NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment

The antitumor functions of NK cells are regulated by the integration of positive and negative signals triggered by numerous membrane receptors present on the NK cells themselves. Among the main activating receptors, NKG2D binds several stress-induced molecules on tumor targets. Engagement of NKG2D by its ligands (NKG2D-Ls) induces NK cell activation leading to production of cytokines and target cell lysis. These effects have therapeutic potential as NKG2D-Ls are widely expressed by solid tumors, whereas their expression in healthy cells is limited. Here, we describe the genetic and environmental factors regulating the NKG2D/NKG2D-L pathway in tumors. NKG2D-L expression is linked to cellular stress and cell proliferation, and has been associated with oncogenic mutations. Tumors have been found to alter their to NKG2D-L expression as they progress, which interferes with the antitumor function of the pathway. Nevertheless, this pathway could be advantageously exploited for cancer therapy. Various cancer treatments, including chemotherapy and targeted therapies, indirectly interfere with the cellular and soluble forms of NKG2D-Ls. In addition, NKG2D introduced into chimeric antigen receptors in T- and NK cells is a promising tumor immunotherapy approach

Les cellules lymphocytaires innées dans l’immunosurveillance des tumeurs métastatiques

Innate lymphoid cells (ILCs) are effectors of innate immunity and play an important role in the regulation of the immune response. NK cells (cytotoxic ILC1) are known for their anti-tumor functions. The immune response of helper-type ILCs (hILC) (ILC1, ILC2 and ILC3) against the tumor is still poorly described in Humans. By their tissue residency, hILCs could play an important role in the immune response to cancer. The development of targeted therapies and immunotherapies has revolutionized the treatment of many cancers. Current therapies are “personalized”, adapted to the mutational profile of the patient and to the quality of the tumor microenvironment. These treatments result in rapid and effective responses in most of the cases. However, resistance mechanisms are frequent, leading to relapses. The mechanisms of resistance are still poorly understood. Current approaches combine therapies targeting specific tumor abnormalities and bypassing immunosuppression to restore effective anti-tumor immunity. My thesis project attempts to address this issue through two projects: a translational research project to analyze the impact of resistance to targeted therapies on NK immunogenicity and a more exploratory project which aims to define the role of lymph node (LN) ILC in tumor immunosurveillance. The first project focuses on the study of cytotoxic ILCs, NK cells, and their interactions with melanoma cells resistant to targeted therapies, directed against the BRAF (Vemurafenib) and MEK (Cobimetinib) tyrosine kinases. We showed that resistant melanoma cells were more sensitive to NK cell lysis due to changes in their phenotype. Resistance notably induces an increase of the expression of the ligands of NKG2D, MICA and ULBP2, which is involved in the activation of NK functions by melanoma cells. We also observed a change in the sensitivity to apoptosis induced by death domain receptors. This study therefore demonstrates the utility of NK cell based immunotherapy in combination with BRAF and MEK inhibitors. We then studied the role of ILCs in the immunosurveillance of lymph node metastases in breast cancer. This work allowed us to identify a CD127+CD56+ ILC3-like cells, identified as an ILC precursor, reduced in metastatic LN compared to healthy LN, in favor of NK cells. Although showing an activated transcriptomic profile, we observe a decrease in the expression of NCRs at the surface of NK cells in metastatic LN, suggesting a decrease in these receptors after contact with tumor cells. For ILC3, we observe an increase in the production of TNFα, which in the context of breast cancer, could participate in tumor progression. This study shows the importance of ILC3 and NK cells in tumor immunosurveillance in the LN. On one hand, ILC3s seem to have pro-tumor effects, so we should consider blocking this population, and on the other hand, NK cells seem to be altered, so they should be reactivated. Immunotherapies targeting ILCs should therefore be considered in cases of LN invasion in breast cancer.Les cellules lymphocytaires innées (ILC) sont des effecteurs de l’immunité innée et jouent un rôle important dans la régulation de la réponse immunitaire. Les cellules NK (ILC1 cytotoxiques) sont connues pour leurs capacités anti-tumorales. La réponse des ILC auxiliaires (ILC1, ILC2 et ILC3) face à la tumeur est encore peu décrite chez l’Homme. Par leur résidence tissulaire, les ILC auxiliaires pourraient jouer un rôle important dans la réponse immunitaire face au cancer. Le développement des thérapies ciblées et des immunothérapies a révolutionné les traitements de nombreux cancers. Les traitements actuels sont « personnalisés », adaptés au profil mutationnel du patient et à la qualité du microenvironnement tumoral. Ces traitements entraînent des réponses rapides et efficaces dans la plupart des cas. Cependant, des mécanismes de résistances sont fréquents, entraînant la rechute de la maladie. Les mécanismes de résistance sont encore mal connus. Les approches actuelles combinent des thérapies ciblant les anomalies spécifiques de la tumeur et un contournement de l’immunosuppression pour restaurer une immunité anti-tumorale effective. Mon projet de thèse s’inscrit dans cette problématique à travers deux projets : un projet de recherche translationnelle pour analyser l’impact de la résistance aux thérapies ciblées sur l’immunogénicité NK et un projet plus exploratoire qui vise à définir le rôle des cellules lymphoïdes innées (ILC) ganglionnaire dans l’immunosurveillance tumorale. Le premier projet est centré sur l’étude des ILC cytotoxiques, les cellules NK, et leurs interactions avec les cellules tumorales de mélanome résistantes aux thérapies ciblées, dirigées contre les tyrosines kinases BRAF (Vemurafenib) et MEK (Cobimetinib). Nous avons montré que les cellules résistantes étaient plus sensibles à la lyse par les cellules NK due à des modifications de leur phénotype. L’induction de la résistance induit notamment une augmentation de l’expression des ligands de NKG2D, MICA et ULBP2, qui est impliqué dans l’activation des fonctions NK par les cellules de mélanome. Nous avons également observé une modification de la sensibilité à l’apoptose induite par les récepteurs à domaine de mort. Cette étude démontre donc l’utilité d’une immunothérapie basée sur les cellules NK en combinaison avec des inhibiteurs de BRAF et MEK. Nous nous sommes ensuite intéressés aux rôles des ILC dans l’immunosurveillance des métastases ganglionnaires du cancer du sein. Ce travail nous a permis d’identifier une population CD127+CD56+ « ILC3-like », identifiée comme précurseur ILC, diminuée dans les ganglions métastatiques par rapport à des ganglions sains, au profit des cellules NK. Bien que présentant un profil transcriptomique activé, nous observons une diminution de l’expression des NCRs à la surface des cellules NK dans les ganglions métastatiques, suggérant une diminution de ces récepteurs après contact avec les cellules tumorales. Pour les ILC3, nous observons une augmentation de la production de TNFα, qui dans un contexte de cancer du sein, pourrait participer à la progression tumorale. Cette étude montre l’importance des ILC3 et des cellules NK dans l’immunosurveillance tumorale dans le ganglion. D’un côté, les ILC3 semblent avoir des effets pro-tumoraux, il faudrait donc envisager de bloquer cette population, et d’un autre côté les cellules NK semblent altérées, il faudrait donc les réactiver. Les immunothérapies ciblant les ILC devraient donc être considérées dans les cas d’invasion ganglionnaire dans le cancer du sein

Metastatic tumors immunosurveillance by innate lymphoid cells

Les cellules lymphocytaires innées (ILC) sont des effecteurs de l’immunité innée et jouent un rôle important dans la régulation de la réponse immunitaire. Les cellules NK (ILC1 cytotoxiques) sont connues pour leurs capacités anti-tumorales. La réponse des ILC auxiliaires (ILC1, ILC2 et ILC3) face à la tumeur est encore peu décrite chez l’Homme. Par leur résidence tissulaire, les ILC auxiliaires pourraient jouer un rôle important dans la réponse immunitaire face au cancer. Le développement des thérapies ciblées et des immunothérapies a révolutionné les traitements de nombreux cancers. Les traitements actuels sont « personnalisés », adaptés au profil mutationnel du patient et à la qualité du microenvironnement tumoral. Ces traitements entraînent des réponses rapides et efficaces dans la plupart des cas. Cependant, des mécanismes de résistances sont fréquents, entraînant la rechute de la maladie. Les mécanismes de résistance sont encore mal connus. Les approches actuelles combinent des thérapies ciblant les anomalies spécifiques de la tumeur et un contournement de l’immunosuppression pour restaurer une immunité anti-tumorale effective. Mon projet de thèse s’inscrit dans cette problématique à travers deux projets : un projet de recherche translationnelle pour analyser l’impact de la résistance aux thérapies ciblées sur l’immunogénicité NK et un projet plus exploratoire qui vise à définir le rôle des cellules lymphoïdes innées (ILC) ganglionnaire dans l’immunosurveillance tumorale. Le premier projet est centré sur l’étude des ILC cytotoxiques, les cellules NK, et leurs interactions avec les cellules tumorales de mélanome résistantes aux thérapies ciblées, dirigées contre les tyrosines kinases BRAF (Vemurafenib) et MEK (Cobimetinib). Nous avons montré que les cellules résistantes étaient plus sensibles à la lyse par les cellules NK due à des modifications de leur phénotype. L’induction de la résistance induit notamment une augmentation de l’expression des ligands de NKG2D, MICA et ULBP2, qui est impliqué dans l’activation des fonctions NK par les cellules de mélanome. Nous avons également observé une modification de la sensibilité à l’apoptose induite par les récepteurs à domaine de mort. Cette étude démontre donc l’utilité d’une immunothérapie basée sur les cellules NK en combinaison avec des inhibiteurs de BRAF et MEK. Nous nous sommes ensuite intéressés aux rôles des ILC dans l’immunosurveillance des métastases ganglionnaires du cancer du sein. Ce travail nous a permis d’identifier une population CD127+CD56+ « ILC3-like », identifiée comme précurseur ILC, diminuée dans les ganglions métastatiques par rapport à des ganglions sains, au profit des cellules NK. Bien que présentant un profil transcriptomique activé, nous observons une diminution de l’expression des NCRs à la surface des cellules NK dans les ganglions métastatiques, suggérant une diminution de ces récepteurs après contact avec les cellules tumorales. Pour les ILC3, nous observons une augmentation de la production de TNFα, qui dans un contexte de cancer du sein, pourrait participer à la progression tumorale. Cette étude montre l’importance des ILC3 et des cellules NK dans l’immunosurveillance tumorale dans le ganglion. D’un côté, les ILC3 semblent avoir des effets pro-tumoraux, il faudrait donc envisager de bloquer cette population, et d’un autre côté les cellules NK semblent altérées, il faudrait donc les réactiver. Les immunothérapies ciblant les ILC devraient donc être considérées dans les cas d’invasion ganglionnaire dans le cancer du sein.Innate lymphoid cells (ILCs) are effectors of innate immunity and play an important role in the regulation of the immune response. NK cells (cytotoxic ILC1) are known for their anti-tumor functions. The immune response of helper-type ILCs (hILC) (ILC1, ILC2 and ILC3) against the tumor is still poorly described in Humans. By their tissue residency, hILCs could play an important role in the immune response to cancer. The development of targeted therapies and immunotherapies has revolutionized the treatment of many cancers. Current therapies are “personalized”, adapted to the mutational profile of the patient and to the quality of the tumor microenvironment. These treatments result in rapid and effective responses in most of the cases. However, resistance mechanisms are frequent, leading to relapses. The mechanisms of resistance are still poorly understood. Current approaches combine therapies targeting specific tumor abnormalities and bypassing immunosuppression to restore effective anti-tumor immunity. My thesis project attempts to address this issue through two projects: a translational research project to analyze the impact of resistance to targeted therapies on NK immunogenicity and a more exploratory project which aims to define the role of lymph node (LN) ILC in tumor immunosurveillance. The first project focuses on the study of cytotoxic ILCs, NK cells, and their interactions with melanoma cells resistant to targeted therapies, directed against the BRAF (Vemurafenib) and MEK (Cobimetinib) tyrosine kinases. We showed that resistant melanoma cells were more sensitive to NK cell lysis due to changes in their phenotype. Resistance notably induces an increase of the expression of the ligands of NKG2D, MICA and ULBP2, which is involved in the activation of NK functions by melanoma cells. We also observed a change in the sensitivity to apoptosis induced by death domain receptors. This study therefore demonstrates the utility of NK cell based immunotherapy in combination with BRAF and MEK inhibitors. We then studied the role of ILCs in the immunosurveillance of lymph node metastases in breast cancer. This work allowed us to identify a CD127+CD56+ ILC3-like cells, identified as an ILC precursor, reduced in metastatic LN compared to healthy LN, in favor of NK cells. Although showing an activated transcriptomic profile, we observe a decrease in the expression of NCRs at the surface of NK cells in metastatic LN, suggesting a decrease in these receptors after contact with tumor cells. For ILC3, we observe an increase in the production of TNFα, which in the context of breast cancer, could participate in tumor progression. This study shows the importance of ILC3 and NK cells in tumor immunosurveillance in the LN. On one hand, ILC3s seem to have pro-tumor effects, so we should consider blocking this population, and on the other hand, NK cells seem to be altered, so they should be reactivated. Immunotherapies targeting ILCs should therefore be considered in cases of LN invasion in breast cancer

Specific Patterns of Blood ILCs in Metastatic Melanoma Patients and Their Modulations in Response to Immunotherapy

Immunotherapy targeting immune checkpoint receptors brought a breakthrough in the treatment of metastatic melanoma patients. However, a number of patients still resist these immunotherapies. Present on CD8+T cells, immune checkpoint receptors are expressed by innate lymphoid cells (ILCs), which may contribute to the clinical response. ILCs are composed of natural killer (NK) cells, which are cytotoxic effectors involved in tumor immunosurveillance. NK cell activation is regulated by a balance between activating receptors that detect stress molecules on tumor cells and HLA-I-specific inhibitory receptors. Helper ILCs (h-ILCs) are newly characterized ILCs that secrete cytokines and regulate the immune homeostasis of tissue. We investigated the modulation of blood ILCs in melanoma patients treated with ipilimumab. Circulating ILCs from metastatic stage IV melanoma patients and healthy donors were studied for their complete phenotypic status. Patients were studied before and at 3, 6, and 12 weeks of ipilimumab treatment. A comparison of blood ILC populations from donors and melanoma patients before treatment showed changes in proportions of ILC subsets, and a significant inverse correlation of CD56dim NK cells and h-ILC subsets was identified in patients. During treatment with ipilimumab, percentages of all ILC subsets were reduced. Ipilimumab also impacted the expression of the CD96/TIGIT/DNAM-1 pathway in all ILCs and increased CD161 and CTLA-4 expression by h-ILCs. When considering the response to the treatment, patients without disease control were characterized by higher percentages of CD56bright NK cells and ILC1. Patients with disease control displayed larger populations of activated CD56dimCD16+ DNAM-1+ NK cells, while anergic CD56dimCD16−DNAM-1− NK cells were prominent in patients without disease control. These results provide original findings on the distribution of ILC subsets in advanced melanoma patients and their modulation through immunotherapy. The effects of ipilimumab on these ILC subsets may critically influence therapeutic outcomes. These data indicate the importance of considering these innate cell subsets in immunotherapeutic strategies for melanoma patients

BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis

BackgroundTargeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine–threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a BRAFV600E/K mutation. However, acquired resistance occurs in the majority of patients, leading to relapse. Immunotherapies that activate immune cytotoxic effectors induce long-lasting responses in 30% of patients. In that context, combination of targeted therapies with immunotherapy (IT) is a promising approach. We considered boosting natural killer (NK) cell tumor immunosurveillance, as melanoma cells express stress-induced molecules and activate NK cell lysis.MethodsHere we have generated vemurafenib (a BRAF inihibitor)-resistant (R) cells from BRAFV600E SK28 and M14-sensitive (S) melanoma cell lines and investigated how resistance interferes with immunogenicity to NK cells. We determined the levels of several soluble molecules including NK ligands in 61 melanoma patients at baseline and 6 months M post-treatment with targeted therapies or immunotherapies.ResultsVemurafenib resistance involved activation of p-AKT in SK28R and of p-MEK/p-ERK in M14R cells and was accompanied by modulation of NK ligands. Compared with S cells, SK28R displayed an increased expression of natural killer group 2 D (NKG2D) receptor ligands (major histocompatibility complex class (MHC) I chain-related protein A (MICA) and UL16-binding protein 2 (ULBP2)) whereas M14R exhibited decreased ULBP2 . SK28R and M14R cells induced higher NK degranulation and interferon gamma secretion and were more efficiently lysed by donor and patient NK cells. SK28R showed increased tumor necrosis factor-related apoptosis-inducing ligand receptor II (TRAIL-RII) expression and TRAIL-induced apoptosis, and TRAIL-induced apoptosis of M14R was decreased. Combined BRAF/MEK inhibitors abrogated the growth of SK28S, M14S, and M14R cells, while growth of SK28R was maintained. BRAF/MEK inhibition attenuated NK activity but R cell lines activated polyfunctional NK cells and were lysed with high efficiency. We investigated the relationship of soluble NK ligands and response to treatment in a series of melanoma patients. Soluble NKG2D ligands known to regulate the receptor function have been associated to cancer progression. Serum analysis of patients treated with target therapies or IT indicates that soluble forms of NK ligands (MICA, B7H6, programmed cell death ligand 1, and carcinoembryonic antigen cell adhesion molecule 1) may correlate with clinical response.ConclusionThese results support strategies combining targeted therapies and NK-based immunotherapies.</jats:sec

BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis

Background Targeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine–threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a BRAFV600E/K mutation. However, acquired resistance occurs in the majority of patients, leading to relapse. Immunotherapies that activate immune cytotoxic effectors induce long-lasting responses in 30% of patients. In that context, combination of targeted therapies with immunotherapy (IT) is a promising approach. We considered boosting natural killer (NK) cell tumor immunosurveillance, as melanoma cells express stress-induced molecules and activate NK cell lysis.Methods Here we have generated vemurafenib (a BRAF inihibitor)-resistant (R) cells from BRAFV600E SK28 and M14-sensitive (S) melanoma cell lines and investigated how resistance interferes with immunogenicity to NK cells. We determined the levels of several soluble molecules including NK ligands in 61 melanoma patients at baseline and 6 months M post-treatment with targeted therapies or immunotherapies.Results Vemurafenib resistance involved activation of p-AKT in SK28R and of p-MEK/p-ERK in M14R cells and was accompanied by modulation of NK ligands. Compared with S cells, SK28R displayed an increased expression of natural killer group 2 D (NKG2D) receptor ligands (major histocompatibility complex class (MHC) I chain-related protein A (MICA) and UL16-binding protein 2 (ULBP2)) whereas M14R exhibited decreased ULBP2 . SK28R and M14R cells induced higher NK degranulation and interferon gamma secretion and were more efficiently lysed by donor and patient NK cells. SK28R showed increased tumor necrosis factor-related apoptosis-inducing ligand receptor II (TRAIL-RII) expression and TRAIL-induced apoptosis, and TRAIL-induced apoptosis of M14R was decreased. Combined BRAF/MEK inhibitors abrogated the growth of SK28S, M14S, and M14R cells, while growth of SK28R was maintained. BRAF/MEK inhibition attenuated NK activity but R cell lines activated polyfunctional NK cells and were lysed with high efficiency. We investigated the relationship of soluble NK ligands and response to treatment in a series of melanoma patients. Soluble NKG2D ligands known to regulate the receptor function have been associated to cancer progression. Serum analysis of patients treated with target therapies or IT indicates that soluble forms of NK ligands (MICA, B7H6, programmed cell death ligand 1, and carcinoembryonic antigen cell adhesion molecule 1) may correlate with clinical response.Conclusion These results support strategies combining targeted therapies and NK-based immunotherapies