Abstract 2403: Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway.
Authors: Byers Lauren Averett , Cortez Maria Angelica , Yang Chao , Wang Jing , Diao Lixia , Fan You Hong , Girard Luc , Gazdar Adi , Wistuba Ignacio , Minna John D. , O'Connor Matthew , Barr Sharon , Heymach John V. 
Published: August 2014

Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets.

Methods: Expression of &gt;140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC50s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1.

Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p&lt;0.0001 by t-test). In agreement with previous studies showing the regulation of mTOR by LKB1, cells with LKB1 loss had significantly greater activation of the mTOR signaling pathway, as indicated by higher levels of phosphorylated (p) mTOR, pS6, and pP70S6K (p&lt;0.014). Other proteins expressed at significantly higher levels in LKB1 deficient NSCLC cell lines included those in the IGF1R pathway (ex., IGF1R and its transcriptional regulator SRC3, p&lt;0.001) and MEK pathway (ex., pERK1/2, pMAPK, p&lt;0.017). Furthermore, we observed increased NRF2 protein levels (0.001) and concomitant downregulation of its suppressor KEAP1 (&lt;0.001) in LKB1 deficient lines, suggesting a potential role in LKB1-mediated regulation of oxidative stress. Inhibition of IGF1R by OSI-906 showed activity in LKB1 deficient lines; however, the combination of IGF1R + mTOR inhibition (OSI-906 + OSI-027) proved to be a more effective therapy, demonstrating synergistic activity at all dose combinations studied. These findings were confirmed in A549 cells stably overexpressing LKB1. Drug combination showed a synergistic effect in A549 control cells compared to A549-LKB1.

Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients.

Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O'Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2013-2403