Zinc finger nucleases were first used to knock out in the pancreatic tumor line Panc 10.05 which harbors a Ametantrone G12D mutation in is sensitive to shRNA-mediated depletion of KRAS (34), and Mouse monoclonal to MAP4K4 maintains high basal levels of autophagic flux (20). Wilcoxon test). IC50 proliferation values for individual cell lines are displayed in Table S1. (value of all 17 shRNAs calculated with the RSA statistic. The waterfall plot is sorted by log values of KRAS shRNAs; cell lines harboring known oncogenic mutations in KRAS are highlighted in red. (values were calculated using the and and values with the RSA statistic. Note that hairpins directed against ATG7, ULK1, and VPS34 are significantly enriched in cells with high GFP levels, in keeping with the inhibition of autophagy and the cellular accumulation of GFP-p62. ATG7 Is Dispensable for KRAS-Driven Cell Proliferation in Vitro. To confirm the results obtained in the shRNA screen, we next applied genome-editing tools to knock out to achieve complete inhibition of the autophagy pathway. ATG7 is essential for the formation of the ATG5CATG12 and LC3CPE conjugates, both of which are required for autophagosome assembly (6). Zinc finger nucleases were first used to knock out in the pancreatic tumor line Panc 10.05 which harbors a G12D mutation in is sensitive to shRNA-mediated depletion of KRAS (34), and maintains high basal levels of autophagic flux (20). Two clonal lines (clones Ametantrone 17 and 47) were identified with undetectable levels of ATG7 and ATG5CATG12 conjugate and an accumulation of free ATG5, nonlipidated LC3 (LC3-I), and p62 (Fig. 2and and and then were lysed and immunoblotted Ametantrone for autophagy pathway components. (were plated, and proliferation assessed after 4 d by cell counting after Trypan Blue exclusion. Macroautophagy Does Not Contribute to KRAS-Dependent Tumor Growth in Vivo. Because macroautophagy deficiency conferred a survival disadvantage under nutrient starvation in vitro (Fig. 2), we evaluated whether this finding would translate to a reduction in tumor growth in vivoWe first assessed whether macroautophagy loss would affect the growth of established tumors by using Panc 10.05 tumor cells harboring doxycycline (DOX)-dependent expression of the dominant-negative protease ATG4BC74A (33, 35) to allow inducible inhibition of macroautophagy in cells subsequent to tumor formation. Inducible expression of ATG4BC74A effectively blocked macroautophagy in Panc 10.05 cells and resulted in a striking accumulation of LC3-I and p62, durable inhibition of macroautophagy, and a small but reproducible decrease in cell growth in vitro (Fig. S5). In vivo, expression of ATG4BC74A for 12 d after tumor formation did not reduce Panc 10.05 tumor xenograft growth (Fig. 3and Table S2). ATG7-deficient cells also were not sensitized to radiation treatment (Fig. 4and Table S2), showed equivalent antiproliferative effects in wild-type and ATG7-deficient A549 cells. This result is surprising, given that chloroquine is broadly used as a chemical probe to investigate the cellular consequences of macroautophagy inhibition. We verified this finding in two additional cellular models and found that chloroquine similarly inhibited the proliferation of wild-type and ATG7-deficient Panc 10.05 and HCT116 cells (Fig. 5 and and and = 0.28) or sunitinib (= 0.67). The addition of chloroquine significantly impacted the IC50 of both erlotinib ( 0.0001) and sunitinib (= 0.0001). ANOVA was performed using the generalized linear models procedure (PROC GLM) of SAS version 9.4. Chloroquine and its analogs are currently being evaluated in clinical trials in combination regimens with other anticancer agents. To determine whether the combinatorial activity of chloroquine is dependent on macroautophagy inhibition, we tested chloroquine in wild-type and ATG7-deficient cells in combination with erlotinib and sunitinib, two tyrosine kinase inhibitors previously reported to synergize with chloroquine (38, 39). Chloroquine, but not ATG7 deficiency, sensitized cells to both erlotinib and sunitinib (Fig. 5 and for 15 min at 4 C. Protein concentrations were quantified using the DC protein assay kit (Bio-Rad) and SDS/PAGE, and immunoblotting was performed as described previously (33). For A549 and PaTu-8988T in vitro samples, total cellular lysates were prepared using NuPAGE-LDS sample buffer (Life Technologies). Cell lysates were water-bath sonicated four times for 30 s each time with the amplitude set at 25% (Qsonica Sonicator). To analyze Ametantrone A549-derived tumor samples, tissue extracts were prepared in RIPA buffer (Teknova) supplemented with protease and phosphatase inhibitor mixtures (Calbiochem). Homogenized samples were sonicated continuously for 3 min and then were cleared by centrifugation for 10 min at 500 at 4 C. Protein concentrations were quantified using the RC DC protein assay kit (Bio-Rad). Equal amounts of proteins were subjected to immunoblotting analysis using the NuPAGE electrophoresis system. Immunoblots were probed with primary and secondary antibodies following the manufacturers instructions and were detected using HRP chemiluminescent substrate (Life Technologies). In Vitro Growth Assays. For Panc 10.05 and HCT116.