Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MET-oncogenic and JAK2-inactivating alterations are independent factors that affect regulation of PD-L1 expression in lung cancer Maria Saigi1*, Juan J Alburquerque-Bejar1*, Anne Mc Leer-Florin2, Carolina Pereira1, Eva Pros1, Octavio A. Romero1, Nuria Baixeras3, Anna Esteve-Codina4,5, Ernest Nadal6,7, Elisabeth Brambilla2and Montse Sanchez-Cespedes1 1Genes and Cancer Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain. 2Département d’Anatomie et Cytologie Pathologiques, Pôle de Biologie et Pathologie, CHU Grenoble Alpes, Grenoble and Université Grenoble Alpes, Grenoble, France. 3Pathology Department, Bellvitge University Hospital, Hospitalet de Llobregat, Spain. 4CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain. 5 Universitat Pompeu Fabra (UPF), 6Department of Medical Oncology, Catalan Institute of Oncology (ICO), Hospitalet de Llobregat, Barcelona, Spain. 7Clinical Research in Solid Tumors (CReST) Group, OncoBell Program, Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain. Corresponding author: Montse Sanchez-Cespedes, Genes and Cancer Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908, Hospitalet de Llobregat, Barcelona, Spain. Email: [email protected] *Both authors contributed equally to this work Disclosure of Potential Conflicts of Interest: E Nadal has received consultancy fees from Pfizer and Roche. No potential conflicts of interest were disclosed by the other authors. Running title: MET activity, JAK2 inactivation and PD-L1 expression in lung cancer Key words: PD-L1, immunotherapy, MET, JAK2, lung cancer, interferon gamma Saigi M, Alburquerque-Bejar JJ et al. 1 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer (LC) to the ability of tumor cells to escape immunosurveillance checkpoints. Experimental Design: Over 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for the levels of HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes and alterations in main LC genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments. Results: MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also up-regulated other immunosuppressive genes (PDCD1LG2 and SOCS1), and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ. Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in LC cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Saigi M, Alburquerque-Bejar JJ et al. 2 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance The ability of tumors to avoid host immune surveillance through the blockade of PD-L1 and PD-1 is being exploited therapeutically. However, few biomarkers are currently well-established predictors of the response to these treatments. Here we found that activation of MET in lung cancer triggers a transcriptional increase of PD-L1. This was framed within a more generic transcriptional activation of immunosuppression-related molecules. We also report recurrent inactivating mutations at JAK2 that impair the up-regulation of immunoresponse-related transcripts when treated with IFNγ. Therefore, MET and JAK2 alterations in lung cancer contribute to promote immunotolerance and may constitute biomarkers for predicting responsiveness to immunotherapies in this type of cancer. Saigi M, Alburquerque-Bejar JJ et al. 3 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction A hallmark of cancer biology is the ability of tumors to avoid host immune surveillance. This characteristic has turned out to be therapeutically exploitable in novel immunotherapies (1). Several alterations in cancer cells are known to impair immune surveillance. These include defects in tumor immunorecognition, such as down-regulation of the HLA-I complex due to gene alterations of its components (either B2M or HLA-A) or at molecules involved in its maturation process (e.g. CALR, TAP1/2, TAPBP) (2-4). An abnormal increase in the expression of negative controllers of the immune response, such as constitutively high levels of programmed death ligand-1 (PD-L1) expression, also contributes to immune tolerance (1). The programmed cell death-1 (PD-1) receptor is expressed in activated T and B cells and regulates the cellular and humoral immune responses by interacting with its cognate ligand, PD- L1, which is expressed in the macrophage lineage and in some carcinomas, including lung cancer (LC) (5). The interaction of PD-1 with PD-L1 attenuates the immune response by inhibiting T-cell proliferation and functions (1,5). The blockade of these checkpoints is the means by which therapeutic agents are intended to enhance tumor immunological responses. In non-small cell lung cancer (NSCLC), anti-PD-1/-PD-L1 therapies have yielded clinical responses and increases in overall survival in subsets of patients (6-8). This clinical benefit makes it crucial to identify factors that determine the sensitivity or refractoriness to immune checkpoint inhibitors (ICIs). Several functionally interrelated tumor biomarkers have been proposed as predictors of the response to ICIs, including high tumor mutational load, DNA mismatch repair deficiency, strong intratumoral T cell infiltration and a high level expression of PD-L1 and of the HLA-I-class complex (4, 6-9). In addition, inactivating mutations of JAK2 in melanoma, involved in IFNγ-signaling, and of B2M in melanoma and in LC, account for primary and acquired resistance to these treatments (4, 10-11). Although somewhat controversial, the levels of PD-L1 protein are among the current main criteria for selecting patients to receive anti-PD-1 or anti-PD-L1 therapies for LC (12-13). Since Saigi M, Alburquerque-Bejar JJ et al. 4 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. these molecules are direct targets of immunotherapy, it is of great interest to understand the intrinsic molecular and genetic events that modulate their expression in cancer cells. Some researchers have proposed that genetic activation of growth factor receptors in LC, such as EGFR, ALK and ROS1, are associated with a high level of expression of immunocheckpoint proteins, including PD-L1 (14-16). Finally, TP53/KRAS-co-mutated tumors have also been proposed to be associated with increased expression levels of PD-L1 and with a notable clinical benefit to those patients who are treated with PD-1 inhibitors (17). However, the results so far are not conclusive (12, 18). Our current work aims to understand the contribution of LC genetics to the ability of tumor cells to escape immunosurveillance. Saigi M, Alburquerque-Bejar JJ et al. 5 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 13, 2018; DOI: 10.1158/1078-0432.CCR-18-0267 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Tumor samples and cancer cell lines Tumor specimens were collected from patients newly diagnosed with NSCLC at