Abstract 2935: The ATP-competitive mTOR inhibitor Torin2 enhances sensitivity of the ALK F1174L mutation to crizotinib in neuroblastoma
Authors: Azarova Anna M. , Bhatnagar Namrata , Olsen-Drake Lauren , Ross Kenneth , Frumm Stacey , Liu Qingsong , Christie Amanda , Sanda Takaomi , Rodig Scott J. , Christensen James G. , Kung Andrew L. , Gray Nathanael , Stegmaier Kimberley , George Rani E. 
Published: August 2012

Abstract
Mutations in the anaplastic lymphoma kinase (ALK) receptor represent an important therapeutic target in neuroblastoma. The most frequently occurring somatic mutation, ALK F1174L, is sensitive to the ALK inhibitor crizotinib only at higher doses in vitro, and not at all in in vivo. Moreover, ALK F1174L mediates acquired resistance to crizotinib in ALK-rearranged cancers, posing a therapeutic challenge in these diseases. To identify critical components of ALK F1174L-associated signaling pathways that contribute to neuroblastoma cell survival and whose simultaneous inhibition with ALK F1174L could increase sensitivity to crizotinib, we exposed neuroblastoma cell lines expressing ALK F1174L (Kelly and SHSY-5Y) to crizotinib (at treatment doses and exposures times titrated to abolish ALK phosphorylation, but in the absence of any detectable commitment to apoptosis), and compared their “ALK-inactive” gene expression signatures with vehicle-treated control cells in which the ALK F1174L signature remained “active”. We noted differential expression of genes involved in the PI3K/AKT/mTOR pathway in cells in which ALK F1174L was inhibited with crizotinib, with downregulation of AKT1, 2 and 3 and elevated expression of mTOR. Immunoblotting confirmed that downregulation of pALK upon exposure to crizotinib was accompanied by downregulation of pAKT473, with unchanged or elevated pRPS6. To test the possibility that the cytotoxicity of crizotinib could be enhanced by simultaneous inhibition of this ALK-driven pathway, we tested crizotinib in combination with the ATP-competitive mTOR inhibitor, Torin2, both in cell lines and in xenograft models of neuroblastoma expressing ALK F1174L. Treatment with crizotinib had no standalone activity as demonstrated by lack of effects on tumor volume or survival. Treatment with Torin2 significantly suppressed tumor growth, but this did not translate into a prolongation of survival. Combined treatment with Torin2 and crizotinib resulted in significant attenuation of tumor growth (p&lt;0.001), and prolongation of survival in comparison to control animals, as well as single agent treatment with either Torin2 or crizotinib. RPS6 remained phosphorylated in neuroblastoma xenografts of mice treated exclusively with crizotinib but was downregulated in cells treated with Torin 2, and in those treated with the combination. Together, these results suggest that single agent treatment with crizotinib at standard doses is less efficacious due to persistent activation of mTOR signaling and that mTOR pathway inhibition should augment the activity of crizotinib in the treatment of patients with ALK F1174L-expressing neuroblastomas and may even delay the onset of resistance in ALK-rearranged cancers in which this pathway is activated.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2935. doi:1538-7445.AM2012-2935