SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.DOI: http://dx.doi.org/10.7554/eLife.17290.001

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

BackgroundSpenic hemangiosarcoma (HSA) in dogs treated with surgery alone is associated with short survival times, and the addition of doxorubicin (DOX) chemotherapy only modestly improves outcome. The purpose of this study was to evaluate the impact of toceranib administration on progression free survival in dogs with stage I or II HSA following splenectomy and single agent DOX chemotherapy. We hypothesized that dogs with splenic HSA treated with adjuvant DOX followed by toceranib would have prolonged disease-free interval (DFI) and overall survival time (OS) when compared to historical dogs treated with DOX-based chemotherapy alone.ResultsDogs with stage I or II splenic HSA were administered 5 cycles of single-agent DOX every 2 weeks beginning within 14 days of splenectomy. Dogs were restaged 2 weeks after completing DOX, and those without evidence of metastatic disease began toceranib therapy at 3.25 mg/kg every other day. Forty-three dogs were enrolled in this clinical trial. Seven dogs had evidence of metastatic disease either before or at re-staging, and an additional 3 dogs were found to have metastatic disease within 1 week of toceranib administration. Therefore 31 dogs went on to receive toceranib following completion of doxorubicin treatment. Twenty-five dogs that received toceranib developed metastatic disease. The median disease free interval for all dogs enrolled in this study (n = 43) was 138 days, and the median disease free interval for those dogs that went on to receive toceranib (n = 31) was 161 days. The median survival time for all dogs enrolled in this study was 169 days, and the median survival time for those dogs that went on to receive toceranib was 172 days.ConclusionsThe use of toceranib following DOX chemotherapy does not improve either disease free interval or overall survival in dogs with stage I or II HSA.

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