Toshifumi Tsukahara1,2. 1. Area of Bioscience and Biotechnology, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan. tukahara@jaist.ac.jp. 2. Division of Transdisciplinary Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan. tukahara@jaist.ac.jp.
Abstract
PURPOSE: Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer. METHODS: We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation. RESULTS: We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis. CONCLUSION: Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.
PURPOSE: Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer. METHODS: We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation. RESULTS: We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis. CONCLUSION: Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.
Authors: Omar O Abudayyeh; Jonathan S Gootenberg; Patrick Essletzbichler; Shuo Han; Julia Joung; Joseph J Belanto; Vanessa Verdine; David B T Cox; Max J Kellner; Aviv Regev; Eric S Lander; Daniel F Voytas; Alice Y Ting; Feng Zhang Journal: Nature Date: 2017-10-04 Impact factor: 49.962
Authors: S Carrera; A Buque; E Azkona; U Aresti; B Calvo; A Sancho; M Arruti; M Nuño; I Rubio; A R de Lobera; C Lopez; G L Vivanco Journal: Clin Transl Oncol Date: 2013-12-04 Impact factor: 3.405
Authors: George Santis; Roger Angell; Guillermina Nickless; Alison Quinn; Amanda Herbert; Paul Cane; James Spicer; Ronan Breen; Emma McLean; Khalid Tobal Journal: PLoS One Date: 2011-09-19 Impact factor: 3.240