate differential roles for TAM kinases in the anti-leukemia immune response. Depletion studies were conducted to evaluate potential roles for T cells and dendritic cells (DCs) in anti-leukemia immunity in Mertk-/- mice. Selective depletion of CD8+ T cells abrogated protection from leukemia in Mertk-/- mice, but survival was still prolonged relative to WT. Thus, while CD8+ T cells were required for complete protection from leukemia, the anti-leukemia response remained partially intact even in the absence of CD8+ T cells, implicating an innate immune mechanism. Indeed, combined depletion of CD8+ T cell and CD8α+ DC subsets completely abrogated the anti-leukemic effects in Mertk-/- mice, revealing a critical immunosuppressive role for MERTK in DCs in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Similarly, single cell RNA sequencing revealed CD8+ DCs with a more mature and antigen-presenting phenotype in Mertk-/- mice compared to WT, while antigen-presenting DCs were not increased in Tyro3-/- mice. Single cell sequencing data also suggest induction of an anti-leukemic DC - T cell axis in WT leukemic mice treated with the MERTK-selective inhibitor MRX-2843. DCs were nearly absent in leukemic bone marrow from saline-treated mice and were dramatically increased in response to treatment with MRX-2843. Treatment with MRX-2843 also decreased the incidence of CD8+ T cells expressing high levels of Tox, which has been associated with T cell exhaustion. These changes coincided with decreased leukemic blasts, even in the context of established disease.Together, our findings support a model whereby MERTK inhibition promotes DC function and CD8+ T cell activity, leading to anti-leukemia immunity. In contrast, anti-leukemia immunity in response to TYRO3 inhibition is less dependent on DCs. Differential roles for the TAM kinases in the leukemia microenvironment provide rationale for development of MERTK and/or TYRO3 targeted immunotherapies to treat acute leukemia. Citation Format: Justus M. Huelse, Swati S. Bhasin, Beena E. Thomas, Madison L. Chimenti, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 240.

Axl Mediates Resistance to Respiratory Syncytial Virus Infection Independent of Cell Attachment.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants and young children. Axl, a TAM family receptor tyrosine kinase, has been demonstrated to be a receptor mediating enveloped virus infection. Here we show that Axl functions as a suppressor of antiviral response during RSV infection. Knockdown of Axl expression in human cells resulted in cell resistance to RSV infection, although the treatment did not significantly affect RSV binding or cell entry. Mice deficient in Axl showed resistance to RSV infection, including reduction in viral load and in pulmonary injury. Although T lymphocyte and macrophage infiltration was reduced, more IFN-γ-producing cells were present in BAL fluid in Axl-/- mice. Fewer alternatively activated alveolar macrophages were found in the lungs of Axl-/- mice. Axl-/- mouse embryonic fibroblasts and siRNA-treated human cells had more robust IFN-β and IFN-stimulated gene induction of antiviral genes. Furthermore, reexpression of Axl using adenovirus-mediated Axl delivery repressed IFN-stimulated gene induction in Axl-null mouse embryonic fibroblasts by RSV infection. The results suggest that Axl, independent of being a virus entry receptor of RSV infection, negatively regulates IFN signaling to modulate host antiviral response against RSV infection.

The Akt-mTORC1 pathway mediates Axl receptor tyrosine kinase-induced mesangial cell proliferation.

Glomerulonephritis (GN), an important pathologic feature of many renal diseases, is frequently characterized by mesangial cell proliferation. We and others have previously shown that the TAM family receptor tyrosine kinases Axl, Mer, and Tyro-3 contribute to cell survival, proliferation, migration, and clearance of apoptotic cells (ACs); that Axl contributes to GN by promoting mesangial cell proliferation; and that small molecule inhibition of Axl ameliorates nephrotoxic serum-induced GN in mice. We now show that stimulation of renal mesangial cell Axl causes a modest increase in intracellular Ca2+ and activates NF-κB, mTOR, and the mTOR-containing mTORC1 complex, which phosphorylates the ribosomal protein S6. Axl-induction of Akt activation is upstream of NF-κB and mTOR activation, which are mutually codependent. Axl-induced NF-κB activation leads to Bcl-xl up-regulation. Axl is more important than Mer at mediating AC phagocytosis by mesangial cells, but less important than Mer at mediating phagocytosis of ACs by peritoneal macrophages. Taken together, our data suggest the possibility that Axl mediates mesangial cell phagocytosis of ACs and promotes mesangial cell proliferation by activating NF-κB and mTORC1.

Targeting the GAS6/AXL Pathway with a Newly Developed AXL-Selective Inhibitor SLC-0211 for the Treatment of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid clonal growth of myeloid lineage cells, with accumulation of immature blasts. Overall survival of AML patients is low and resistance to frontline chemotherapy is a major cause of treatment failure, highlighting the need for new therapies. It has been reported that AXL, a TAM family receptor tyrosine kinase, along with its ligand growth arrest-specific gene 6 (GAS6), mediates proliferation and survival of many types of cancer cells. An increasing body of evidence suggests that the GAS6/AXL pathway plays a major role in resistance to targeted therapies and conventional cytotoxic drugs. Particularly, increased expression levels of AXL and GAS6 have been reported in some AML and chronic myeloid leukemia (CML) patients, which are associated with poor prognosis. Several AXL inhibitors have recently been developed and some of them are in clinical trials. However, many of these small molecules are multi-kinase inhibitors and off-target effects and/or toxicity on healthy hematopoietic cells remain challenging. Moreover, it is still not known if the GAS6/AXL pathway is specifically activated in subgroups of AML patients carrying specific chromosomal abnormalities or mutations, and if AXL inhibitors can sensitize AML stem/progenitor cells to chemotherapy drugs and targeted therapeutics, since these cells are highly resistant to current anti-cancer therapies. We have therefore investigated expression changes in AXL and GAS6 in 15 AML cell lines and CD34+ stem/progenitor cells obtained from 11 primary AML patient samples. Interestingly, the transcript levels of AXL and GAS6 were found to be significantly increased in several AML cell lines carrying MLL fusion genes as compared to cells without MLL fusions (~6-fold and 8-fold). This result was further confirmed in these cells by Western blot analysis. Moreover, AXL transcripts were highly increased in primary CD34+ stem/progenitor cells as compared to more mature CD34- cells from nine AML patient samples studied (~7-fold). Transcript levels of GAS6 were slightly increased in CD34+ cells compared to CD34- cells from the same patient samples. These results indicate that both AXL and GAS6 are highly expressed in AML cell lines harboring MLL fusion genes, as well as in patient stem/progenitor cells.Interestingly, we have further demonstrated that AML cells with high expression of GAS6/AXL were more sensitive to the highly selective and novel AXL inhibitor SLC-0211, as compared to AML cells with low expression of GAS6 and/or AXL, as assessed by viability and apoptosis assays (2-3 fold). Notably, AML cells with high expression of both GAS6 and AXL, but without FLT3-ITD mutation, were highly sensitive to SLC-0211 treatment compared to other AXL inhibitors (>10-fold). Additive effects of SLC-0211 with chemotherapeutic drugs cytarabine or daunorubicin were also observed. In addition, SLC-0211 modestly reduced the colony forming ability of CD34+ AML cells, but the inhibitory effect of SLC-0211 was strikingly enhanced in re-plating assays, resulting in a significant reduction in re-plating efficiency of more primitive AML cells (75-97% inhibition). Importantly, the drug concentrations used to inhibit AML CFCs were not toxic to normal human CD34+ stem/progenitor cells. At the molecular level, treatment with SLC-0211 greatly reduced phosphorylation of AXLY779, AKTS473 and ERKT202/Y204 in AML cells in a dose-dependent manner as compared to control cells, as demonstrated by Western blot analysis. Taken together, these results indicate that inhibition of AXL by SLC-0211 specifically targets primitive AML cells where the AXL/GAS6 pathway is highly activated. Thus, targeting AXL may provide improved therapies for specific subgroups of AML. DisclosuresNo relevant conflicts of interest to declare.

Biparatopic single-domain antibodies against Axl achieve ultra-high affinity through intramolecular engagement

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.

Mononuclear phagocytes drive Mertk-dependent T cell regulation at autoimmune and tumor sites

Abstract Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes (MNPs) in regulating effector T cells in type 1 diabetes and melanoma. MNPs in the islets impaired T cell responsiveness to antigen by preventing T cell arrest. MNPs in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. T cell arrest was accompanied by increased T cell-antigen presenting cell interactions in the islets. Antigen experience and effector function by T cells was increased with Mertk inhibition in the islets, but not in lymph nodes. Single cell RNA-seq analysis of islet MNPs identified multiple myeloid subsets. Inhibition of Mertk induced the most significant changes in gene expression among the islet resident macrophages including upregulation of inflammatory cytokine and adhesion genes. In the NOD mouse model, inhibition of Mertk-dependent T cell regulation culminated in the rapid acceleration of autoimmune pathology and disease. In human islets, the number of Mertk-expressing cells were increased in remaining insulin-containing islets from type 1 diabetic patients, suggesting that they might have a protective role in human disease. These data indicate that Mertk signaling in MNPs drives T cell regulation that functions specifically at the disease site in peripheral tissues through a mechanism that prevents T cell arrest and hinders response to antigen.

TAM family receptors in conjunction with MAPK signalling are involved in acquired resistance to PI3K\u03b1 inhibition in head and neck squamous cell carcinoma

BackgroundAberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs).MethodsFive unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance.ResultsProlonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20–35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib.ConclusionsWe have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.

Mononuclear phagocytes drive Mertk-dependent T cell regulation at autoimmune and tumor sites

Abstract Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes (MNPs) in regulating effector T cells in type 1 diabetes and melanoma. MNPs in the islets impaired T cell responsiveness to antigen by preventing antigen-mediated T cell arrest. MNPs in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. T cell arrest was accompanied by increased T cell-antigen presenting cell interactions as well as increased antigen experience and effector function by T cells in the islets. Single cell RNA-seq analysis of islet MNPs identified multiple myeloid subsets. Inhibition of Mertk induced the most significant changes in gene expression among the islet resident macrophages including upregulation of inflammatory cytokine and adhesion genes. In the NOD mouse model, inhibition of Mertk-dependent T cell regulation culminated in the rapid acceleration of autoimmune pathology and disease. In human islets, the number of Mertk-expressing cells were increased in remaining insulin-containing islets from type 1 diabetic patients, suggesting that they might have a protective role in human disease. These data indicate that Mertk signaling in MNPs drives T cell regulation that functions specifically at the disease site in peripheral tissues through a mechanism that prevents T cell arrest and response to antigen.

Abstract B024: Clinical, genomic, and immune landscape of the receptor tyrosine kinase AXL in non-small cell lung cancer

Abstract Therapeutic resistance limits effective treatment of non-small cell lung cancer (NSCLC) and a better understanding of mechanisms contributing to resistance and strategies to overcome these are urgently needed. AXL, a TAM family receptor tyrosine kinase, has emerged as a key determinant of resistance to chemotherapy, radiation and targeted therapies in NSCLC and other cancers, through its roles in mediating epithelial-mesenchymal transition (EMT) and immune escape. As several small-molecule AXL inhibitors and anti-AXL biologics are currently in clinical testing, AXL has emerged as a target of interest in treatment-resistant NSCLC. Here, we investigated AXL expression in NSCLC by analyzing genomic, transcriptomic and proteomic profiles across 3 treatment-naïve (1095 tumors) and 2 previously treated (245 tumors) clinical cohorts of lung adenocarcinoma and squamous cell carcinoma. While AXL expression did not vary with disease stage, tumor samples from patients with prior systemic treatment (and subsequent relapse) had significantly higher AXL expression and were more likely to have undergone EMT (based on our published EMT score), as compared to treatment-naïve NSCLC patients (p<0.001). Although AXL-high tumors were seen in all histologic subtypes, AXL levels were 1.4-fold higher in lung adenocarcinoma than in squamous cell carcinoma (p<0.001). Further analysis of driver genes in adenocarcinoma revealed that treatment-naïve NSCLC tumors with ALK translocations frequently had high expression of AXL at baseline (p=0.07), while approximately half of EGFR mutant NSCLC expressed high AXL mRNA. In contrast, tumors bearing mutations in STK11 and KEAP1 had relatively lower AXL expression (1.8-fold and 1.6-fold respectively, both p<0.001). Similarly, the KRAS/STK11 co-mutation subgroup, which is associated with higher rates of inactivating KEAP1 mutations and an immune suppressed phenotype, had lower AXL levels as compared to the KRAS/TP53 or KRAS/CDKN2A/B co-mutation subgroups (p<0.001). As AXL has been described to play a role in immune escape, we next investigated the association between AXL expression and immune response genes. AXL expression was strongly correlated with expression of immune suppressive mediators (TIM3, FOXP3, PD-L1 and PD-L2) as well as macrophage-recruiting factors (CCL2 and CSF-1). Furthermore, a significant correlation (rho > 0.3; p<0.001) between AXL expression and CXCL10 and CCL5, effector chemokines of STING-mediated innate immune response, was also observed in lung adenocarcinoma. In summary, our results show that AXL, which is associated with resistance to therapy, is present at higher levels in a significant subset of NSCLC tumors, including in treatment-naïve patients with ALK translocations and EGFR mutations. These findings suggest that early co-targeting of AXL along with standard therapies could improve therapeutic outcomes. Citation Format: Kavya Ramkumar, Carminia M. Della Corte, Lixia Diao, Robert J. Cardnell, Vali A. Papadimitrakopoulou, John V. Heymach, Jing Wang, Don L. Gibbons, Lauren A. Byers. Clinical, genomic, and immune landscape of the receptor tyrosine kinase AXL in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B024. doi:10.1158/1535-7163.TARG-19-B024

Oncogenic role of TYRO3 receptor tyrosine kinase in the progression of pancreatic cancer.

The expression and functions of TYRO3, a member of the TAM receptor tyrosine kinase family, in pancreatic cancer (PC) have not been specifically elucidated. In this study, we confirmed TYRO3 expression in five human PC cell lines (PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and PK-9) using Western blotting. TYRO3 silencing and overexpression studies have revealed that TYRO3 promotes cell proliferation and invasion in PC via phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). Using a mouse xenograft model, we showed that tumor growth was significantly suppressed in mice subcutaneously inoculated with TYRO3-knockdown PC cells compared with mice inoculated with control PC cells. Furthermore, TYRO3 expression was examined in PC tissues obtained from 106 patients who underwent pancreatic resection for invasive ductal carcinoma through immunohistochemical staining. TYRO3-positive patients had poor prognoses for overall survival and disease-specific survival compared with TYRO3-negative patients. Multivariate analysis revealed that TYRO3 expression is an independent prognostic factor for overall survival. Our study demonstrates the critical role of TYRO3 in PC progression through Akt and ERK activation and suggests TYRO3 as a novel promising target for therapeutic strategies against PC.

ACTR-48. CLINICAL DEVELOPMENT TARGETING AXL IN RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a lethal disease without effective therapies. Previously we reported that the TAM receptor tyrosine kinase family member AXL is upregulated in mesenchymal GBM and its knockdown induces apoptosis of mesenchymal but not proneural glioma sphere cultures (GSCs). In this study, we report that BGB324, a novel small molecule inhibitor for AXL, prolongs the survival of immunocompromised mice bearing mesenchymal GSC-derived GBM-like intracranial tumors by inhibition of AXL activation in tumor cells. Mechanistically, we identified that protein S (PROS1), known as a ligand for other TAM receptors, is secreted by tumor-associated macrophages, thereby physically associates and activates AXL in mesenchymal GSCs. The PROS1-driven phosphorylation of AXL resulted in the NF-kB activation in mesenchymal GSCs and this activation was eliminated by BGB324 treatment. In addition, BGB324 downregulated the immune checkpoint protein PD-L1 both in vitro and in vivo. Combination of BGB324 with Nivolumab - the neutralizing antibody for PD-1 (a receptor for PD-L1) was effective to prolong the survival of immunocompetent mice bearing syngeneic GBM models. Clinically, both AXL and PROS1 expression indicated poorer prognosis of GBM patients. These data suggest that the PROS1/AXL pathway regulates intrinsic mesenchymal signaling as well as extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. To translate the pre-clinical data to human GBM patients, we designed a surgical PK/PD clinical trial with BGB324 for recurrent GBM. As of May 2019, this trial, termed ABTC1701, is in pending approval by the Brain Malignancy Steering Committee in the National Cancer Institute.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Abstract 276: Targeting AXL sensitizes non-small cell lung cancer to ATR inhibitors by enhancing replication stress

Abstract Therapeutic resistance limits effective treatment of non-small cell lung cancer (NSCLC) and a better understanding of mechanisms contributing to resistance and strategies to overcome these are urgently needed. AXL, a TAM family receptor tyrosine kinase, has emerged as a key determinant of intrinsic and acquired resistance to chemotherapy, radiation and targeted therapies in NSCLC and other cancers, through its roles in mediating epithelial-mesenchymal transition (EMT) and immune escape. We previously showed that AXL may also play a role in DNA damage repair and that AXL overexpression mediated primary as well as acquired resistance to inhibitors of WEE1, a replication stress response kinase, in small cell lung cancer. In the present study, we further investigated the role of AXL in replication stress response. We found that, in NSCLC cell lines, AXL inhibition with the selective small-molecule AXL inhibitor BGB324 caused replication stress checkpoint activation, as indicated by increased RPA32 hyper-phosphorylation and ATR-mediated CHK1 phosphorylation. We further screened ATR inhibitors, VX-970 and AZD6738, in a panel of 20 NSCLC cell lines and correlated drug sensitivity with baseline expression of over 200 phosphorylated and total proteins, measured by reverse phase protein array. Notably, AXL was one of the biomarkers of resistance to the ATR inhibitor VX-970 (rho=0.52, p<0.05). Therefore, we hypothesized that AXL plays a unique role in regulating replication stress and targeting AXL will sensitize NSCLC cells to ATR inhibitors. Combination of BGB324 and ATR inhibitors (VX-970 and AZD6738) significantly decreased cell proliferation in a panel of human and GEMM-derived NSCLC cell lines as compared to single agents alone. In NSCLC cells with primary resistance to ATR inhibition, co-targeting AXL and ATR significantly increased RPA32 hyper-phosphorylation, concomitantly with increased DNA double strand breaks and inactivated G2/M checkpoint, resulting in mitotic catastrophe. AXL knockdown in a GEMM-derived Kras/Trp53 mutant NSCLC model also showed similar results. Notably, NSCLC cell lines with low levels of SLFN11 (a DNA/RNA helicase that induces replication arrest following DNA damage independently of ATR) were more sensitive to AXL/ATR co-targeting. In conclusion, these findings suggest that AXL may play a novel and unexpected role in regulating replication stress. Furthermore, our results show that targeting AXL sensitizes NSCLC cell lines with primary resistance to ATR inhibitors and that AXL/ATR inhibitor combinations could be useful in treating platinum- and PARP inhibitor-resistant SLFN11-low tumors. Citation Format: Kavya Ramkumar, Pan Tong, You-Hong Fan, David Peng, John V. Heymach, Don L. Gibbons, Jing Wang, Lauren A. Byers. Targeting AXL sensitizes non-small cell lung cancer to ATR inhibitors by enhancing replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 276.