C.M. Della Corte

51PD Effect of MEK inhibition on PDL1 and and on cytokinesproduction profilein NSCLC cell lines and in human lymphocites

Background: Preclinical models suggest that MAPK pathway is implicated in the immune-resistance of tumors and MEK-inhibition can increase the CD8+ T-cell infiltration and the efficacy of PD-1/PD-L1 blockade. Methods: First, we evaluated PD-L1 mRNA expression by Real Time qPCR and its protein production, togheter with MAPK proteins in a panel of non-small cell lung cancer (NSCLC) cell lines. Then, we studied the changes in PD-L1 and major histocompatibility complex class-I (MHC-I) expression and cytokines’ production, after inhibition with selumetinib or stimulation of MAPK signalling by phorbol 12-myristate 13-acetate (PMA). In addition, we explored the effect of MEK inhibition on T-cell function by using Peripheral blood mononuclear cells (PBMC) from healthy volunteers. Results: A consistent correlation between PD-L1 mRNA and protein expression across cell lines suggested that expression mainly depends on trascriptional regulation, and it is regulated by MAPK signal, through the bindng of p65 to the PD-L1 promoter. Moreover, MEK inhibition resulted in an increased expression of MHC-I on cancer cells and increased mRNA expression levels of IFN gamma, IL-6, IL-1B, and TNFalpha, all molecules involved in the activation and differentiation of TCD8+ cytotoxic lymphocytes (CTL) subset. In this scenario, we also tested the effect of MEK inhibitor on activated T-lymphocytes from PBMC of healthy volunteers. After five days of treatment, RT-qPCR analysis revealed a significant increase of mRNA expression of some typical CD8+ T cell pro-inflammatory cytokines, like IL-12, TNFalpha and IFNgamma. Conclusions: These results further support the idea that MEK inhibitor reduces PD-L1 expression and this allows the establishment of a pro inflammatory microenvironment. On the other side, pheripheral T cells, treated with selumetinib, produce pro inflammatory cytokines typical of CTL subset, that seems more involved in immune response against cancer. In this context, MEK inhibition may represent a potential mechanism to convert otherwise resistant cancers and suggest new potential treatment combination strategies of MEK-inhibitors with anti-PD-L1 antibodies in NSCLC. Legal entity responsible for the study: University of Campania “L. Vanvitelli” Funding: Has not received any funding Disclosure: All authors have declared no conflicts of interest.

1604PROLE OF HEDGEHOG PATHWAY IN MEDIATING RESISTANCE TO EGFR-INHIBITORS IN NON-SMALL CELL LUNG CANCER

ABSTRACT Aim: Tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) are treatment choices in the management of EGFR wild-type (WT) non-small cell lung cancer (NSCLC) patients who progressed on previous chemotherapy. However, development of resistance, even in responders, is sooner or later observed. Recently our group demonstrated the acquisition of a mesenchymal phenotype in an in vitro model of NSCLC cell lines with acquired resistance to anti-EGFR TKIs. The Hedgehog (Hh) signaling pathway has emerged as an important mediator of carcinogenesis and cancer metastases. The aim of this work is to explore the Hh role in EGFR TKIs resistence. Methods: We studied the activation of Hh signaling (Shh, Gli1, PTCH, Smo) in a panel of NSCLC cell lines (H460, H1299, A549, GLC82, CALU-3, H322, H358), harbouring WT EGFR, and with different sensitivity to two EGFR TKIs, gefitinib and erlotinib. Immunohistochemistry of PTCH and Shh was performed on 33 histological samples of NSCLC patients treated with erlotinib in second/third line of therapy. Results: Experiments showed a strong expression of Shh, Smo, PTCH and Gli1 in NSCLC cell lines resistant to EGFR TKIs, indicating activation of Hh signalling in such cells. Treatment with different Smo inhibitors, cyclopamine, LDE225 or vismodegib, in combination with EGFR inhibitors, strongly inhibited the proliferation of resistant NSCLC cell lines and induced apoptosis. Furthermore, combined treatment blocked the invasive and migratory behavior of resistant cells along with a complete inhibition of PI3K/Akt and MAPK phosphorylation. Preliminary data, obtained from the immunohistochemical analysis of Hh related molecules, showed higher expression of Shh and PTCH in cancer cells as compared to surrounding normal tissue. Among 20 patients, which received EGFR TKI in second/third line of therapy, those presenting a lower Shh and PTCH expression experienced a trend toward a better progression free survival. Conclusions: Our study should provide the opportunity to better understand the role of HH pathway in mediating resistance to anti-EGFR TKIs and to design new strategies to be easily transferred into clinical practice. Disclosure: All authors have declared no conflicts of interest.

1609PROLE OF EGFR AND AXL PATHWAYS IN MEDIATING PROLIFERATION AND INVASIVENESS OF HUMAN MESOTHELIOMA CELL LINES

ABSTRACT Aim: Preclinical data identified Axl, a tyrosine kinase receptor, as a key mediator of tumor progression and epithelial mesenchymal transition in several types of cancers. It was also demonstrated his role in mediating epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) resistance in lung adenocarcinoma. Recent findings revealed that Axl overexpression in tissue specimes correlated with patients prognosis and survival in malignant pleural mesothelioma (MPM). So, the aim of this work is to investigate EGFR and Axl role in mesothelioma. Methods: Protein levels and activation status of EGFR and Axl were analyzed in a panel of mesothelioma cell lines (H2452, H2052, H28, MSTO211H). Single agent and combined treatment with foretinib (a multitarget inhibitor of Met, Axl, and others) and gefitinib were tested in vitro to assess synergistic effects on cell proliferation and activation/inhibition of downstream pathways. Results: By using phospho-receptor tyrosine kinase (RTK) arrays we screened 49 RTKs activation in human mesothelioma cell lines. H2452 and H28 cell lines had both increased level of phosphorylated/activated Axl and EGFR along with increased levels of GAS6 mRNAs levels, indicating an activation of these pathways. Combined treatment with foretinib and gefitinib inhibited cell proliferation, and to a greater extent, migration and invasion capabilities, indicating a potential role of these pathways in the acquisition of mesenchymal phenotype. Response to combined treatment in these cell lines was characterized by inhibition of PI3-K/AKT/mTOR and MAPK signaling. Conclusions: Our data suggest that both Axl and EGFR pathways may exert a role in proliferation and survival of mesothelioma cells and imply their role as potential new therapeutic targets warranting further clinical evaluation in mesothelioma. Disclosure: All authors have declared no conflicts of interest.

643PCELL GROWTH INHIBITION OF HER2 POSITIVE TRASTUZUMAB RESISTANT GASTRIC CANCER CELL LINES BY COMBINED INHIBITION OF PI3K/AKT/MTOR AND MAPK

ABSTRACT Aim: HER2 is a key genetic driver in gastric cancer development. The anti-HER2 antibody, trastuzumab, in combination with standard chemotherapy,increases survival in HER2 positive gastric cancer. However it is still unclear why HER2 overexpressing tumours are initially and subsequently nonresponsive to trastuzumab treatment. Our aim is to investigate the possible mechanisms involved in trastuzumab resistance. Methods: A panel of human gastric and oesophago-gastric cancer cell lines(NCI-N87, KATO III, OE 19) were screened for EGFR, HER2, HER3, MAPK, PI3K/Akt/mTOR and 4EBP-1 expressions by Western Blot analysis. Cells were treated with increasing concentrations of selective inhibitors such as trastuzumab (HER2 inhibitor), erlotinib (EGFR inhibitor), MEK inhibitor (BAY-86-9766) or PIK3CA/mTOR inhibitor (GDC-0980) as a single agent and/or in combination. MTT analyses were performed to evaluate the inhibitory effect of each compound on cell growth. Results: In all cancer cells EGFR, HER2, MAPK, AKT and 4EBP1 were expressed at different degree. Although high HER2 expression levels, trastuzumab caused growth inhibition only in OE19 cancer cell line with no activity showed in KATO III and NCI-N87, suggesting a trastuzumab intrinsic resistance. The combination of MEK and PIK3CA/mTOR inhibitors in cancer cell lines was able to inhibit cell growth of NCI-N87, KATOIII and OE19 with an IC50 of 0.25 µM, 0.01 µM and 0.25 µM, respectively. The role of such therapeutic doublet in achieving cell growth inhibition was accompanied by significant reduction in the expression of AKT, pAKT, MAP, pMAP, 4EBP1 and p4EBP1 compared to control and trastuzumab treatment as revealed by Western Blot analysis. Conclusions: The results of our in vitro study suggest that in HER2 trastuzumb resistant gastric cancer cell lines the double blockade of MAP and PI3K/Akt/mTOR pathways is able to induce significant growth inhibition suggesting a relevant crosstalk between the two pathways in this setting. On the basis of these preclinical data, new potential approaches could be explored in gastric cancer HER2 patients not responding to trastuzumab. Disclosure: All authors have declared no conflicts of interest.

1586PCO-ACTIVATION OF HEDGEHOG AND MET PATHWAYS IN MEDIATING RESISTANCE TO GEFITINIB IN NON-SMALL CELL LUNG CANCER CELL LINE HARBORING ACTIVATING MUTATION OF EGFR

ABSTRACT Aim: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective treatments in the management of patients harboring EGFR activating mutations. However, all initially responsive patients experience relapse as a result of acquired drug resistance. The epithelial-to-mesencymal transition (EMT) is a critical event in the development of metastases and represents an important mechanism of acquired resistance to molecularly targeted agents in non-small cel lung cancer (NSCLC). Among the various molecular pathways, the Hedgehog (Hh) signaling cascade has emerged as an important mediator of EMT. Methods: We studied the activation of Hh signaling in a NSCLC model of acquired resistance to gefitinib (HCC827-GR) obtained from a cell line harboring an activating mutation (del exon 19) of EGFR (HCC827). Results: HCC827-GR cells displayed an EMT phenotype, along with higher expression aand increased activation of MET, MAPK and AKT. Asignificantly increased expression of Shh, Smo and Gli1 mRNAs and proteins were evidenced as compared to parental HCC827 cells. Combined inhibition of MET and Hh pathways strongly inhibited cell proliferation, migration, invasion, reverted the mesenchymal phenotype and induced apoptosis at higher level than single inhibition of each pathway. MET and Smo proteins co-precipitated in HCC827-GR cells suggesting a reciprocal activation with a canonical activation of Gli1. Furthermore, MET inhibition strongly reduced Gli1 trascriptional activity, indicating a direct activity of MET-activated downstream pathways on Gli1 activation suggetsing also a cMET-mediated non canonical activation of Gli1. Conclusions: Our data suggest that both MET and Hh pathways provide alternative proliferation and survival mechanisms for NSCLC cancer cells harboring EGFR activating mutations in which EGFR is blocked by gefitinib. Disclosure: All authors have declared no conflicts of interest.