Molecular determinants of response to PI3K/akt/mTOR and KRAS pathways inhibitors in NSCLC cell lines.

Despite the impressive results obtained in the preclinical setting, all the inhibitors targeting two central cascades in cancer, the PI3K/akt/mTOR and the KRAS/MEK/ERK pathways, have shown, apart from very few exceptions, disappointing efficacy when translated to the clinic. One of the main reasons of their clinical failure seems to be the lack of a clear molecular determinant of response to these drugs. In this study, we tried to address this point by evaluating the cytotoxic activity of different inhibitors targeting the two pathways at different levels in a panel of ten NSCLC cell lines harboring alterations in PI3K, KRAS or both. We were not able to highlight a correlation between the presence of KRAS and PI3K mutations and a specific sensitivity to the different drugs used. Molecular analyses performed after equimolar treatments showed that, independently from the entity of the response, the drugs are able to modulate the activation of their targets. Interestingly, we found that p53 mutational status separates the cell lines according to their sensitivity to PI3K pathway inhibitors treatments. The alterations considered in the PI3K/akt/mTOR and in the KRAS/MEK/ERK pathways in the different NSCLC cell lines are not sufficient to drive treatment choice but rather p53 status is a potential biomarker for the activity of this class of drugs.

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

INTRODUCTION: Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy. METHODS: Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo. RESULTS: In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. CONCLUSIONS: This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.

Generation and characterization of MEK and ERK inhibitors- resistant non-small-cells-lung-cancer (NSCLC) cells.

BACKGROUND: The RAS/RAF/MEK/ERK pathway is one of the most downregulated pathway in cancer. Inhibitors of RAF and MEK have established clinical use while ERK inhibitors recently faced the clinic. We aimed to generate resistant cell lines which could be helpful for defining new combinations able to overcome resistance. METHODS: the human NSCLC cell line NCI-H727, sensitive to both MEK and ERK inhibitors, was treated with increasing concentrations of MEK162 (as MEK inhibitor) or SCH772984 as ERK inhibitor. RESULTS: we successfully obtained a MEK resistant subline (H727/MEK, after 40 passages) as well as an ERK resistant subline (H727/SCH, after 18 passages). The two resistant sublines H727/MEK and H727/SCH were cross-resistant to ERK and MEK inhibitors, respectively, but not to RAF inhibitors. The sublines maintained the responsiveness to inhibitors of the parallel PI3K/akt/mTOR pathway as well as to agents with different mechanism of action. Mechanistically, treatment of sensitive and resistant cells with MEK or ERK inhibitors was able to induce a similar inhibition of ERK phosphorylation, while only in parental cells the drugs were able to induce a downregulation of S6 and RSK phosphorylation. CONCLUSIONS: these resistant cells represent an important tool for further studies on the mechanisms of resistance and ways to overcome it.

Wee1 inhibitor MK1775 sensitizes KRAS mutated NSCLC cells to sorafenib.

Non-Small-Cell Lung Cancer (NSCLC) is a poorly chemosensitive tumor and targeted therapies are only used for about 15% of patients where a specific driving and druggable lesion is observed (EGFR, ALK, ROS). KRAS is one of the most frequently mutated genes in NSCLC and patients harboring these mutations do not benefit from specific treatments. Sorafenib, a multi-target tyrosine kinase inhibitor, was proposed as a potentially active drug in KRAS-mutated NSCLC patients, but clinical trials results were not conclusive. Here we show that the NSCLC cells' response to sorafenib depends on the type of KRAS mutation. KRAS G12V cells respond less to sorafenib than the wild-type counterpart, in vitro and in vivo. To overcome this resistance, we used high-throughput screening with a siRNA library directed against 719 human kinases, and Wee1 was selected as a sorafenib response modulator. Inhibition of Wee1 by its specific inhibitor MK1775 in combination with sorafenib restored the KRAS mutated cells' response to the multi-target tyrosine kinase inhibitor. This combination of the Wee1 inhibitor with sorafenib, if confirmed in models with different genetic backgrounds, might be worth investigating further as a new strategy for KRAS mutated NSCLC.

Activity of Pan-Class I Isoform PI3K/mTOR Inhibitor PF-05212384 in Combination with Crizotinib in Ovarian Cancer Xenografts and PDX.

The Phosphatidyl inositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and c-Met signaling pathways are often deregulated in cancer. The two pathways are interconnected and at least c-Met has been implicated in drug resistance. The aim of the study was to assess in ovarian cancer preclinical models, the efficacy and tolerability of a dual PI3K mTOR inhibitor (PF-05212384 or gedatolisib) and a c-Met inhibitor (crizotinib) either as single agents or in combination. In vitro, both PF-05212384 and crizotinib showed a concentration dependent activity in the two ovarian cancer cell lines. The combination of the two did not result in synergistic activity. A subline resistant to gedatolisib was obtained and showed an increased expression of MDR-1 gene. In vivo results show that crizotinib alone did not display any activity in all the tumors investigated, while PF-05212384 alone had some marginal activity. The combination of the two resulted in all the experiments superior to single agents with a good tolerability. Considering that crizotinib did not show activity in the models used, the results indicate that crizotinib is able to potentiate the activity of PF-05212384. Although the activity of the combination was not striking in these three models of ovarian cancer, due to the good tolerability of the combination, the results would suggest the possibility to combine the two drugs in settings in which gedatolisib or crizotinib alone have already some significant activity.