E.M., G.M., J. Bartek and F.C. conceived the study and designed experiments. G.M., P.D., C.D.S., M.C. and V. Cesarini performed all analyses regarding the development of Ambra1 cKO mice. E.M., G.M., C.M. and S.G.H. carried out the biochemical and microscopy experiments linking AMBRA1 to genomic instability and synthetic lethality. S.G.H., J. Bartkova, V. Cianfanelli, D.D.Z. and A.B. carried out the experiments and analysis of the Ambra1-deficient KRAS lung model. F.N. performed the experiments involving N-MYC. S.G.H., E. Pupo and L.L. performed analyses of mitotic cells. E.M., S.R. and L.d.L., evaluated U2OS-FUCCI dynamics upon AMBRA1 deficiency. E.M., M.D.M., F.R. and E. Papaleo performed all related bioinformatics. R.R. performed traffic light experiments. E.M. and M.R. performed the experiments and analysis of xenograft SKUT-1B experiments. M.L., E.G., N.S. and G.V. carried out the xenograft experiments with transformed MEFs. C.J.D. and R.C.S. generated and validated the MYC(pS62) antibody used in the immunohistochemistry experiments. A.M.-M. and J.M.M.-M. performed experiments and analysis regarding fork speed and symmetry. D.S., G.R., Y.-T.J. and M.P. provided key information about AMBRA1 substrates, as well as some cDNAs. A.O. helped with some biochemical experiments. R.E.H. and D.R.P. gave experimental support for lung cancer cell models. M.B., S.C., A.G., G.F., L.L., P.H., A.B., C.S., M.P., E. Papaleo, D.D.Z., A.M.-M. and F.L. provided critical support, key data analyses and conceptual advice. E.M., G.M., J. Bartek and F.C. wrote the original draft. All authors took part in writing, reviewing and editing the final manuscript. All authors read and accepted the manuscript.