Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer.

BACKGROUND: We aimed to understand the dependence of MEK and m-TOR inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cell lines. METHODS: In a panel of KRAS(M) and KRAS(WT) NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively. RESULTS: GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P<10(-4)). There was no significant difference in GI caused by maximal m-TOR compared with maximal m-TOR+MEK inhibition. However, GI caused by the combination was significantly greater in the KRAS(M) cell lines (79% vs 61%, P=0.017). CONCLUSIONS: m-TOR inhibition was more critical to GI than MEK inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cells. The combination of MEK and m-TOR inhibition was most effective in KRAS(M) cells.

Characterisation of the Phosphatidylinositol 3-Kinase Pathway in Non-Small Cell Lung Cancer Cells Isolated from Pleural Effusions.

OBJECTIVES: We hypothesised that it was possible to quantify phosphorylation of important nodes in the phosphatidylinositol 3-kinase (PI3K) pathway in cancer cells isolated from pleural effusions of patients with non-small cell lung cancer (NSCLC) and study their correlation to somatic mutations and clinical outcomes. MATERIALS AND METHODS: Cells were immunomagnetically separated from samples of pleural effusion in patients with NSCLC. p-AKT, p-S6K and p-GSK3ß levels were quantified by ELISA; targeted next-generation sequencing was used to characterise mutations in 26 genes. RESULTS: It was possible to quantify phosphoproteins in cells isolated from 38/43 pleural effusions. There was a significant correlation between p-AKT and p-S6K levels [r = 0.85 (95% confidence interval 0.73-0.92), p < 0.0001], but not p-AKT and p-GSK3ß levels [r = 0.19 (95% confidence interval -0.16 to 0.5), p = 0.3]. A wide range of mutations was described and p-S6K was higher in samples that harboured at least one mutation compared to those that did not (p = 0.03). On multivariate analysis, p-S6K levels were significantly associated with poor survival (p < 0.01). CONCLUSION: Our study has shown a correlation between p-AKT levels and p-S6K, but not GSK3ß, suggesting differences in regulation of the distal PI3K pathway by AKT. Higher p-S6K levels were associated with adverse survival, making it a critically important target in NSCLC.

Percentage Change in Plasma Cytokeratin 18 Is Associated with Clinical Outcomes in Patients Receiving Pemetrexed and Carboplatin for the Adenocarcinoma Subtype of NSCLC.

BACKGROUND: The adenocarcinoma subtype of non-small cell lung cancer (adeno-NSCLC) is routinely treated with chemotherapy if patients do not have molecular aberrations such as epidermal growth factor receptor mutations or anaplastic lymphoma kinase rearrangements. There are currently no validated biomarkers that can predict if patients will gain clinical benefit from chemotherapy, leading to a majority of patients receiving many cycles of unnecessary chemotherapy. We hypothesized that the percentage rise in plasma caspase-cleaved cytokeratin 18 (cCK18) and total cytokeratin 18 (tCK18) assessed before and after chemotherapy correlates with the radiological response to chemotherapy. METHODS: Plasma samples from 40 patients with stage IV adeno-NSCLC, treated with first-line chemotherapy with carboplatin (AUC5) plus pemetrexed (500 mg/m(2)), were collected prior to chemotherapy and 48 h after treatment. ELISA was used to quantify cCK18 and tCK18. RESULTS: The male-to-female ratio was 3:1, and the median age of patients was 63 years. Patients who had a clinical benefit (complete response, partial response or stable disease) at the first radiological assessment following chemotherapy had a significantly higher percentage change in plasma tCK18 levels compared to those who had no clinical benefit, i.e. progressive disease (69.5 ± 75.1 vs. 25.3 ± 30.9%, respectively; p = 0.042). The receiver operating characteristic area was 0.712 (p = 0.039). There was an increase in the percentage change in cCK18 in patients with clinical benefit compared to those without clinical benefit but this was not statistically significant (57.6 ± 112.8 vs. 24.38 ± 45.1%, respectively; p = 0.85). CONCLUSIONS: The percentage change in plasma tCK18 levels before and after the first cycle of pemetrexed and carboplatin chemotherapy is associated with clinical benefit. If validated in larger cohorts, this test can be used to identify patients unlikely to respond to treatment who can thus be offered alternative treatments or entry into clinical trials.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments.

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models.

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer.

Low-dose IFN-gamma induces tumor MHC expression in metastatic malignant melanoma.

Specific antitumor immune responses require expression of MHC class I or II molecules on tumor cells, and MHC antigen down-regulation is a presumed tumor growth promoting mechanism. Because IFN-gamma up-regulates tumor MHC antigen expression in vitro, in this Phase II trial of an immunologically active dose and schedule we evaluated whether this was the case in vivo. Twenty-three patients with metastatic melanoma were treated with IFN-gamma 100 microg/m(2) s.c. once weekly for a maximum of 6 months. There were three complete responses, now maintained for 53, 36, and 25 months. The remainder had progressive disease. The treatment was well tolerated, with no toxicity exceeding National Cancer Institute Common Toxicity Criteria grade II. Immunohistochemical analysis of tumor biopsies during treatment was performed using monoclonal antibodies to HLA class I (W/632) and class II (CR3/43) monomorphic determinants. HLA class I was down-regulated in 2 of 19 patients pretreatment and up-regulated by IFN-gamma in both. HLA class II was down-regulated pretreatment in 14 of 18 patients and up-regulated by IFN-gamma in 6 (43%). The HLA up-regulation persisted throughout the study. IFN-gamma induced significant but short-lived up-regulation of surrogate markers of monocyte activation (serum neopterin) and class I up-regulation (serum beta-2-microglobulin) in most patients. There was no consistent relationship between surrogate marker up-regulation, tumor antigen up-regulation, and responses. The study shows that the significant immune modulation induced by IFN-gamma does not correlate with tumor responses and that the serum surrogate marker changes do not reflect tumor events. The durable and long-lived responses, clear demonstration of tumor MHC up-regulation, and low toxicity suggest that weekly IFN-gamma 100 microg/m(2) would be a useful addition to chemoimmunotherapeutic regimens.

The association of PI3 kinase signaling and chemoresistance in advanced ovarian cancer.

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3ß by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3ß. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN.