The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors. To examine the effects of Ambra1 loss on lung adenocarcinoma development in in vivo model, we integrated CRISPR/Cas9 genome editing with tumor barcoding and high-throughput barcode sequencing (Tuba-seq), a highly quantitative method that controls for the variability associated with viral infection. In this approach, pooling lenti-sgRNA-Cre vectors that target different genes allows for a direct comparison of each gene’s effect on tumor growth relative to inert controls in a cohort of mice. KPTC and KTC (p53 wild-type) mice were infected with lenti-sgRNA-Cre pools consisting of guides against Ambra1 and three other known lung cancer tumor suppressors (Rb1, Apc, and Rbm10), as well as 5 inert sgRNAs (sgRNAs targeting Neomycin loci or non-targeting). KT mice (lacking Cas9) were also infected with the same lentiviral pool to control for differences in virus titer and/or infectivity. 14-15 weeks after tumor initiation, we harvested the lungs and quantified cell number per tumor by sequencing the integrated barcodes (sgID-BCs).