The Role of Axl Signaling in Anti-Tumor Immune Evasion
Prosjekt
- Prosjektnummer
- 912034
- Ansvarlig person
- Jing Kang
- Institusjon
- Helse Bergen HF
- Prosjektkategori
- Doktorgradsstipend
- Helsekategori
- Cancer
- Forskningsaktivitet
- 1. Underpinning
Rapporter
AXL, MERTK and TYRO3 make up the TAM family of receptor tyrosine kinases. They are pleiotropically expressed receptors in both healthy and diseased tissues, and can regulate multiple cellular processes, including like cell growth, differentiation, proliferation, migration, and invasion. Ligands growth arrest-specific factor 6 (Gas6) or protein S (PROS1) acts as bridging molecules by binding to phosphatidylserine (PS), thereby activating the TAM receptor. Although GAS6 preferentially activates AXL, PROS1 has the highest affinity for MERTK and TYRO3. In this project, we elucidated AXL receptor tyrosine kinase cell signaling in cancer. The central hypothesis is that AXL receptor signaling drives metastasis.
In our study, we found that pharmacological AXL inhibition can prevent cell migration, while CRISPR knocking out AXL in some cell lines cannot inhibit cell migration. To further study this mechanism, we established different cell lines in melanoma and breast cancer cell lines by knocking out AXL by CRISPR and compared the downstream signal transduction and cell migration of selective AXL and pan-TAM kinase inhibitor treatment. In summary, our results show that MERTK can compensate for the loss of AXL signal under the condition of AXL knockout in cell lines expressing autocrine GAS6. The compensation effect of MERTK is closely related to the expression and activation of AKT3 and the function of cell migration. Our research explains the underlying mechanism of reporting conflicts and highlights the fundamental difference between CRISPR gene knockout and therapeutic targeting. These results will form the manuscript "TAM Receptor Dynamics in Therapeutic Resistance" currently being prepared.
On the other hand, mechanistical study on how AXL clustering processes affect signal transduction and contributes tumor metastasis is vital. In this study we developed a CRISPR-based complementation approach for functional mapping of AXL signal transduction derived from combined retroviral expression system. We established 13 AXL-complemented cell lines with AXL receptor mutations on specific tyrosine residues both in melanoma and breast cancer cell lines. After AXL receptor tyrosine kinase was activated in dimerization way or clustering way, SH2 domain binding proteins will be assessed by Mass spectrometry; and the downstream signaling proteins will be measured by CyTOF; blocking metastasis will be assessed by xcelligence assay. We hypothesize that AXL receptor signaling contributes to metastasis in melanoma and breast cancer. Clustering activation of AXL creates a stronger and longer signaling transduction, and the specific tyrosine residue Y866 of AXL shows important roles in AXL signaling pathway. Collectively, our results support a therapeutic rationale for clinical AXL pathway inhibitors in tumor metastasis treatment in melanoma and breast cancer. These results will form the paper “GAS6-mediated AXL receptor clusters activate unique cell signaling pathways underlying EMT and metastasis ”,a manuscript currently in preparation.
Defense of thesis is planned to April 2021.
Although targeted therapy related to the TAM receptor family have made great progress in recent years, both laboratory data and clinical data shows none of them are 100% effective. We found that pharmacological AXL inhibition can prevent cell migration, while CRISPR knocking out AXL in some cell lines cannot inhibit cell migration. Based on our research, the AXL inhibitor Bemcentinib that blocks the kinase region and BMS777607 that can simultaneously inhibit all members of the TAM receptor family, both can stably inhibit the cell migration of melanoma NZM17 cells. However, knocking out AXL by CRISPR improved cell migration. The possible explanation for our data is that the compensatory activation of MERTK under the condition of autocrine GAS6 leads to the improvement of cell migration, and we also proved that inhibiting AKT3 can reduce cell migration. Overall, our research explains the underlying mechanism of reporting conflicts and highlights the fundamental difference between CRISPR gene knockout and therapeutic targeting, supports a therapeutic rationale for clinical AXL pathway inhibitors in tumor metastasis treatment in melanoma and breast cancer, and has guiding significance for the future clinical combination of AXL inhibitors and AKT3 inhibitors to break through AXL resistance.
Cancer is a major health problem throughout the world including breast cancer and melanoma. AXL is a member of the TAM (TYRO3-AXL-MERTK) family of receptor tyrosine kinases, which, when activated, can regulate tumor cell survival, proliferation, migration and invasion, angiogenesis, and tumor-host interactions. In this project, we elucidated the function of AXL receptor tyrosine kinase cell signaling in metastasis in melanoma and breast cancer.The related AXL, TYRO3 and MERTK receptor tyrosine kinases (RTKs) are increasingly being implicated in a host of discrete cellular responses including cell survival, proliferation, migration and phagocytosis. In this project, we want to elucidate AXL receptor tyrosine kinase cell signaling in cancer. Our specific aims are including functional mapping of AXL phosphotyrosine-mediated signal transduction and cell migration using a genetic (CRISPR complementation) approach in melanoma and breast cancer cell lines; characterize the mechanism of GAS6-mediated receptor clustering activation of AXL; and investigate the role of AXL-mediated activation of AKT3 in metastasis.
The AXL receptor tyrosine kinase binds vitamin K-dependent ligands growth arrest-specific factor 6 (GAS6), and acts as bridging molecules to promote phosphatidylserine (PS)-mediated signal transduction required for cancer cell metastasis in melanoma and breast cancer. In this study we developed a CRISPR-based complementation approach for functional mapping of AXL signal transduction derived from combined retroviral expression system. We hypothesize that AXL receptor signaling contributes to metastasis in melanoma and breast cancer. Clustering activation of AXL creates a stronger and longer signaling transduction, and the specific tyrosine residue Y866 of AXL shows important roles in AXL signaling pathway. Collectively, our results support a therapeutic rationale for clinical AXL pathway inhibitors in tumor metastasis treatment in melanoma and breast cancer.
Besides, GAS6 is a common ligand for the AXL, TYRO3 and MERTK (TAM) receptor tyrosine kinase family. We found that pharmacological AXL inhibition blocked cell migration while CRISPR knockout of AXL did not in certain cell lines. In this study we showed that MERTK and TYRO3 can compensate for loss of AXL signaling only under conditions of AXL gene knockout in cell lines with autocrine GAS6 expression. Our study explains a mechanism underlying conflicting reports and highlights a fundamental difference between CRISPR knockout and therapeutic targeting.
The receptor tyrosine kinase AXL and its ligand GAS6 regulate fundamental biological processes, including cell proliferation, survival and motility, through multiple downstream signaling pathways include AKT. AXL-AKT3 are often co-expression in cancer gene expression datasets and in this study, we elucidate a mechanism for how AXL-AKT3 signaling affects metastasis in melanoma and breast cancer. We demonstrated that it is AKT3 regulates cell proliferation, and we also show that knock out AKT3 can significantly reduce cell migration in melanoma and breast cancer. Our results provide therapeutic rationale to target the AXL-AKT3 axis in melanoma and breast cancer.
The Axl receptor tyrosine kinase has an important role in tumor metastasis and anti-tumor immune evasion. The aim of this project is to characterize Axl in metastatic melanoma, and elucidate the mechanism for how Axl receptor cell signaling leads to anti-tumor immune evasion.Immunotherapies and targeted therapies are associated with improved disease control and significantly increased survival times in metastatic melanoma, however, both single agent responses and acquired drug resistance limited the therapeutic effect. Therefore melanoma has still significantly contributed to the burden of cancer in all over the word. The Axl receptor tyrosine kinase is an important driver of epithelial to mesenchymal transition (EMT) and tumor progression, and has been implicated in promoting in cell adhesion, survival, proliferation, motility and invasion. Furthermore the Axl receptor tyrosine kinase is also a regulator of anti-tumor immunity, and used by carcinoma cells to escape the anti-tumor immune response. For these reasons, it is still urgent to further investigate the molecular mechanisms of melanoma in order to develop novel strategies combining immunotherapy and anti-AXL therapies for its early diagnosis and treatment.
The hypothesis of this project is that Axl receptor signaling contributes to metastatic melanoma via Axl-Akt3 signaling mechanism; and Axl receptor signaling in tumors contributes to immune evasion and resistance to immunotherapeutics.
We have evaluated that Axl expression level contributes to proliferation, which is even more in physiological oxygen tension than in atmospheric oxygen tension; we also have found that simply knockout Axl can not restrain proliferation but even lead it to increase; besides we also learned that knocking out Axl leads to Akt3, Akt expression and Akt activation increase. However, when knocking out both Axl and Akt3 simultaneously, tumor proliferation was restrained. So combining Axl inhibitor and Akt3 inhibitor may restrain tumor development. Besides, as vitamin K shows important effect in Gas6/Axl signaling pathway, we measured migration among some of these carrying single loss-of-function mutations on the Axl receptor cell lines, and the results show that vitamin K can increase Akt expression but reduce Akt activation level; Vitamin K can influence Axl related migration, and at the same time we found that Y866F can cause reduced migration with Vitamin K, which indicated that Y866 might be an important site for Axl in migration.
We will during 2019 first update our data of how knocking out Axl influence melanoma migration and invasion, moreover we will in vivo measure the results of combing Axl inhibitor and Akt3 inhibitor in treating metastatic melanoma, all these results will contribute to the first paper. Besides we will continue functional in vivo mapping of the Axl signal transduction required for cancer cell immune evasion using optical imaging-based neo-antigen-mediated anti-tumor immune response orthotopic murine models of melanoma and breast cancer.
The Axl receptor tyrosine kinase has emerged as an important target of anti-tumor treatment. We hypothesize that Axl affects melanoma metastasis via activation of Akt3, and Axl contributes to immune evasion and resistance to immunotherapeutics. Using CRISPR gene editing we are studying how Axl-Akt3 signaling affects metastasis in melanoma and how Axl receptor cell signaling leads to anti-tumor immune evasion.Malignant melanoma is the most aggressive form of skin cancer, and its incidence has increased faster than other tumors. Axl signaling enhances many essential biological functions for cancer progression by influencing cell transformation, proliferation, invasion, metastasis, survival, angiogenesis, and resistance to chemotherapeutic and targeting drugs. Besides, Akt3 from Akt kinase family is the predominant isoform in melanomas where it plays a critical role in invasion, metastasis and therapeutic resistance. A hypothesis model of Axl-dependent EMT gene program maintenance via activation of nuclear Akt3 was previously proposed by our research group indicating that Axl-Akt3 signaling may play an important role in cancer progression and Axl-Akt3 signaling pathway may become a novel therapeutic target to treat cancer.
Recently, checkpoint immunotherapy is quickly entering first line treatment of metastatic disease for multiple malignancies, but both single agent responses and acquired drug resistance limited the therapeutic effect. The receptor tyrosine kinase Axl was identified as a regulator of anti-tumor immunity, and used by carcinoma cells to escape the anti-tumor immune response. Genetic deletion of Axl resulted in sensitization of tumor cells to checkpoint immunotherapy. With the potential to target the epithelial to mesenchymal transition, stimulate antigen presentation, and block immune suppressive macrophage recruitment to the tumor microenvironment clinically significant responses may be achieved by combining immunotherapy and anti-Axl therapies.
To understand how Axl signaling pathway effect melanoma metastasis, we knock out Axl in several cancer cell lines by CRISPR (Clustered regularly interspaced short palindromic repeats), and verified them by flow cytometry, western blot, surveyor nuclease assay, DNA sequencing. We measured changes in cell proliferation, migration and invasion, and found that knocking out Axl increased proliferation, at the same time we also found that Akt3 expression level and Akt activation level increased; but both knock out Axl and akt3 can decrease proliferation indicating that combine Axl inhibitor and Akt3 inhibitor may restrain tumor development.
We developed a new CRISPR-based complementation approach. Eleven Axl gene expressions constructs carrying single loss-of-function mutations of the catalytic kinase domain and intracellular tyrosine phosphorylation sites on the Axl receptor were transfected into Axlnull cell lines by retrovirus transfection. As vitamin K shows important effect in Gas6/Axl signaling pathway, we measured migration among some of these carrying single loss-of-function mutations on the Axl receptor cell lines, and the results show that vitamin K may increase cell migration when cells express Axl, besides we also found that Akt activation level was redused; Y702F and Y703F may cause migration improve, but Y866F can cause decreased migration. Experiments are still ongoing to measure the effect of specific Axl receptor mutations on anti-tumor immune evasion.
Next we will further functional in vivo mapping of the Axl signal transduction required for cancer cell immune evasion using optical imaging-based neo-antigen-mediated anti-tumor immune response orthotopic murine models of melanoma and breast cancer; evaluation of new therapeutic strategies based on Axl signaling inhibition to improve efficacy of immune checkpoint inhibitor therapy in breast carcinoma and melanoma; measurement of Axl signaling-based biomarkers in clinically-annotated patient biopsy material to determine correlation with clinical outcome following checkpoint inhibitor therapy.
This Project is a basic mechanism theoretical Research, so until now, there is no ueser participation.
The Axl receptor tyrosine kinase has emerged as an important regulator of anti-tumor immunity. We hypothesize that Axl contributes to immune evasion and resistance to immunotherapeutics. Using CRISPR gene editing we are studying how Axl receptor cell signaling leads to anti-tumor immune evasion.The recent clinical success of anti-tumor immunotherapeutics such as the immune checkpoint inhibitors, demonstrated that the immune system may be mobilized to attack tumors and provide unprecedented durable clinical benefit for some patients. This revealed that the major barrier to immune-mediated tumor rejection is local anti-tumor immune evasion fueled by the tumor microenvironment that induces intrinsic tumor cell plasticity and recruits immune suppressive cells. The Axl receptor tyrosine kinase is a regulator of anti-tumor immunity, and used by carcinoma cells to escape the anti-tumor immune response. Recent results show that blocking Axl signaling enhances immune checkpoint inhibitor efficacy in a murine cancer models.
The central hypothesis of this project is that Axl receptor signaling in tumors contributes to immune evasion and resistance to immunotherapeutics. To study of how Axl receptor cell signaling contributes to immune evasion we generated of CRISPR-complement cell lines.
We used CRISPR (Clustered regularly interspaced short palindromic repeats) a gene editing technique to make specific changes in genes, to delete Axl from several cancer cell lines. These cell lines were characterized (flow cytometry, western blot, surveyor nuclease assay, DNA sequencing) to verify that the new CRISPR-Axlnull cell lines were stable. We measured changes in cell proliferation, migration, and invasion confirming that CRISPR-Axlnull cells had decreased migration and invasion, but higher ability in proliferation.
During 2016, we developed a new CRISPR-based complementation approach. Eleven Axl gene expressions constructs carrying single loss-of-function mutations of the catalytic kinase domain and intracellular tyrosine phosphorylation sites on the Axl receptor were transfected into Axlnull cell lines by retrovirus transfection. Cell lines chorts comprising different Axl receptor mutations were isolated by flow cytometry. Experiments are ongoing to measure the effect of specific Axl receptor mutations on anti-tumor immune evasion.
Deltagere
- Jing Kang Ph.d.-kandidat
- Oddbjørn Straume Medveileder
- James Lorens Hovedveileder
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
Alle henvendelser rettes til Faglig rapportering, Helse Vest