D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer¹, but the mechanisms that regulate their turnover are still being debated^2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4^AMBRA1 (also known as CRL4^DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR–Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

D 型细胞周期蛋白是细胞分裂周期的中心调节剂，是人类癌症中最常解除管制的治疗靶点之一¹，但调节其营业额的机制仍存在争议^2,3。在这里，通过结合体细胞中的生化和遗传学研究，我们将 CRL4 ^AMBRA1（也称为 CRL4 ^DCAF3）鉴定为靶向所有三种 D 型细胞周期蛋白进行降解的泛素连接酶。在开发过程中，Ambra1丢失诱导 D 型细胞周期蛋白和视网膜母细胞瘤 (RB) 过度磷酸化和过度增殖的积累，并导致通过用 FDA 批准的 CDK4 和 CDK6 (CDK4/6) 抑制剂 abemaciclib 治疗怀孕小鼠来减少神经系统缺陷。此外，AMBRA1 在小鼠模型中充当肿瘤抑制因子，低AMBRA1 mRNA 水平预示着癌症患者的存活率低。D 型细胞周期蛋白中的癌症热点突变消除了它们与 AMBRA1 的结合并诱导其稳定。最后，全基因组 CRISPR-Cas9 筛选将AMBRA1鉴定为对 CDK4/6 抑制反应的调节剂。AMBRA1的丢失通过促进 D 型细胞周期蛋白与 CDK2 形成复合物来降低对 CDK4/6 抑制剂的敏感性。总的来说，我们的研究结果揭示了在细胞周期进程、发育和人类癌症中控制 D 型细胞周期蛋白稳定性的分子机制，并暗示 AMBRA1 是 RB 通路的关键调节因子。