Abstract 3272: LKB1 loss is associated with resistance to VEGF inhibitors in non-small cell lung cancer (NSCLC)
Authors: Guijarro Irene , Poteete Alissa , Yang Chao , Roarty Emily , Nilsson Monique , Sun Huiying , Tong Pan , Chang Edward , Rodriguez-Canales Jaime , Mino Barbara , Parra Edwin , Wistuba Ignacio , Wang Jing , Heffernan Timothy , Heymach John V. 
Published: July 2016

Abstract
Background
LKB1 is a protein kinase that is mutated and down-regulated in 20-30% of NSCLC. LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival, and frequent metastases. Because LKB1 activates AMPK, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating tumor progression, metastasis and angiogenesis, likely as a compensatory strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the anti-VEGF antibody improves PFS in NSCLC patients combined with chemotherapy but the benefit is modest and transient and often patients develop resistance. Our laboratory has identified alterations in cell metabolism and in vasculature of LKB1 deficient tumors when compared to LKB1 wild type. These findings may indicate that loss of LKB1 could alter the tumor vasculature in NSCLC.
Methods
mRNA expression of angiogenesis related genes were analyzed in wild type and LKB1 deficient NSCLC tumors (TCGA). In vitro validation was performed by qPCR and western blot. CD31 IHC was performed to analyze microvasculature density (MVD) in Krasmut and Krasmut LKB1f/f mice tumors. HUVEC tube formation and migration assays were performed with conditioned medium of LKB1 expressing and deficient NSCLC isogeneic cell lines. Xenograft NSCLC models were established via s.c. injection of H460 (LKB1 deficient) and H460 LKB1 (LKB1 expressing) cells in nude mice. Treatment consisting of human and/or mouse bevacizumab and nintedanib were administrated. Tumor volumes were measured and vasculature analysis was performed.
Results
In vitro HUVEC cells exhibited an increase migration and differences in endothelial network formation when incubated with conditioned medium from LKB1 deficient cells compared to LKB1 expressing cells medium (p&lt;0.05). LKB1 deficient cells upregulated hypoxia and energetic stress related genes (HIF1AN, EGLN1, HIF3A, EPAS1 and CA12) and increased the secretion of angiogenesis related factors (VEGF, IL8, endoglin). IHC analysis of baseline CD31 expression of tumors from Krasmut and Krasmut LKB1f/f mutant mice showed no significant differences in MVD. However, anti-angiogenic therapy significantly inhibited tumor progression in LKB1 expressing xenografts but did not show any therapeutic effect in LKB1 deficient tumors. LKB1 expressing xenografts treated with human or mouse bevacizumab, or the combination of both and nintedanib, resulted in a significant decrease in tumor volume (p&lt;0.05). The blockade of human and mouse VEGF showed an optimal therapeutic effect with approximately 70% reduction of tumor volume (p&lt;0.001).
Conclusions
LKB1 deficiency may promote resistance to anti-angiogenic therapy by regulating compensatory angiogenic pathways along metabolic adaptations to energetic stress in NSCLC.
Citation Format: Irene Guijarro, Alissa Poteete, Chao Yang, Emily Roarty, Monique Nilsson, Huiying Sun, Pan Tong, Edward Chang, Jaime Rodriguez-Canales, Barbara Mino, Edwin Parra, Ignacio Wistuba, Jing Wang, Timothy Heffernan, John V. Heymach. LKB1 loss is associated with resistance to VEGF inhibitors in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3272.