Among these two pathways, the JAK/STAT pathway played a dominant role, with combinatorial inhibition by IKK-16 and ruxolitinib affording no additional benefit beyond ruxolitinib treatment alone (determine 7ACC)

Among these two pathways, the JAK/STAT pathway played a dominant role, with combinatorial inhibition by IKK-16 and ruxolitinib affording no additional benefit beyond ruxolitinib treatment alone (determine 7ACC). analysis. Results Single-cell analysis using mass and flow cytometry exhibited that IFN consistently induced PD-L1 and MHC class Cetylpyridinium Chloride I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFN treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFN, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFN treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. Conclusions These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies. strong class=”kwd-title” Keywords: immunology, interferon, tumours Background In the USA, lung cancer has an incidence of 225?000 patients every year leading to approximately 160?000 deaths.1 Over the last several years, the development of targeted therapeutics for the treatment of patients with non-small-cell lung cancer (NSCLC) with specific genetic changes, such as epidermal growth factor receptor (EGFR) mutations, echinoderm microtubule-associated protein like-4-anaplastic lymphoma kinase (EML4/ALK) fusion Cetylpyridinium Chloride and c-ros oncogene 1 (ROS1) fusions, have led to exciting new treatment options for these patients. Unfortunately, virtually all lung cancers with driver mutations ultimately develop resistance to targeted therapies.2 Another recent development in the treatment of NSCLC involves the use of antibodies targeting immune checkpoint molecules including PD-1 or its ligand PD-L1, which can lead to durable responses in around 15%C20% of unselected patients with advanced NSCLC.3 4 Despite promising results with these immunotherapy-based therapies, the majority of patients with lung cancer fail to respond to this intervention. Extensive ongoing research efforts continue to define predictors of response to checkpoint therapy, with tumor mutational burden and characteristics of the tumor microenvironment (TME), including lymphocytic infiltration and an interferon gamma (IFN) responsive gene signature (ie, PD-L1 expression and the induction of antigen presentation machinery, MHC class I and class II molecules) positively correlated with response to therapy.5C9 Additional studies have further identified mechanisms of resistance to checkpoint therapy, including mutations in genes associated with IFN signaling [janus kinase (JAK)1 and JAK2] and antigen presentation (beta-2-microglobulin).10 Our group has previously analyzed determinants of response to immune checkpoint blockade, using orthotopic implantation of KRAS mutant NSCLC lines into syngeneic hosts.11 These studies have demonstrated that this CMT167 cell line is both sensitive to checkpoint blockade with PD-1/PD-L1 antibodies and exhibits an IFN responsive phenotype in vivo. Conversely, the Lewis lung carcinoma (LLC) cell line is usually resistant to checkpoint blockade and has a blunted Neurog1 IFN-inducible gene signature in vivo. Notably, while both CMT167 and LLC cells show induction of some IFN-inducible genes in vivo, including PD-L1, CMT167 cells showed a unique induction of genes encoding the MHC class II antigen presentation and processing pathway. 12 In this study, we aimed to Cetylpyridinium Chloride characterize the determinants of divergent IFN responsiveness between these tumor lines in vitro in order to gain mechanistic insights that may lead to novel approaches to enhance IFN sensitivity and thus make more tumors susceptible to PD-1/PD-L1 checkpoint blockade, with an emphasis on MHC II regulation. Our studies point to a critical requirement for epigenetic and post-translational-dependent mechanisms that actively restrict IFN induction of the MHC class II machinery in NSCLC. Methods Cell lines Murine cell lines: CMT167 and LLC cells stably expressing firefly luciferase were used in this study.11 Luciferase-expressing LLC cells were purchased from Caliper Life Sciences (LL/2-luc-M38). CMT167 cells were originally obtained from the lab of Alvin Malkinson.