Abstract 1074: Combining forces: Synergy of erlotinib and crizotinib in a wild-type squamous non-small cell lung cancer cell line
Authors: Steen Nele Van Der , Rolfo Christian , Pauwels Patrick , Peters Godefridus J. , Giovannetti Elisa 
Published: July 2017

Abstract
Oncogenic drivers are often overexpressed in adenocarcinoma non-small cell lung cancer (NSCLC) patients, so this tumor type is often sensitive to targeted therapies. Sensitizing mutations in the epidermal growth factor receptor (EGFR) are good predictive biomarkers for response to EGFR-small molecule inhibitors, eg erlotinib. Amplification or exon 14 skipping of cMET are good biomarkers for cMET-small molecule inhibitors, eg crizotinib. In other subtypes of NSCLC, eg squamous, targeted therapy is not considered to be active.
We tested the combination of erlotinib and crizotinib on a panel of squamous NSCLC cell lines that were wild-type for both EGFR and cMET. The effect of this drug combination was tested with different assays. The sulforhodamine B-assay was used to determine growth inhibition and combination indexes (CI) were calculated with the method of Chou&Talalay. A CI&lt; 0.8 was synergistic, 0.8&lt;CI&lt;1.2 was additive and CI&gt;1.2 was antagonistic. With flow cytometry the cell cycle was studied. With the wound-healing assay we determined the effect on cell migration. A 3D spheroid assay was performed to determine the effect of limited nutrient availability and hypoxia. Protein phosphorylation was studied with a pathscan array and phospho-specific western blotting.
The LUDLU cell line gave a synergistic CI of 0.39±0.07, whereas the Calu1 (CI = 0.81±0.04), H520 (CI = 0.87±0.06) and SKMES1 (CI = 0.81±0.02) were additive and the H1703 cell line (CI = 1.20±0.15) was antagonistic. Cell cycle analysis revealed that erlotinib monotherapy caused a G1 phase arrest (38 to 76%) and crizotinib caused a G2/M (13 to 64%) phase arrest. In cells treated with the combination, the effect of crizotinib was dominant (13 to 52%). In the wound-healing assay the LUDLU cells were not migratory. The pathscan revealed a decrease in the phosphorylation of pERK1/2 (Thr202/204), pPRAS40 (Thr246) and pGSK3β (Ser9). Western blotting showed that the levels of pPRAS40 were decreased in the LUDLU cells after treatment with erlotinib or crizotinib, and almost gone when treated with the combination. In the SKMES1 cells only the combination decreased pPRAS40. H1703 cells showed no change in pPRAS40.
These changes in phosphorylation make us hypothesize that Her3 and PI3K/Akt signaling may be important in the interaction of both drugs. Her3 is a promiscuous receptor that is able to heterodimerize with EGFR and cMET. When blocking one of these receptors, Her3 is still able to continue downstream PI3K/Akt signaling. When both EGFR and cMET are blocked, Her3 and PI3K/Akt signaling is inhibited, leading to synergy. The expression of total-Her3 was lowest in the antagonistic cell line. The combination of both inhibitors lead to a complete inhibition of pHer3 (Tyr1289).
In conclusion: Blocking both EGFR and cMET signaling causes inhibition of Her3 downstream signaling through PI3K/Akt, leading to synergy in wild-type squamous NSCLC cells.
Citation Format: Nele Van Der Steen, Christian Rolfo, Patrick Pauwels, Godefridus J. Peters, Elisa Giovannetti. Combining forces: Synergy of erlotinib and crizotinib in a wild-type squamous non-small cell lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2017-1074