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. Overall design: 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).

细胞分裂的启动过程整合了大量胞内与胞外信号输入。D型细胞周期蛋白（D-type cyclins，下文简称cyclin D）将这些信号输入与DNA复制启动过程相耦联¹。cyclin D水平升高可通过激活细胞周期蛋白依赖性激酶4和6（cyclin-dependent kinases 4 and 6，下文简称CDK4/6）促进细胞分裂，后者可进一步磷酸化并灭活视网膜母细胞瘤抑癌蛋白。据此，cyclin D-CDK4/6复合物的水平与活性异常升高，与不受控的细胞增殖及癌症紧密相关²,³。然而，调控cyclin D水平的分子机制尚未完全阐明⁴,⁵。

本研究证实，自噬与beclin 1调控因子1（autophagy and beclin 1 regulator 1, AMBRA1）是调控cyclin D降解的核心因子。我们通过全基因组筛选探究CDK4/6抑制剂应答的遗传机制时，鉴定出了AMBRA1。AMBRA1缺失会导致细胞与小鼠体内cyclin D水平升高，进而促进细胞增殖，并降低癌细胞对CDK4/6抑制剂的敏感性。从分子机制来看，AMBRA1作为cullin 4 E3泛素连接酶复合物（cullin 4 E3 ligase complex）的底物受体，介导cyclin D的泛素化修饰与蛋白酶体降解。在小鼠肺腺癌模型中，AMBRA1缺失可促进肿瘤生长；而在肺腺癌患者队列中，AMBRA1低表达与不良预后显著相关。综上，AMBRA1可调控细胞内cyclin D的蛋白水平，参与癌症发生发展过程，并影响癌细胞对CDK4/6抑制剂的应答敏感性。

总体实验设计：为探究AMBRA1缺失对体内肺腺癌发生发展的影响，我们将CRISPR/Cas9基因组编辑技术与肿瘤条形码标记高通量测序（tumor barcoding and high-throughput barcode sequencing, Tuba-seq）相结合——该方法具备高精度定量特性，可有效控制病毒感染过程中产生的实验变量差异。该实验策略通过混合靶向不同基因的慢病毒sgRNA-Cre载体，可在同一小鼠队列中，直接比较各基因相对于惰性对照的肿瘤生长调控效应。我们将靶向Ambra1、另外3种已知肺腺癌抑癌基因（Rb1、Apc及Rbm10）以及5种惰性sgRNA（靶向新霉素基因位点或非靶向性sgRNA）的慢病毒sgRNA-Cre混合载体，感染KPTC与KTC（p53野生型）小鼠。同时，我们以不表达Cas9的KT小鼠感染同一慢病毒混合载体，作为病毒滴度与感染性差异的对照。肿瘤启动后14~15周，我们摘取小鼠肺部，通过对整合的条形码标记（sgID-BCs）进行高通量测序，定量每个肿瘤的细胞数量。