Exploration of targeting GPR56 and its cancer cell ligand as a novel immune checkpoint inhibition strategy

Project Information

GPR-ICI

Grant agreement ID: 101123379

DOI

10.3030/101123379

Project closed

EC signature date 30 May 2023

Start date 1 June 2023

End date 30 November 2024

Funded under

• European Research Council (ERC)

Total cost

No data

EU contribution

€ 150 000,00

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - GPR-ICI (Exploration of targeting GPR56 and its cancer cell ligand as a novel immune checkpoint inhibition strategy)

Reporting period: 2023-06-01 to 2024-11-30

Summary of the context and overall objectives of the project

ICI has revolutionized cancer therapy over the last decade by harnessing the immune system to target cancer cells. It blocks immuno-suppressive receptors and their ligands, which normally reduce T cell activity and pro-inflammatory cytokine secretion, like interferon γ (IFNγ). This blockade lifts immunosuppression and enhances T-cell anti-tumor activity. Initial ICI therapies using anti-CTLA-4 and anti-PD-1 monoclonal antibodies (mAbs) showed promising results in melanoma and lung cancer. However, primary or acquired resistance limits their long-term effectiveness, benefiting only a fraction of patients. To address these limitations, new immune checkpoints (ICs) are being explored in clinical studies. Combining novel and established ICIs has shown significant efficacy, making it crucial to expand the ICI therapy repertoire to improve cancer treatment and patient survival, especially for those with resistance. Recently, GPR56 was identified as a novel IC that reduces T cell migration. GPR56 is an adhesion GPCR with a large extracellular domain that undergoes autoproteolytic cleavage. The N terminal fragment (NTF) remains non-covalently attached to the C terminal fragment (CTF) but is shed extracellularly upon ligand stimulation. Shedding the NTF increases the constitutive activity of the CTF, negatively regulating receptor activity. GPR56 acts as an inhibitory receptor on T cells and natural killer cells, influencing cell adhesion, migration, and development in the CNS and hematopoietic systems. GPR56 is also a negative prognostic marker in cancer, promoting cancer cell adhesion, proliferation, and progression. Unpublished data from our group show an interaction between GPR56 and HERV-K, a member of the HERV family linked to ancient retroviral infections. HERV-K, usually silenced in healthy tissues, is highly expressed in various cancers, including breast cancer, melanoma, and ovarian cancer. It is associated with neurodegeneration and CNS tumors, suggesting a dependency of these cancers on HERV-K. It is a critical regulator in certain CNS tumors and essential for tumorigenesis and metastasis in breast cancer. Although ICI has revolutionized cancer therapy and improved clinical outcome, primary and acquired resistance limit the success. Thus, only a fraction of cancer patients can benefit long-term from ICI, with some cancers not responding to available therapies. To overcome this, novel ICs have been identified, and subsequent clinical studies showed remarkable efficacies. Still, expanding the ICI repertoire is essential for improving therapy and patient survival. Here, our innovative approach could give oncologists and clinicians a new therapeutic option when treating the end users of our innovative ICI therapy – cancer patients, especially with primary or acquired resistance to other ICIs.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first aim, we wanted to investigate the potential of blocking the GPR56-HERV-K ENV interaction to prevent immune-suppressive signalling that we have observed before. The initial studies were performed in a simple NFAT-Luciferase reporter system, where NFAT transcription factor activation would lead to gene expression of a luciferase gene under control of a NFAT binding site. We wanted to use a more advanced system to assess NFAT signalling, using a dual luciferase plasmid where NFAT activation would increase Firefly luciferase expression, while Renilla luciferase expression would be constitutive, thereby serving as a proxy for transfection efficiency and cell viability. We were not able to detect the previous increase in NFAT signalling using this system. Hence, we were interested if the possible interaction of GPR56 with HERV-K ENV is physical or if downstream signalling of the two receptors led to an increase in NFAT signalling. For this, we applied the NanoBiT system to our receptors. This system consists of a split luciferase that has two parts. These two parts (Large BiT, small BiT) have no strong luciferase function on their own, but upon close proximity they reconstitute a functional luciferase enzyme and luminescence can be measured upon substrate addition. We tagged GPR56 with the large and HERV-K ENV with the small part. Next, we co-expressed or co-cultured cells expressing either one (co-culture) or both (co-expression) membrane proteins to probe for a physical proximity of GPR56 and HERV-K ENV. We also used a membrane bound Large BiT as negative control, since HERV-K ENV should not have any affinity to this membrane anchor. We were not able to confirm a physical interaction with this assay, since GPR56 co-culture or co-expression gave the same results as co-expression/co-culturing with the membrane anchor. We also evaluated this interaction assay for a reported interactor of HERV-K, CD98hc, but also here we were not able to confirm the reported interaction, indicating that the tags might interfere with the interaction.
As mapping physical interactions was not possible we tried investigating HERV-K ENV function, since its reported to mediate cell fusion and is supposed to be the main protein responsible for the ancient HERV-K retrovirus. We applied a simple cell fusion assay previously used in HIV research for this. It consists of two parts: effector cells, expressing the HERV-K ENV together with a plasmid expressing a Gal4-VP16 transcription factor, and target cell expressing the entry receptor (here constitutively) together with a luciferase gene that is under the Gal4 promoter. Cell fusion will lead to diffusion of the Gal4-VP16 transcription factor from the effector cell part to the target cell, where it induces luciferase gene expression from the target cell reporter. We could observe a dose-dependent increase in cell fusion with increasing amounts of HERV-K ENV. This indicates that the HERV-K entry receptor is expressed constitutively on HEK293 cells. Also, we could see an increase in cell fusion by lowering the pH and thereby simulating the environment in an endosome where HERV-K ENV is thought to function. We were also not able to increase the cell fusion by (over)expressing GPR56 or CD98hc, indicating that these two proteins do not cause HERV-K cell entry.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Regarding the potential interaction between GPR56 and HERV-K, more research needs to be conducted to confirm or refute this interaction. Since for a – according to literature – positive control with CD98hc, where we could also not see a positive signal in any of the tested assay systems, it is possible that another approach to assess this interaction might be more insightful than the previous approaches tested here. Since we were able to set up a cellular assay suitable for monitoring HERV-K ENVs cell fusion activity, this could be exploited in other ways to find potential inhibitors of HERV-K by applying them in the cell fusion assay in a more HTS manner and explore the properties of HERV-K particles.