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AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Abstract

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel—the MYC pathway and the cyclin D–cyclin-dependent kinase (CDK)–retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D–CDK–RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1–cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

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Fig. 1: AMBRA1 regulates cell proliferation by affecting the stability of D-type cyclins through interaction with DDB1 and CLR4.
Fig. 2: Depletion of AMBRA1 causes replication stress.
Fig. 3: AMBRA1 is a tumour suppressor and its loss is synthetic lethal with CHK1 inhibition.

Data availability

Data from the Kaplan–Meier analysis in Extended Data Fig. 6g, h referenced during the study are available in a public repository from the websites (http://kmplot.com/ and http://gepia2.cancer-pku.cn/#analysis). AMBRA1 expression data and the stemness score (RNA-based) were downloaded from the Xena platform (http://xena.ucsc.edu/). The graph and map of AMBRA1 mutations in TCGA Pan-Cancer Atlas studies were downloaded from cBioPortal (https://www.cbioportal.org/). The original uncropped immunoblot data that support the findings of this study are available in Supplementary Fig. 1. A representative gating strategy for fluorescence-activated cell sorting (FACS) analysis is included in Supplementary Fig. 2Source data are provided with this paper.

Code availability

All of the computer scripts and source codes used to generate and analyse the results from The Cancer Genome Atlas (TCGA) analyses presented in Extended Data Figs. 6a, 8a are available at https://github.com/ELELAB/AMBRA_low.

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Acknowledgements

E. Maiani is an Adjunct Professor at UniCamillus—Saint Camillus International University of Health Sciences. The F.C. laboratory is supported by grants from the Danish Cancer Society (KBVU R72-A4408, R146-A9364, R231-A14034 to F.C.; R146-A9471 to V. Cianfanelli; R146-A9414 to G.F.; R204-A12424 to D.D.Z.), the Novo Nordisk Foundation (NNF13OC0007559, NNF16OC0022544), the Lundbeck Foundation (R233-2016-3360 to F.C.; R209-2015-3505 to V. Cianfanelli), the LEO Foundation (LF17024 to F.C. and E. Papaleo; LF-OC-19-000004 to D.D.Z.), the Associazione Italiana per la Ricerca sul Cancro (AIRC project IG 2019 #23543 to F.C.; #22811 to L.L.; 5x1000 #9962 and AIRC IG 2018 #21724 to F.L.), the Italian Ministry of Research (MIUR, project PRIN 2017 FS5SHL Radius) and the Italian Ministry of Health (Ricerca Corrente to F.L. and F.N.). This work was also supported by the European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant agreement 642295 (MEL-PLEX)). D.D.Z. is supported by the Melanoma Research Alliance (MRA 620385). The F.C., J. Bartek and E. Papaleo laboratories in Copenhagen are part of the Center of Excellence for Autophagy, Recycling and Disease (CARD), funded by the Danmarks Grundforskningsfond (DNRF125). L.L. is supported by FPRC 5x1000 Ministero della Salute 2015. V. Cianfanelli, C.M. and M.B. are supported by the Fondazione Umberto Veronesi. M.P. is funded by grants from the National Institute of Health (R01-CA76584 and R35-GM136250) and is an investigator with the Howard Hughes Medical Institute; the work of E. Papaleo is supported by the Carlsberg Foundation Distinguished Fellowship (CF18-0314); work in the G.V. group is supported by the PI15/00339 grant, integrated into the State Plan for R&D + I2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (ERDF), by the Marie Skłodowska-Curie Innovative Training Network (ITN) action TRAIN (GA 721532) funded by the European Commission (H2020) and by grants from Voices Against Brain Cancer and ‘Fundació La Marató de TV3’ (20134031). J. Bartek, J. Bartkova and A.M.-M. are supported by grants from the Danish Cancer Society (R204-A12617-B153), the Novo Nordisk Foundation (16854 and 0060590), the Danish Council for Independent Research (DFF-7016-00313), the Lundbeck Foundation (R266-2017-4289), the Swedish Research Council (VR-MH 2014-46602-117891-30) and the Swedish Cancerfonden (170176). We thank P. Bonaldo and P. Braghetta for the generation of the Ambra1flox/flox mouse model, V. Turcanova for help with cloning and mutagenesis, Plaisant S.r.l. (Castel Romano) and the Danish Cancer Society animal facilities for help with in vivo experiments, V. Tocco and the FACS facility members for technical help in the flow cytometry analysis and C. Rodolfo for his help and support. G.M. is grateful to A. M. Gatta and V. Milletti for their support.

Author information

Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Jiri Bartek or Francesco Cecconi.

Ethics declarations

Competing interests

M.P. is a consultant for and has financial interests in Coho Therapeutics, CullGen, Kymera Therapeutics and SEED Therapeutics. M.P. is a cofounder of Coho Therapeutics, is on the Scientific Advisory Board of CullGen and Kymera Therapeutics, and is a consultant for Santi Therapeutics. The other authors declare no competing interests.

Additional information

Peer review information Nature thanks Piotr Sicinski and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 AMBRA1 regulates cyclin D stability in NSCs.

a, Schematic for production of the conditional knockout mouse model. b, c, Images of wild-type and Ambra1 cKO P21 mice (b) and brains (c). c, bottom, representative image of PCR amplification of Tm1c, Ambra1 and Cre. d, Wild-type and Ambra1 cKO olfactory bulbs in sagittal sections of E18.5 embryos, stained for Ki67 antibody and Hoechst (n = 3). e, Quantification of Ki67+ cell area in the whole brain of wild-type and Ambra1 cKO E13.5 embryos (sagittal sections shown in Fig. 1b) (n = 5). P value by two-tailed unpaired t-test. f, Representative scheme of NSCs extraction and cell culturing from mouse embryo medial ganglionic eminences (MGE). LGE, lateral ganglionic eminences. g, Densitometry quantification of normalized protein levels in wild-type and Ambra1 cKO NSCs shown in Fig. 1c (n = 4). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. h, Left, representative images of NSCs extracted from mouse embryo medial ganglionic eminences. Right, violin plot of clonal neurosphere diameters in wild-type and Ambra1 cKO NSCs (n = 3; total of 128 neurospheres analysed for each condition). P value by two-tailed unpaired t-test. i, Whole-brain quantification of E13.5 wild-type and Ambra1 cKO cyclin D1 staining normalized over DAPI, represented in Fig. 1e. P value by two-tailed unpaired t-test (n = 5). j, Wild-type and Ambra1 cKO olfactory bulbs in sagittal sections of E18.5 embryos, stained for cyclin D1 antibody and Hoechst (n = 3). k, Sagittal sections of wild-type and Ambra1 cKO E13.5 embryos, stained for cyclin D2 (n = 3). l, Left, representative images of sagittal sections of the mesencephalic ventricular zone in wild-type and Ambra1 cKO E13.5 embryos, stained for RB(pS807/811) (n = 5). Right, quantification of RB(pS807/811-positive area in the mesencephalic ventricular zone of E13.5 wild-type and Ambra1 cKO embryos (n = 5). P value by two-tailed unpaired t-test. m, Left, representative images of RB(pS807/811) in sagittal sections of the olfactory bulb in wild-type and Ambra1 cKO E18.5 embryos. Right, quantification of the number of RB(pS807/811)-positive cells (n = 3). P value by two-tailed unpaired t-test. n, Immunoblot of N-MYC after cycloheximide treatment in wild-type and Ambra1 cKO NSCs (n = 3). o, Quantitative PCR with reverse transcription (qRT–PCR) of NSCs; the investigated genes are at the bottom of the graph (n = 5). P values by two-tailed unpaired t-test. p, Immunoblot of control and AMBRA1-silenced SH-SY5Y cells (n = 3). q, Immunoblot of AMBRA1 immunoprecipitation in SH-SY5Y cells. r, Immunoblot for AMBRA1, PP2AC and N-MYC in SH-SY5Y cells silenced for the indicated genes (n = 3). Unless otherwise stated, n refers to biologically independent samples. For immunoblots, actin was used as loading control. Data are mean ± s.e.m. Scale bars, 250 μm.

Source data

Extended Data Fig. 2 Ambra1 deficiency affects the cell cycle, cell death and neuronal differentiation.

a, Densitometric quantification of cyclin D1 and D2 protein levels in the cycloheximide time course normalized over actin (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. b, Immunoblot of wild-type or Ambra1 cKO NSCs treated with cycloheximide and/or MG132 for the indicated times. (n = 3). c, Distribution of cell-cycle phases in NSCs after release from nocodazole treatment (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. d, Six-hour BrdU incorporation of passage-2 wild-type and Ambra1 cKO NSCs with or without abemaciclib treatment (n = 3). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. e, Percentage of apoptotic cells in wild-type and Ambra1 cKO NSCs (n = 3). EA, early apoptotic; LA, late apoptotic. P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. f, Left, immunoblot of the indicated proteins in NSCs after abemaciclib treatment. Right, densitometry quantification of the indicated proteins (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. g, Left, representative images of sagittal sections from wild-type and Ambra1 cKO E13.5 embryos, stained for SOX2 and TBR2. Right, quantification of immunostained positive area (SOX, n = 6; TBR2, n = 4). P values by two-tailed unpaired t-test. h, Left, representative images of sagittal sections of wild-type and Ambra1 cKO E18.5 embryos, stained for TBR2. Right, quantification of immunostained positive area (n = 6). P value by two-tailed unpaired t-test. Arrows indicate TBR2+ cells in the subventricular zone. i, j, Representative images of sagittal sections of wild-type and Ambra1 cKO E18.5 embryos, stained for the neuronal marker NeuN. i, Left, higher magnification of the mesencephalic alar plate. Right, quantification of immunostained positive cells (n = 3). P value by two-tailed unpaired t-test. j, Lower magnification to better appreciate the uncropped quantified area (n = 3). Scale bar, 500 μm. Unless otherwise stated, n refers to biologically independent samples. For immunoblots, actin was used as loading control. Data are mean ± s.e.m. Unless otherwise noted, scale bars represent 250 μm.

Source data

Extended Data Fig. 3 The AMBRA1–cyclin D1 axis affects the cell cycle.

a, Immunoblot of control or AMBRA1-silenced U87-MG cells for the indicated proteins (n = 3). b, Immunoblot of cyclin D1 in control or AMBRA1-silenced U87-MG cells treated with cycloheximide and/or MG132 for the indicated times (n = 3). c, Analysis of densitometry for the cyclin D immunoblot in U87-MG cells, silenced for the indicated genes, shown in Fig. 1g (n = 4). P values by one-way ANOVA followed by Dunnett’s multiple comparisons test. d, Left, immunoblot of cyclin D1 in U87-MG cells silenced for AMBRA1 expression and overexpressing empty vector (pcDNA), wild-type AMBRA1 or AMBRA1(ΔWD40). Right, analysis from densitometry (n = 3). P values by one-way ANOVA followed by Tukey’s multiple comparisons test. e, Immunoblot analysis of cyclin D1 immunoprecipitation from protein extracts of control and AMBRA1-silenced U87-MG cells (n = 3). f, Co-immunoprecipitation of AMBRA1 in U87-MG cells transiently overexpressing empty vector, cyclin D1–Flag or cyclin D1(T286A)–Flag. Cells were treated with MG132 for 3 h before lysis (n = 3). g, Fold change in the number of cells in control or AMBRA1-silenced U87-MG cells (n = 11). P value by two-tailed unpaired t-test. h, Immunoblot of the indicated proteins of U87-MG, BJ-hTERT and U2OS cells that were untreated or treated with MLN4924 for 4 h (n = 3). i, j, Cells immunostained with cyclin D1, EdU antibody and counterstained with Hoechst. i, Scatter plots reporting single-cell total nuclear intensities of EdU versus Hoechst (cells examined over three independent experiments: siSCR, n = 3,279; siAMBRA1, n = 3,608 cells). j, Box plots (centre line, median; box limits, 25th and 75th percentile) indicating total cyclin D1 nuclear intensities (siSCR, n = 3,279; siAMBRA1, n = 3,608 cells. median siSCR = 169,654; siAMBRA1 = 429,623). k, l, Immunoblot of cell-cycle markers in control and AMBRA1-silenced BJ-hTERT cells (k) and cell-cycle-sorted U2OS-FUCCI cells (l) (n = 3). m, Immunoblot of the indicated proteins in AMBRA1-silenced BJ-hTERT cells synchronized by 24-h serum starvation. Cells were collected after the indicated starvation recovery time points (n = 3). n, Representative images of live-cell imaging of control and AMBRA1-silenced U2OS-FUCCI cells from 0 to 14 h with a 2-h interval between different images. The length of the G1 phase is shown in Fig. 1k (n = 3). Scale bar, 5 μm. o, Cell proliferation in control or AMBRA1-silenced BJ-hTERT cells (24 h and 48 h n = 6; 72 h siSCR n = 6, siAMBRA1 n = 5). p, q, Control or AMBRA1-silenced U2OS-FUCCI cells. p, Representative contour plot. q, Fold increase of cells present in S–G2 phase in AMBRA1-downregulated cells with respect to control cells (n = 10). r, Box plots (centre line, median; box limits, 25th and 75th percentile; whiskers, minimum and maximum) showing the cell-cycle length of siSCR (n = 65; median = 13) or siAMBRA1 (n = 65; median = 8.5) U2OS-FUCCI cells examined over three independent experiments. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (g, o, q) or two-tailed Mann–Whitney test (j, r). For immunoblots, actin or β-tubulin were used as loading control.

Source data

Extended Data Fig. 4 AMBRA1 deficiency causes replication stress.

a, Total γH2AX nuclear intensity in the different cell-cycle phases of BJ-hTERT cells (n = 3). Data are mean ± s.d. b, Average number of γH2AX foci in control or AMBRA1-silenced U2OS cells (n = 3). c, Alkaline comet assay of control, AMBRA1- and ATG7-silenced U2OS cells (n = 3). d, Scatter plots showing γH2AX versus Hoechst total nuclear intensities from immunostainings of control, AMBRA1- and ATG7-silenced BJ-hTERT cells. The proportion of γH2AX-positive cells (red, arbitrary cut-off) is indicated (siSCR, n = 721; siAMBRA1, n = 725; siATG7, n = 733 cells examined over 3 independent experiments). e, Immunoblot of γH2AX in control, AMBRA1- and ATG7-silenced BJ-hTERT cells (n = 3). f, Homologous recombination (HR) efficiency in control, AMBRA1- and ATG7-silenced U2OS cells (n = 3). Data are mean ± s.d. g, Number of BRCA1 foci per nucleus in control and AMBRA1-silenced U2OS cells either untreated or treated with 3-Gy irradiation, stained against BRCA1 (n = 500 cells examined over 3 independent experiments, centre indicates the mean). h, Time in mitosis in control (n = 91 cells examined over 3 independent experiments) or AMBRA1-silenced (n = 72 cells examined over 3 independent experiments) cells. Bars represent median and interquartile range. i, Dying cells upon mitotic exit as evaluated by time-lapse imaging (n = 2 independent experiments; more than 60 cells per condition). j, Distribution of 53BP1 nuclear foci in G1 U2OS cells (n = 3). k, Total γH2AX versus Hoechst intensity in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with 2 mM hydroxyurea (HU) for 2 h (siSCR, n = 2,481; siAMBRA1, n = 2,237; siSCR + HU, n = 2,484; siAMBRA1 + HU, n = 2,281 cells; scatter plots are representative of n = 3 independent experiments). l, Quantification of normalized protein levels of CHK1 represented in Fig. 2e (n = 3). m, n, BJ-hTERT cells as in Extended Data Fig. 4k treated with cycloheximide or with cycloheximide and 2 mM hydroxyurea. m, Immunoblot analysis of the indicated proteins in total cell lysates. n, Quantification of normalized CHK1 protein expression levels (n = 4). o, p, qRT–PCR analyses of the indicated genes in control or AMBRA1-silenced BJ-hTERT (o) and U2OS (p) cells, respectively (CCNA2, E2F1 and RAD51 n = 5; BRCA1 n = 4; CHEK1 n = 3). q, r, Immunoblot analysis of the indicated proteins in control or AMBRA1-silenced U2OS (q) and BJ-hTERT (r) cells (n = 3 in both conditions). s, Gene ontology (GO) biological processes (2018) from enrichment analysis of DEA (Differential Expression Analysis) genes from RNA sequencing (RNA-seq) experiments. DEA originating from three RNA-seq independent experiments was used as input for the web-based software EnrichR34,35. P values computed using Fisher’s exact test; clearer bars show a smaller P value. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (a, b, c, f, j, l, n, o, p) or two-tailed Mann–Whitney test (g, h). For immunoblots, β-tubulin, SOD1 or GADPH were used as loading control.

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Extended Data Fig. 5 AMBRA1 deficiency causes replication stress.

a, Analysis of DEA genes (from n = 3 independent RNA-seq experiments) predicting the transcription factor activated after depletion of AMBRA1. b, qRT–PCR analyses of the indicated genes in control or AMBRA1-silenced U2OS-FUCCI cells sorted for the different cell-cycle phases (n = 3) c, Immunoblot for the indicated proteins in U2OS cells interfered for the indicated autophagy regulators (n = 3). d, Left, violin plot of γH2AX nuclear mean intensity in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with 0.1 μM abemaciclib for 48 h. Right, representative scatter plot of single-cell γH2AX nuclear mean intensity versus Hoechst, and cell cycle phase gating strategies from control and AMBRA1-silenced BJ-hTERT cells treated with abemaciclib (n = 643 cells). e, Cell count of control U87-MG cells or U87-MG cells with inducible cyclin D1 expression, three days after stimulation with dox (n = 3). f, Cell count of control BJ-hTERT cells or BJ-hTERT cells with inducible cyclin D1 expression at the indicated time points after stimulation with dox, normalized over non-induced cells (1-d V15+, n = 6; 3-d V15+, 3-d E30+, n = 4; 1-d E30+, 4-d V15+, 4-d E30+, 6-d V15+ and 6-d E30+, n = 5). V15+: dox-treated control cells; E30+: dox-treated cyclin D1-inducible cells. g, h, Percentage of cells in each cell-cycle phase in U87-MG (g) and BJ-hTERT (h) cells, control or with inducible cyclin D1 expression, untreated or 48 h after doxycycline stimulation (n = 3). i, Immunoblot for the indicated proteins in control U87-MG cells or U87-MG cells with inducible cyclin D1 expression at the indicated time points with or without dox stimulation (n = 3). j, k, Mean fork speed (j) (kb min−1) and fork symmetry analysis (k) of DNA fibres from control BJ-hTERT cells and BJ-hTERT cells with inducible cyclin D1 expression treated as in Fig. 2d (scored forks: −dox, n = 312; 3-d dox, n = 449; 4-d dox, n = 429; 6-d dox, n = 426). Data are mean ± s.d. l, Quantification of immunohistochemistry staining in Fig. 2g (n = 3 mice). m, Immunoblot for the indicated proteins in wild-type or Ambra1 cKO NSCs (n = 3). Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (b, l), two-tailed Mann–Whitney test (d, j, k), two-way ANOVA followed by Sidak’s multiple comparisons test (e, g, h) or one-way ANOVA followed by Sidak’s multiple comparisons test (f). Exact P values are provided in the ‘Statistical analysis and data reproducibility’ section of the Supplementary Methods.

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Extended Data Fig. 6 Bioinformatics analysis of AMBRA1 in cancer.

a, Bioinformatics analysis of expression data from the TCGA database. Pie charts show the percentage of AMBRA1-low cancers (light blue) with respect to the total (grey) in the indicated datasets. BLCA, bladder urothelial carcinoma; COAD, colon adenocarcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; UCEC, uterine corpus endometrial carcinoma. b, Xena correlation analysis of AMBRA1 mRNA expression and stemness score. The shaded area in the plot indicates the confidence interval (95%). c, Lolliplots showing the distribution of AMBRA1 mutations annotated in TCGA Pan-Cancer Atlas Studies datasets. d, Frequency of AMBRA1 mutations (expressed as a percentage) in TCGA Pan-Cancer Atlas Studies datasets. The cut-off was selected at 2%. e, Oncoprint of AMBRA1 alterations (homodeletions, shallow deletions, mutations), and TP53 and EGFR mutations from TCGA Pan-Lung Cancer datasets. f, Mutual exclusivity and co-occurrence analysis of the indicated genes from TCGA Pan-Lung Cancer datasets. P values derived from one-sided Fisher’s exact test. g, Kaplan–Meier analysis of patients in the Pan-Cancer Atlas Studies database was generated based on the expression level of AMBRA1 (low, below 20%; high, above 80%). Plot was downloaded from the online database GEPIA36 (http://gepia2.cancer-pku.cn/#analysis). P values derived from one-sided log-rank Mantel–Cox test). h, Kaplan–Meier analysis of overall survival based on RNA-seq analysis of AMBRA1 mRNA levels using the KM-plotter37 lung adenocarcinoma database.

Extended Data Fig. 7 AMBRA1 controls tumour growth in a mouse model of lung cancer.

a, Schematic representations of the mouse model and initial testing of the system. The KrasG12D transgenic mouse is mated with the conditional Ambra1flox/flox mouse to produce the Ambra1+/+::KrasG12D/+ and the Ambra1flox/flox::KrasG12D/+ genotypes. Lung-specific expression of oncogenic KrasG12D and deletion of Ambra1 is induced by intranasal inoculation with defective adenoviral particles carrying the Cre recombinase. b, Immunoblot analysis of AMBRA1 immunoprecipitation from tissue lung samples from Ambra1flox/flox::KrasG12D/+ mice 16 weeks after administration of AdenoCre (n = 3). c, The expression of the Ambra1 floxed allele after Cre administration was verified by RT–PCR performed in lung tissue samples as in c (n = 3). Primers were designed to distinguish wild-type and floxed alleles. d, Representative examples of H&E images of fixed lungs. Bottom, Magnification of the bronchus, highlighting the tumour initiation site. Scale bar, 1 mm. e, Quantification of immunohistochemistry staining in Fig. 3b (Ki67, n = 3; γH2AX, n = 3; RPA(pS4/8), n = 3; cyclin D1, n = 4; c-Myc(pS62), n = 3 in two independent tumours for each condition). Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. P values for γH2AX and cyclin D1 by two-tailed Welch t-test; P values for Ki67, c-MYC(pS62), RPA(pS4/8) by two-tailed unpaired t-test.

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Extended Data Fig. 8 AMBRA1 deficiency is synthetic lethal with CHK1 inhibitors.

a, Ratio between CHEK1 expression in the AMBRA1-low subpopulation of cancers with respect to normal tissue. b, Gating strategy for Fig. 3c. Bottom, scatter plots of total nuclear DNA intensity versus γH2AX intensity (siSCR DMSO, n = 1,850; siSCR AZD, n = 1,716; siAMBRA1 DMSO, n = 1,866; siAMBRA1 AZD, n = 1,731 cells; representative of three independent experiments). The γH2AX-positive cells (arbitrary cut-off) are indicated in red. Top, Hoechst nuclear intensity versus counts. γH2AX-positive cells are indicated by the red line. c, Immunoblot of AMBRA1, RPA(pS4/8) and β-tubulin in control or AMBRA1-silenced BJ-hTERT cells that were untreated or treated with AZD7762. d, Left, gating strategy for the quantification on the right. Top, Hoechst nuclear intensity versus counts. Bottom, scatter plots reporting single-cell total nuclear DNA intensity versus TUNEL intensity. Right, TUNEL-positive cells in the different cell phases calculated based on Hoechst intensity (n = 3). P values by two-tailed unpaired t-test. e, Viability analysis of control and AMBRA1-silenced BJ-hTERT cells treated with the indicated concentrations of LY2603618 for 24 h (n = 3). P values by two-stage step-up (Benjamini, Krieger and Yekutieli). f, Cell viability in control, AMBRA1- and ATG7-silenced BJ-hTERT cells that were untreated or treated with AZD7762 for 24 h (n = 4 for Control and treatments with 100 nM AZD7762). P value by two-tailed unpaired t-test. g, Cell viability in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with olaparib for 24 h (n = 3). Analysis by two-tailed unpaired t-test. h, Fork symmetry analysis from BJ-hTERT cells treated for 24 h with 100 nM AZD7762 or 5 μM LY2603618 (scored forks: siSCR DMSO, n = 533; siSCR AZD, n = 560; siSCR LY, n = 548; siAMBRA1 DMSO, n = 601; siAMBRA1 AZD, n = 543; siAMBRA1 LY, n = 548). P values by two-tailed Mann–Whitney test. Data are mean ± s.d. i, Cell viability analysis of control and AMBRA1-silenced A549, H11299 and HCC827 lung cancer cell lines treated with the indicated concentrations of AZD7762 and LY2603618 (n = 3) for 24 h. P values by two-stage step-up (Benjamini, Krieger and Yekutieli). Data are mean ± s.d. j, Immunoblot of the indicated proteins in control or AMBRA1-silenced A549, HCC827 and H1299 cells (n = 3). k, Immunoblot of sarcoma cell lines (n = 3). l, Immunoblot of SKUT-1B cells treated with the inhibitor MLN4924 for 4 h (n = 3). m, Left, immunoblot of SKUT-1B cells reconstituted with wild-type AMBRA1 or mutant AMBRA1(ΔWD40) or AMBRA1(PXP). Right, densitometry quantification of the indicated normalized protein levels (n = 3). P values by two-sided one-way ANOVA followed by Sidak’s multiple comparisons test. n, Late apoptosis analysis in SKUT-1B cells reconstituted with wild-type AMBRA1, AMBRA1(ΔWD40) or AMBRA1(PXP) and treated with 200 nM AZD7762 for 24 h (n = 3). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are provided in the ‘Statistical analysis and data reproducibility’ section of the Supplementary Methods.

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Extended Data Fig. 9 AMBRA1 deficiency is synthetic lethal with CHK1 inhibitors in vivo.

a, Cell viability of Ambra1+/+ and Ambra1gt/gt MEFs treated with AZD7762 or vehicle for 24 h (n = 4 independent experiments). P values by two-tailed unpaired t-test. b, Box plots (centre line, median; box limits, 25th and 75th percentile; whiskers, minimum and maximum) indicating weight of Ambra1+/+ and Ambra1gt/gt MEF xenografts referred to in Fig. 3f (Ambra1+/+ + vehicle, n = 8; Ambra1+/+ + AZD7762, n = 8; Ambra1gt/gt + vehicle, n = 10; Ambra1gt/gt + AZD7762, n = 11 mice). P values by two-tailed unpaired t-test. c, Cell death percentage in control U87-MG cells or overexpressing cyclin D1, either untreated or treated with AZD7762 for 24 h; mean ± s.e.m. (n = 3 independent experiments). P values by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. Unless otherwise stated, data are mean ± s.d.

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Supplementary information

Supplementary Information

This file contains Supplementary Methods and References.

Reporting Summary

Supplementary Figure 1

Original uncropped Immunoblot Data.

Supplementary Figure 2

Representative gating strategy of experiments shown in Fig. 1d, Extended Data Fig. 2c, 2d, 3l, 3f, 5b, 5g, 5h, 8n.

Supplementary Table 1

Raw count of all samples analysed by RNASeq.

Supplementary Table 2

RNASeq differential analysis of all siAMBRA1 tRNA vs all siSCR samples.

Supplementary Table 3

Bioinformatical analysis of AMBRA1 and CHEK1 expression levels from TCGA database.

Video 1

Time-lapse imaging of mitotic H2B-GFP U2OS control cells.

Video 2

Time-lapse imaging of mitotic H2B-GFP U2OS cells depleted for AMBRA1.

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Maiani, E., Milletti, G., Nazio, F. et al. AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity. Nature 592, 799–803 (2021). https://doi.org/10.1038/s41586-021-03422-5

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