CDK4 Regulates Lysosomal Function and Mtorc1 Activation to Promote Cancer Cell Survival Laia Martínez-Carreres1, Julien Puyal2, Lucía C

Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708

Cancer Molecular Cell Biology Research

CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival Laia Martínez-Carreres1, Julien Puyal2, Lucía C. Leal-Esteban1, Meritxell Orpinell3, Judit Castillo-Armengol1, Albert Giralt1, Oleksandr Dergai1, Catherine Moret1, Valentin Barquissau1, Anita Nasrallah1, Angelique Pabois4,5, Lianjun Zhang4, Pedro Romero4, Isabel C. Lopez-Mejia1, and Lluis Fajas1

Abstract

Cyclin-dependent kinase 4 (CDK4) is well-known for its models. Importantly, the use of CDK4 inhibitors in therapy is role in regulating the cell cycle, however, its role in cancer known to cause senescence but not cell death. To overcome metabolism, especially mTOR signaling, is undefined. In this this phenomenon and based on our findings, we increased the study, we established a connection between CDK4 and lyso- autophagic flux in cancer cells by using an AMPK activator in somes, an emerging metabolic organelle crucial for mTORC1 combination with a CDK4 inhibitor. The cotreatment induced activation. On the one hand, CDK4 phosphorylated the tumor autophagy (AMPK activation) and impaired lysosomal func- suppressor folliculin (FLCN), regulating mTORC1 recruit- tion (CDK4 inhibition), resulting in cell death and tumor ment to the lysosomal surface in response to amino acids. regression. Altogether, we uncovered a previously unknown On the other hand, CDK4 directly regulated lysosomal func- role for CDK4 in lysosomal biology and propose a novel tion and was essential for lysosomal degradation, ultimately therapeutic strategy to target cancer cells. regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at Significance: These findings uncover a novel function of the lysosomal surface, led to the accumulation of undigested CDK4 in lysosomal biology, which promotes cancer progres- material inside lysosomes, which impaired the autophagic flux sion by activating mTORC1; targeting this function offers a and induced cancer cell senescence in vitro and in xenograft new therapeutic strategy for cancer treatment.

Introduction HER2-negative breast cancer. Clinical studies using CDK4/6 inhibitors to treat other malignancies are being conducted (3). Cyclin-dependent kinase 4 (CDK4) has a well-established role Research from our group and others has shown that the role of in cell-cycle control (1) and CDK4–cyclin complexes are com- CDK4 is not limited to the control of the cell cycle. Indeed, CDK4 monly deregulated in tumorigenesis (2). These complexes are of is also a major regulator of energy homeostasis (4–6) through great interest as therapeutic targets, and the FDA has approved the E2F1–RB complex (7), AMPK (8), and IRS2 (9). Importantly, the speciﬁc CDK4/6 kinase inhibitors PD0332991 (palbociclib), CDK4 pathway has been shown to cross-talk with the mTOR LEE011 (ribociclib), and LY2835219 (abemaciclib) for treating pathway, which is a major regulator of cell growth and metab- advanced or metastatic hormone receptor (HR)-positive and olism (10, 11). CDK4/6 inhibition attenuates mTOR Complex 1 (mTORC1) activity in some cancer models (12, 13), yet the effects of CDK4/6 inhibitors on mTORC1 seem to be cell-type speciﬁc 1Center for Integrative Genomics, University of Lausanne, Lausanne, because opposite results were observed in other cancer types (14). Switzerland. 2Department of Fundamental Neurosciences, University of The exact mechanism underlying the CDK4–mTOR cross-talk in Lausanne, Lausanne, Switzerland. 3Department of Physiology, University of mammals is unknown, although in Drosophila it occurs via 4 Lausanne, Lausanne, Switzerland. Department of Fundamental Oncology, the phosphorylation of TSC2 (15). Given that mTOR activity is University of Lausanne, Epalinges, Switzerland. 5Ludwig Institute for Cancer increased in numerous cancers and participates in the transla- Research, University of Lausanne, Epalinges, Switzerland. tional regulation of several oncogenic proteins, mTOR inactiva- Note: Supplementary data for this article are available at Cancer Research tion constitutes an attractive strategy for cancer treatment (16). Online (http://cancerres.aacrjournals.org/). Lysosomes, considered for years as only the digestive system Current addresses for L. Zhang: Center for Systems Medicine, Institute of Basic of the cell, have emerged as key effectors in cell metabolism, due Medical Sciences, Chinese Academy of Medical Sciences and Peking Union to their role as platforms in the activation of mTOR pathway Medical College, Beijing, China; or Suzhou Institute of Systems Medicine, Suzhou, – Jiangsu, China. (17 19). mTORC1 is recruited to the surface of lysosomes in a complex amino acid (AA)-dependent manner (17). Among the Corresponding Author: Lluis Fajas, Center for Integrative Genomics, University multiple regulators of this process, we focused on folliculin of Lausanne, Lausanne 1015, Switzerland. Phone: 41766111675; E-mail: [email protected] (FLCN), a tumor suppressor that functions as a complex with FNIP. The FLCN–FNIP complex interacts with Rag GTPases in the Cancer Res 2019;79:5245–59 absence of AAs repressing their activity. When AAs are sensed, doi: 10.1158/0008-5472.CAN-19-0708 FLCN–FNIP complexes dissociate from Rag GTPases eliciting 2019 American Association for Cancer Research. their activation. The activation of Rag GTPases is crucial for

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mTORC1 recruitment to lysosomes (20). Importantly, mTORC1 Plasmid transfection was performed using X-tremeGENE HP. activation is also triggered by the accumulation of AAs in the pRK5-FLAG-FLCN was obtained from Addgene (#72290; ref. 30). lysosomal lumen (21). Therefore, alterations in the lysosomal MIG-human RagC WT and MIG-human RagC S75N plasmids function directly impact mTORC1 activity (22, 23). Additionally, were kindly provided by Alejo Efeyan (CNIO). pCMV-R24C these organelles play roles in cell survival and cell proliferation, plasmid was a gift from Mariano Barbacid (CNIO). thus becoming emerging targets for cancer therapy (24–26). For insulin pathway stimulation, cells were first cultured in FBS- In this study, we demonstrate that CDK4 is capable of mod- free media for 15 hours and subsequently treated with IGF-1 for ulating mTORC1 activity in a direct manner, through the phos- 20 minutes before lysis or fixation. phorylation of FLCN, and indirectly, by promoting lysosomal For AA stimulation, after a 15 hours treatment with FBS-free function. When CDK4 inhibitors are used, the lack of lysosomal media with or without CDK4/6 inhibitor, the cells were incubated function induces senescence in triple-negative breast cancer for 2 hours in KRBB media containing 111 mmol/L NaCl, (TNBC) cells and impairs tumor growth in a mouse xenograft 25 mmol/L NaHCO3, 5 mmol/L KCl, 2.5 mmol/L CaCl2,1 model. Moreover, a combination of AMPK activation and CDK4 mmol/L MgCl2, 25 mmol/L HEPES, 25 mmol/L glucose, inhibition was used in an attempt to trigger autophagy in con- and dialyzed FBS with or without CDK4/6 inhibitor. For the last ditions when lysosomes are dysfunctional and resulted in cell 20 minutes, 2X MEM amino acids solution and 2 mmol/L death and tumor regression. This finding is of high relevance in glutamine were added to the cells. TNBC, a highly invasive and aggressive cancer type that does not For acute CDK4/6 inhibition (3 hours), LY2835219 was dilut- have a clear therapeutic strategy yet (27). ed in RPMI complete media at the indicated concentrations.

Western blotting Materials and Methods Western blotting was performed as described previously (7). Materials The band intensities were quantified using the Fiji image- LY2835219, PD0332991, and LEE011 were purchased from processing package (31). The following antibodies were MedChem Express and used at 0.5 mmol/L, unless otherwise used: anti-CDK4 (H22), anti-CDK6 (C21), and anti-RB indicated. R3 human IGF-1 (I11146; Sigma) was used at 30 ng/mL. (C15) from Santa Cruz Biotechnology; anti-phospho Rb- Minimal essential media (MEM) amino acids solution (50X; S780 (D59B7), anti-phospho P70S6K-T389 (108D2), anti- Gibco) was used at the indicated concentrations. Rapamycin 4E-BP1 (53H11), anti-phospho AKT-T308 (244F9), anti- (LC Laboratories) was kindly provided by Pedro Romero phospho AKT- S472/3 (D9E), anti-p70S6 kinase (49D7), (University of Lausanne) and used at the indicated concentra- anti-LC3B (polyclonal), anti-SQSTM1 (polyclonal), anti- tions. Bafilomycin A1 (BafA1) was purchased from Enzo Life AcetylCoA Carboxylase (ACC; polyclonal), anti-phospho Acet- Sciences (ALX-380-030-M001) and used at 0.3 mmol/L. Cell perme- ylCoA Carboxylase (p-ACC), anti-ULK1 (D8H5), and anti- able aKG analog DMKG (349631; Sigma) was used at 5 mmol/L. phospho ULK-S757 from Cell Signaling Technology; and anti-a-tubulin (DM1A) from Sigma. Cell culture and transfection Site-specific rabbit polyclonal antibody against phospho-FLCN MDA-MB-231, CCRF-CEM, HTC116, IB115, HT29, SKOV, was a gift from Kei Sakamoto (Nestle Institute of Health Sciences, MCF7, and PC3 cell lines, all obtained from ATCC, were cultured Lausanne, Switzerland). It was generated by YenZym Antibodies in RPMI1640 þ GlutaMAX containing, 10 mmol/L HEPES, 1 by immunization and affinity purification with a phosphorylated mmol/L sodium pyruvate (All from Gibco), and 10% FBS (PAA peptide of the human sequence (CQMNSRMRAH-S-PAEG-amide Laboratories). Thawed cells were allowed one passage to for pS62 FLCN, the prefix denotes the phosphorylated residue). reach exponential growth phase before being used. Cells were used during maximum of 10 passages in the experiments per- Mass spectrometry formed in this study. PCR-based mycoplasma tests were done Protein samples were separated on SDS-PAGE gels. Bands routinely using specific primers: 50-TGCACCATCTGTCACTCTGT- corresponding to FLCN-GST or FLCN-FLAG were excised and TAACCTC-30 and 3-GGGAGCAAACAGGATTAGATACCCT-50, the digested with trypsin. Peptide mixtures were analyzed by last test was performed in June 2019. HPLC/MS-MS. MS data were analyzed using Mascot software MDA-MB-231 CDK4 knockout (KO), E2F1 KO, and FLCN KO 2.6 (Matrix Science). Scaffold software (version 4.8.4; Prote- stable cell lines were generated with CRISPR/Cas9 technology. omeSoftwareInc.)wasusedtovalidateMS/MS-basedpeptide The lentiCRISPR v2 plasmid was a gift from Feng Zhang (MIT; and protein identifications,andtoperformdatasetalignment. Addgene, plasmid #52961; ref. 28). The pMD2.G plasmid was a MsViz software (32) was used for comparison of sequence gift from Didier Trono (EPFL; Addgene, #12259). The pCMV- coverage and phosphorylation of FLCN protein. A full descrip- dR8.91 plasmid and the guide RNA for FLCN were gifts from tion of the methods can be found in Supplementary Materials Christian Widmann (University of Lausanne). The target sequences and Methods. for the guide RNAs are shown in Supplementary Table S1. Synthetic oligonucleotides were purchased and cloned into the Flow cytometry digested LentiCrispR vector as described in Shalem and collea- Cells were trypsinized and stained with Annexin V-PE (Bio- gues (28). Lentiviral production was based on the standard Legend, 640907) according to the manufacturer's protocol. The protocol established by Salmon and Trono (29). The resulting cells were analyzed on either a Gallios (Beckman Coulter) or lentivirus (2 mL) was then used to infect MDA-MB-231 cells for LSR II (BD Biosciences) flow cytometer. For intracellular Ki67 72 hours. Infected cells were selected with 5 mg/mL puromycin for staining, cells were fixed and permeabilized according to the 5 consecutive days. Western blotting was performed to ensure that manufacturer's protocol (BioLegend). Cells were then incubat- the protein of interest was no longer expressed. ed with anti-Ki67-FITC (BioLegend) for 30 minutes on ice.

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After washing twice with permeabilization buffer, cells were formulated in 1% HEC in distilled water, was administrated resuspended in PBS prior to analysis. For each sample, at least orally. A-769662 (20 mg/kg) was administrated by intraperi- 10,000 events were acquired. toneal injection in NaCl 0.9%. Body weight and tumor growth For LysoTracker experiments, fluorescence was analyzed with were monitored every other day. After 8-days treatment, an ImageStream III Flow Cytometer. After treating the cells, 100 the animals were anesthetized with isoflurane (3%) and sacri- nmol/L Lysotracker Green DND-26 (Life Technologies) was ficed by cervical dislocation. Tumors from 10 mice per added to live cells for 1 hour. The cells were then trypsinized group were dissected and cut into two pieces; one piece was and fixed with 4% PFA for 15 minutes at room temperature, fixed in PFA for histological staining, and the other piece washed twice with PBS, and resuspended with 2% FBS. Nuclear was snap-frozen in liquid N2 for protein and RNA analysis. staining was performed with DAPI. Ten thousand events were Tumors from 5 mice per group were processed for TEM analysis acquired per condition at a magnification power equivalent to and SA-b-Gal staining. All animal care and treatment proce- 60. dures were performed in accordance with Swiss guidelines Lysosomal Intracellular Activity Assay Kit (Cell-Based; Biovi- and were approved by the Canton of Vaud, Service de la sion) was used according to the manufacturer's instructions. Ten Consommation et des Affaires Veterinaires (SCAV; authoriza- thousand events were acquired with an ACCURI C6 flow tion VD 2797.1). cytometer. All flow cytometry analyses were performed with FlowJo soft- Quantification and statistical analyses ware (Version 7.6.5). The results are expressed as the means SEM. Comparisons between 2 groups were performed with unpaired 2-tailed Student Cathepsin B Activity Assay Kit (fluorometric) t tests, and multiple group comparisons were performed by Cathepsin B activity was assessed with a Fluorometric Kit unpaired 1-way ANOVA and 2-way ANOVA, both followed (ab65300; Abcam). Fluorescence was measured with a Tecan by Tukey test or otherwise indicated. All P values below 0.05 plate reader (Ex/Em ¼ 400/505 nm). were considered significant. Statistically significant values are re- presented by asterisks corresponding to , P < 0.05; , P < 0.01; Colorimetric detection of senescence-associated , P < 0.001; and , P < 0.0001. b-galactosidase For senescence-associated b-galactosidase (SA-b-Gal) analy- Supplementary methods sis, cells in culture were fixed with 2% PFA and 0.2% glutar- Additional details about the methods used in this paper can be aldehyde for 5 minutes at room temperature, washed with found in the Supplementary Materials and Methods and in PBS, and stained with a solution containing 40 mmol/L citric Supplementary Tables S2 to S5. acid Na phosphate buffer, 5 mmol/L K4 [Fe(CN)6]3H2O, 5 mmol/L K3 [Fe(CN)6], 150 mmol/L NaCl, 2 mmol/L MgCl2, and 1 mg/mL X-gal in distilled water for 15 hours at 37C. After Results staining, the cells were washed twice with PBS and once with CDK4 activity is required for mTORC1 localization at methanol, and the plates were allowed to air dry. Bright-field lysosomes images were obtained with an upright light microscope (Zeiss) CDK4/6 inhibition was previously shown to decrease mTORC1 with a 20 objective. Positive cells and the total number of activity in some models of human cancer (12–14). To determine cells per field were counted manually. the cell-type specificity of this cross-talk, we activated mTORC1 in For SA-b-Gal analysis of mouse tumors, fresh tissues were 8 human cancer cell lines with IGF-1 in the presence of CDK4/6 frozen embedded in OCT, and stored at 80C. Tumors were inhibitor LY2835219. The efficiency of CDK4/6 inhibition was cut into 8-mm-thick sections and mounted onto glass slides. measured by RB phosphorylation. Treatment with LY2835219 After air-drying for 30 minutes, the sections were fixed with 2% caused a decrease in p70S6K and 4E-BP1 phosphorylation (2 PFA and 0.2% glutaraldehyde and stained as previously. The well-known targets of mTORC1), both in the unstimulated and in sections were counterstained with 0.1% Nuclear Fast Red the IGF-1 stimulated conditions (Supplementary Fig. S1A). (Sigma). Despite showing considerable mTORC1 inhibition, some cell lines showed only a mild decrease in AKT phosphorylation in Electron microscopy the presence of LY2835219 (HCT116, IB115, MDA-MB-231, For transmission electron microscopy (TEM), a standard fixa- SKOV3, PC3), suggesting that the effects observed in mTORC1 tion, embedding, and staining protocol of mice tumors and cells activity were at least partially independent of decreased AKT was established. Ultrathin sections were prepared and imaged signaling. using a Philips CM100 at 80 kV acceleration voltage equipped We focused our experiments on the TNBC cell line MDA-MB- with a TVIPS TemCam-F416 digital camera. For further details see 231 because it was one of the most responsive to CDK4/6 the Supplementary Materials and Methods. inhibition. The treatment with PD0332991 and LEE011, 2 other CDK4/6 inhibitors, also resulted in decreased mTORC1 activity in Animal studies this cell line (Supplementary Fig. S1B and S1C). MDA-MB-231 cells were injected into the fourth mammary We next found that MDA-MB-231 wild-type (WT) cells treated gland of 8-week-old female NSG mice (NOD.Cg-Prkdcscid with LY2835219, or CDK4 KO MDA-MB-231 cells showed Il2rgtm1Wjl/Sz strain; The Jackson Laboratory). Tumor growth impaired translocation of mTORC1 to the lysosomes in response and body weight were measured twice per week until the tumor to AAs, a key step for mTORC1 activation (Fig. 1A and B), which size reached 50 mm3 per mouse. Mice were randomized into correlated with decreased phosphorylation of 4E-BP1, p70S6K, 2 groups for the 8-days treatment. LY2835219 (75 mg/kg), and ULK (Fig. 1C and D). Inhibition of glutaminolysis has been

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Figure 1. CDK4 inhibition or depletion prevents the recruitment of mTOR to the lysosomal surface in response to amino acid stimulation. A, Confocal immunofluorescence analysis showing mTOR colocalization with lysosomes (LAMP1 and mTOR staining) in WT and CDK4 KO MDA-MB-231 cells, with or without AA stimulation and in the presence of 0.5 mmol/L of the CDK4/6 inhibitor LY2835219 or DMSO. B, Quantification of mTOR–LAMP1 colocalization from A using Pearson coefficient. At least 40 fields per treatment condition from three independent experiments were analyzed. C, Western blot analysis showing levels of phospho-RB and total RB, CDK4, and mTORC1 target genes (phospho-p70S6K and total p70S6K, phospho-ULK and total ULK, and 4E-BP1) in WT and CDK4 KO MDA-MB-231 cells with or without AA stimulation and with or without 0.5 mmol/L LY2835219. a-Tubulin was used as a loading control. D, Quantification of phospho-p70S6K normalized to a-tubulin from three independent experiments as in C. , P < 0.05; , P < 0.001; , P < 0.0001.

shown to prevent lysosomal recruitment and subsequent activa- mTORC1 activity might be independent of E2F1 transcriptional tion of mTORC1 (33). The effects of LY2835219 could not be activity. rescued, however, with a-ketoglutarate (aKG) stimulation, indicating that CDK4 inhibition or depletion does not affect mTORC1 CDK4 regulates mTORC1 activity through phosphorylating translocation to the lysosomal surface by impairing glutamine FLCN metabolism (Supplementary Fig. S2A and S2B). Interestingly, We hypothesized that CDK4 potentially regulates mTORC1 MDA-MB-231 cells lacking E2F1 showed normal mTORC1 trans- pathway through phosphorylation of one of its regulators. A location to the lysosomal surface and increased mTORC1 activa- bioinformatics search of the proteins of mTORC1 pathway iden- tion (Supplementary Fig. S2C–S2F) but were still sensitive to tiﬁed proteins containing the putative phosphorylation site for CDK4 inhibition. These suggest that the effects of CDK4 on CDK4: (S/T)PX(K/R/P) using phosphosite.org (Supplementary

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Figure 2. CDK4 regulates mTORC1 activity through phosphorylating FLCN. A, Representation of the phosphorylation sites detected by mass spectrometry analysis in the in vitro kinase assay with FLCN-GST and CDK4/CyclinD3. Green rectangles represent protein coverage. Blue dots represent phosphorylation sites, and their size correlates with their abundance. B, Confocal immunofluorescence analysis showing the colocalization of overexpressed FLCN with lysosomes (LAMP1 and FLCN staining) in WT and CDK4 KO MDA-MB-231 cells, with or without AA stimulation and in the presence of 0.5 mmol/L of LY2835219 or DMSO. C, Quantification of FLCN-LAMP1 colocalization from B using Pearson coefficient. At least 40 fields per treatment condition from three independent experiments were analyzed. D, Western blot analysis of WT and FLCN KO MDA-MB-231 cells, treated with DMSO or LY2835219 for 3 hours. Phospho-FLCN and total FLCN, phospho-p70S6K and total p70S6K, phospho-RB and total RB, and a-tubulin as a loading control. E, Quantification of phospho-FLCN levels normalized to a-tubulin from D. F, Immunofluorescence of phospho-S6 from WT and FLCN KO MDA-MB-231 after 20 minutes of AA stimulation, in the presence or absence of LY2935219. G, Quantification of phospho-S6 intensity from F. , P < 0.05; , P < 0.01; , P < 0.0001.

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Fig. S3A). The corresponding peptides were used for a kinase nutrient-rich conditions, TFEB is phosphorylated by mTORC1, competition assay versus RB consensus peptide. The peptides causing its retention in the cytoplasm. By contrast, when belonging to 4E-BP1, LARP1, and FLCN, triggered a decrease of mTORC1 is inactivated, unphosphorylated TFEB translocates to RB phosphorylation, indicating a competition with RB for CDK4 the nucleus and promotes the transcription of numerous genes phosphorylation (Supplementary Fig. S3B). In vitro kinase assays encoding lysosomal and autophagic proteins (19). To test wheth- using CDK4/CycD3 recombinant protein and glutathione S- er CDK4 inhibition or depletion affected TFEB target genes, we transferase (GST) fusion of full-length FLCN followed by mass treated WT and CDK4 KO MDA-MB-231 cells with complete or spectrometry revealed that FLCN could be phosphorylated by serum-starvation media, with or without LY2835219. As recombinant CDK4/CycD3 at 4 different sites (S62, S73, T227, expected, under starvation conditions, WT MDA-MB-231 cells and S571; Fig. 2A). Importantly, S62, S73, and S571 phos- showed increased expression of genes regulated by TFEB (Fig. 3A). phorylation sites were found when FLCN was immunopreci- Moreover, we found that CDK4/6 inhibition synergized with pitated from MDA-MB-231 cells upon AA and IGF-1 stimula- starvation to further increase the expression of those genes. CDK4 tion (Supplementary Fig. S3C). Radioactive in vitro kinase KO cells also presented increased expression of those genes under assays confirmed that CDK4/CycD3 can phosphorylate the basal conditions, but no further increase was found upon CDK4/6 full-length FLCN in vitro (Supplementary Fig. S3D). When inhibition (Fig. 3A). FLCN S62, S73 and S571 were mutated to alanine (nonphos- We next used LysoTracker staining and flow cytometry to phorylatable forms), the intensity of the autoradiography look at the percentage of lysosome-positive cells upon CDK4 bands was reduced, indicating a decrease in FLCN phosphor- inhibition or depletion. The percentage of LysoTracker positive ylation. This result was even more striking in the triple mutant cells and the size of the LysoTracker positive particles were (Supplementary Fig. S3D). consistently and markedly increased in the absence of Importantly, FLCN is retained at the lysosomes in the presence CDK4 activity (Fig. 3B–D). Similarly, when we quantified the of AAs upon CDK4 inhibition or depletion (Fig. 2B and C), which size of lysosomal-associated membrane protein 1 (LAMP1)- is consistent with the absence of mTOR. To further study the positive particles using immunofluorescence, we found a sig- effects of CDK4 on FLCN, we generated an MDA-MB-231 FLCN nificant increase in lysosomal density in CDK4 KO cells com- KO stable cell line. We used a phospho-specific antibody directed pared to WT MDA-MB-231 cells (Fig. 3E and F). Consistently, against phospho-S62 FLCN, one of the phosphorylation sites that PD0332991 and LEE011 treatments also increased LysoTracker we found in CDK4-FLCN in vitro kinase assays. Phosphorylation intensity in MDA-MB-231 cells (Supplementary Fig. S5A). of FLCN decreases significantly in WT cells treated with Immortalized MEFs treated with LY2835219 also showed LY2835219 for only 3 hours (Fig. 2D and E), indicating an acute increased LysoTracker intensity (Supplementary Fig. S5B). control of CDK4 over FLCN, potentially resulting in mTORC1 These results suggest a new function of CDK4 in the control regulation. FLCN KO cells were still sensitive to mTORC1 inac- of lysosomal biology. tivation by CDK4/6 inhibition, as shown by decreased p70S6K phosphorylation, but to a lesser extent than WT cells (Fig. 2D). CDK4 is required for lysosomal function Moreover, WT cells showed a 2-fold decrease in the phosphory- It is well known that the inhibition of mTORC1 results in the lation of S6 when treated with LY2835219, whereas FLCN induction of autophagy, a conserved catabolic process that KO cells showed a milder, yet significant, reduction of levels of triggers the degradation of intracellular constituents and orga- S6 ribosomal protein phosphorylation. S6 phosphorylation nelles in the lysosome (35). Serum-starvation conditions, as remained significantly higher in LY2835219-treated FLCN KO well as mTOR inhibition by rapamycin, increased the amounts cells, compared with LY2835219-treated WT cells (Fig. 2F and G). of the autophagosome marker LC3-II (Fig. 4A and B, long Our results suggest that complementary mechanisms to FLCN exposure). CDK4/6 inhibition similarly increased LC3-II levels. phosphorylation account for the effects of CDK4 on mTORC1 Moreover, CDK4 KO cells showed increased levels of LC3-II in activation. basal conditions and were more sensitive to starvation- Interestingly, the overexpression of the R24C CDK4 mutant, mediated autophagic stimuli (Fig. 4A and B). These indicate which abolishes the ability p16(INK4a) to inhibit CDK4 (34), either an increase of autophagosome biogenesis or an highly increased phospho-p70S6K levels under FBS starvation impairment of the autophagic flux in the absence of CDK4. conditions as compared with untransfected cells (Supplemen- Indeed, these effects could be secondary to mTOR inactivation, tary Fig. S4A and S4B). Increased pS62-FLCN/FLCN ratio was because the decreased mTORC1 activity caused by CDK4 inhi- also observed with R24C overexpression (Supplementary bition or depletion shown in Fig. 1 could ultimately induce Fig. S4A and S4C), indicating that the R24C mutation bypasses lysosomal biogenesis. the effect of starvation on the activation of the mTORC1 In WT cells, under starvation conditions and in the presence of pathway. rapamycin treatment, BafA1, which is a potent V-ATPase inhibitor Additionally, the overexpression of RAGC S75N mutant that blocks autophagosome-lysosome fusion, further increased confers insensitivity to mTORC1 activity upon acute treatment LC3-II levels, indicating that rapamycin induces autophagosome with LY2835219 (Supplementary Fig. S4D and S4E). This biogenesis. In contrast, in CDK4 KO cells or cells treated with reinforces the link between CDK4 and mTORC1, not only CDK4 inhibitor, BafA1 failed to cause any additional increase in through FLCN, but also through other mechanisms of nutrient LC3-II levels (Fig. 4A–C). These results suggest that CDK4 does sensing. not directly participate in autophagosome biogenesis. On the other hand, no abnormal SQSTM1 accumulation was observed CDK4 inhibition or depletion increases lysosomal mass after CDK4 inhibition or depletion, despite observing a consistent Lysosomal biogenesis is a biological process coordinated by the SQSTM1 increase with BafA1 (Fig. 4A). SQSTM1 protein levels are transcription factor TFEB, which is repressed by mTORC1. Under often negatively correlated with autophagic degradation.

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Figure 3. CDK4 inhibition or depletion increases lysosomal mass. A, qPCR analysis showing the expression of transcription factor EB (TFEB)–regulated genes that are involved in lysosomal or autophagic processes (cathepsins B and D and SQSTM1) in WT or CDK4 KO MDA-MB-231 cells in complete (þFBS) or serum-starvation media (FBS), treated with or without 0.5 mmol/L LY2835219. Each condition was assessed in duplicates for three independent experiments. B, Representative flow cytometry images of WT or CDK4 KO MDA-MB-231 cells treated with or without LY2835219 and stained with LysoTracker Green DND-26 under the indicated conditions. C, Proportions of cells containing lysosomes, estimated by quantification of fluorescence from LysoTracker Green DND-26–stained cells from two independent experiments. At least 10,000 events were acquired. Data are subdivided into categories of the number of lysosomes per cell. Significant differences between WT and KO MDA-MB-231 cells are indicated by: , total lysosomal particles (P < 0.05); $, lysosomal particles per cell (3–5 particles; P < 0.05); &, lysosomal particles per cell (>5 particles; P < 0.05); 2-way ANOVA followed by Tukey multiple comparisons test. D, As in C, but subdivided into categories of the sizes of lysosomes per cell. Significant differences between WT and KO MDA-MB-231 cells are indicated by: , lysosomal particles <0.5 mm2 (P < 0.05); $, lysosomal particles per cell (<0.5 mm2; P < 0.05). E, Immunofluorescence analysis showing LAMP1 and CDK4 expression in WT and CDK4 KO MDA-MB-231 cells in complete media. F, Quantification of the total volume of LAMP1-positive particles in z-stack images. At least 30 cells in total from three independent experiments were analyzed. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 4. Absence of CDK4 alters lysosomal function. A, Western blot analysis of autophagosomal markers LC3-I, LC3-II, and SQSTM1 in WT and CDK4 KO MDA-MB-231 cells treated 24 hours with complete media (þFBS) or serum-starvation media (FBS), presence/absence of 0.5 mmol/L rapamycin and of 0.5 mmol/L LY2835219 and presence/absence of 0.3 mmol/L BafA1, 6 hours before the treatment. B, Quantification of protein expression of LC3-II protein levels normalized to a-tubulin from three independent experiments as in A (only the conditions without BafA1). C, Quantification of LC3-II protein levels normalized to a-tubulin from three independent experiments as in A (only the conditions with BafA1). Statistical analysis was performed using a paired t test. D, Representative electron micrographs of WT and CDK4 KO MDA-MB-321 cells showing that cells lacking CDK4 have increased autolysosome (arrows) density and size. In CDK4 KO cells, autolysosomes accumulated nondegraded material, such as undegraded autophagosomes (arrowheads), in both complete (þFBS) and serum-starvation media (FBS). In cell cultures without FBS, an increase in autophagosomes (arrowheads) was also observed in CDK4 KO cells as compared with WT cells. N, nucleus. E–G, Quantification of the autophagosome (E) and autolysosome (F) area per cell, and of the percentage of autolysosomes containing degraded material (G), for the conditions shown in D. n ¼ 20 cells per condition. H, Quantification of intracellular lysosomal activity of WT and CDK4 KO MDA-MB-231 cells in complete media. I, Quantification of cathepsin B activity in WT and CDK4 KO MDA-MB-231 cells in complete media. , P < 0.05; , P < 0.01; , P < 0.001.

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However, it has been already observed that the expression of starvation induced autophagosome and lysosome formation SQSTM1 does not always inversely correlate with autophagic in MDA-MB-231 WT cells. CDK4 KO MDA-MB-231 cells dis- activity, given that they can be restored during prolonged played higher densities of autophagosomes and lysosomes. starvation (36). Moreover, TEM analysis revealed that the lysosomes in CDK4 We next used TEM to analyze the ultrastructure of KO cells were ﬁlled with electron-dense undigested material lysosomes in WT and CDK4 KO MDA-MB-231 cells incubated (Fig. 4D–G; Supplementary Fig. S5C). LY2835219-treated WT with serum-starvation media to trigger autophagy. Serum- cells mimicked CDK4 KO cells (Supplementary Fig. S5D–S5H).

Figure 5. Dysfunctional lysosomes, but not mTORC1 inhibition, induce senescence. A, Proliferation of WT MDA-MB-231 cells cultured in either complete (þFBS) or serum- starvation (FBS) media, with or without LY2835219, shown as the percentage of Ki67-positive cells. B, Apoptosis under the same conditions, shown as the percentage of Annexin V-positive cells. C, Quantification of the percentage of SA-b-Gal-positive WT and CDK4 KO MDA-MB-231 cells from triplicates, at least 5 fields per replicate. D, Quantification of the fold induction of SA-b-Gal induced by LY2835219 or rapamycin treatment from triplicates, at least 5 fields per replicate. E, mTOR-independent induction of senescence by LY2835219, visualized by colorimetric senescence-associated SA-b-Gal staining of WT and CDK4 KO MDA-MB-231 cells cultured in /þ FBS media for the last 16 hours after 8 days of DMSO, 0.5 mmol/L LY2835219, or 0.5 mmol/L rapamycin treatment. F, qPCR analysis showing expression of genes usually upregulated in senescence in WT MDA-MB-231 cells treated for 8 days with DMSO, LY2835219, or rapamycin in complete media. Triplicates were analyzed for each condition. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. ns, nonsignificant.

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Consistently, the intracellular lysosomal activity and cathepsin treatment also decreased the activity of mTORC1 (Fig. 6H and I). B activity were decreased in the CDK4 KO cells (Fig. 4H and I). TEM analysis proved that tumors from LY2835219 treated mice Overall, these results suggest that CDK4 is fundamental for the had higher densities of autophagosomes and lysosomes, and that activity of lysosomes and that its absence impairs autophagic those lysosomes accumulated nondigested material (Fig. 6J–M; flux at the lysosomal degradation step. Supplementary Fig. S6C). Furthermore, we showed that the tumors of these mice had typical features of senescence Dysfunctional lysosomes, but not mTORC1 inhibition, induce (Fig. 6N; Supplementary Fig. S6D). Together, these results further senescence demonstrate that CDK4 plays an essential role in the regulation of Lysosomal processes are associated with aging and longev- lysosomal function in vivo and that alterations in lysosomal ity (37), and the increase of lysosomal content is a characteristic function lead to tumor cell senescence in a mouse model of breast of senescence progression (38). This led us to investigate the cancer. fate of cells lacking CDK4 activity. Analysis of Ki-67 levels showed that CDK4/6 inhibition significantly decreased the The CDK4 inhibitor LY2835219 in combination with the AMPK proliferation rate of cells in complete medium (Fig. 5A). Yet, activator A769662 induces cell death in breast cancer cells and when the cells were serum-starved, no differences were tumors observed in response to LY2835219 (Fig. 5A). Apoptosis, Lysosomes are essential for autophagy. We speculated that measured by Annexin V staining, was not significantly induced triggering autophagy in LY2835219-treated mice, which have by LY2835219 in complete medium (Fig. 5B). A slight increase impaired lysosomal function, would trigger the collapse of the in apoptosis was seen when CDK4/6 inhibition was combined system and result in cell death. In vivo, the allosteric AMPK with serum starvation, but the overall percentage of apoptotic activator A769662, which is a potent inducer of autophagy, in cells was extremely low (Fig. 5B). Interestingly, 8 days of combination with LY2835219, resulted in tumor regression, in treatment with LY2835219 resulted in higher senescence, as contrast to the treatment with LY2835219 or A769662 alone measured by senescence-associated beta-galactosidase (SA- (Fig. 7A–D). Indeed, almost 50% of the tumors decreased their bGal) staining. CDK4 KO cells also showed increased senes- size upon cotreatment, although no major differences were cence (Fig. 5C–E). No differences were observed under serum found in proliferation (Fig. 7E and F). In contrast, cleaved- starvation conditions or upon rapamycin treatment, indicating caspase-3 staining revealed a 6-fold induction of apoptosis in that the induction of senescence in the absence of CDK4 was mice cotreated with A769662 and LY2835219 (Fig. 7E–G), not due to mTORC1 inhibition (Fig. 5C–E). suggesting that increased apoptosis was underlying the Consistent with the SA-bGal data, LY2835219 treatment decrease in tumor size in these mice. CDK4 inhibition resulted induced the expression of most of the senescence-related genes in decreased RB phosphorylation, but no further decrease was evaluated (Fig. 5F). However, CDKN1A and RBL2, which are observed in cotreated tumors (Fig. 7H and I). As for mTORC1 p53-regulated genes, did not respond to CDK4/6 inhibition in activity, LY2835219 treatment, as well as AMPK activation, the CDKN2A and p53 mutant MDA-MB-231 cell line. This decreased p70S6K phosphorylation (Fig. 7H and J). Interest- reinforces the idea that the induction of senescence by CDK4 ingly, CDK4 inhibition triggered AMPK activation, as measured inhibition or depletion is more likely related to the lysosome than by the phosphorylation of ACC, a known target of AMPK to any effect on p53. With the exception of RKHD3 and IGFBP5, (Fig. 7H and K). mTOR inhibition by rapamycin also failed to induce the expres- In vitro, CDK4 inhibition and AMPK activation as single treat- sion of genes related to senescence (Fig. 5F). ments failed to induce cell death after 1-week treatment in MDA- These results suggest that the lysosomal dysfunction induced MB-231 cells as shown by the low levels of Annexin V-positive by CDK4 inhibition or depletion is the direct cause of the cells (Supplementary Fig. S7A). Only the combination of senescent phenotype in these cells, independent of mTORC1 LY2835219 and A769662 increased notably the percentage of inhibition. Annexin V-positive cells (Supplementary Fig. S7A). TEM analysis of the tumors revealed that the percentage of The CDK4 inhibitor LY2835219 alters lysosomal function, the autophagosome and lysosome area per cell was increased in attenuates mTORC1 activity, and decreases tumor growth in a the single treatments, as well as in the A769662 and breast cancer xenograft mouse model LY2835219 cotreated group (Supplementary Fig. S7B–S7D). To investigate the effects of CDK4/6 inhibition on lyso- Mice cotreated with both A769662 and LY2835219 still dis- somal function in vivo, we used a breast cancer xenograft played a significant decrease in lysosomes with digested mate- model by injecting MDA-MB-231 cells into the mammary rial (Supplementary Fig. S7B and S7E). The underlying mech- glands of NSG mice. Intratumoral decrease in RB phosphor- anism of this apparent paradox could be that A769662 induced ylation in response to LY2835219 confirmed that CDK4/6 was cell death only in cells in which LY2835219 treatment impaired indeed inhibited in the tumors (Fig. 6A and B). Consistent lysosomal function. Indeed, some of the cotreated cells dis- with previous reports (39), LY2835219-treated mice showed played mixed morphological features of apoptotic cell death halted tumor growth with reduced cell proliferation (Fig. 6C– (highly condensed chromatin, shrinkage of the cytoplasm) and E), compared with mice treated with vehicle. The lack of autophagic cell death (numerous autophagosomes and auto- immune cell infiltration in the tumors suggested that the lysosomes, focal swelling of the perinuclear membrane effects were independent of the immune system (Supplemen- [Supplementary Fig. S7B (indicated by )]. tary Fig. S6A). Taken together, these results show that a combination treat- The tumors of LY2835219-treated mice had increased expres- ment using A769662 and LY2835219 provides a better outcome sion of the lysosomal marker LAMP1 (Fig. 6F and G) and of TFEB than LY2835219 alone, inducing cell death and tumor regression target genes (Supplementary Fig. S6B). In addition, LY2835219 in the MDA-MB-231 breast cancer xenograft model.

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Figure 6. The CDK4/6 inhibitor LY2835219 alters lysosome morphology in a breast cancer xenograft mouse model. A, Western blot analysis for RB and phospho-RB in xenograft protein extracts from both groups. B, Quantification of A as arbitrary levels of phospho-RB normalized to total RB. C, Volume of MDA-MB-231 tumors from mice (n ¼ 8–9 mice per condition). D, IHC with Ki67 antibody and DAPI for tumor sections from DMSO- or LY2835219-gavaged mice. E, Percentage of Ki67- positive cells from D. Three images per section were analyzed for 8 or 9 mice. F, IHC with LAMP1 antibody and DAPI of tumor sections from mouse xenograft models treated with either LY2835219 or DMSO. G, Quantification in arbitrary units of the area of LAMP1 staining normalized to the number of nuclei per field. Three images per section were analyzed for 8 or 9 mice. H, Western blot analysis for the mTOR target protein p70S6K and phospho-p70S6K from tumor lysates, following gavage with either DMSO or LY2835219. I, Quantification of H showing arbitrary levels of phospho-p70S6K normalized to total p70S6K. J, Representative electron micrographs of xenografts showing that LY2835219 treatment notably increases the density of autolysosomes (arrows) and that these autolysosomes accumulate nondegraded materials, such as undegraded autophagosomes (arrowheads). N, nucleus. K–M, Quantification of conditions shown in J as the percentage of autophagosome area per cell (K), percentage of autolysosome area per cell (L), and the percentage of autolysosomes containing degraded material per cell (M). n ¼ 30 cells per condition. N, SA-b-Gal staining of tumor cell senescence in tumor xenograft sections taken from mice gavaged with LY2835219 or DMSO. , P < 0.05; , P < 0.001; , P < 0.0001.

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Figure 7. The CDK4 inhibitor LY2835219 in combination with the AMPK activator A769661 induces cell death in breast cancer cells and tumors. A, Tumor volume of breast cancer xenografts in NSG mice was monitored throughout the whole experiment. Treatment started on day 21 and lasted for 8 days (n ¼ 9–10). B, Representative images from tumors at the day of sacrifice, after the corresponding treatment. C, Increment of tumor volume per mouse: Vol day 29/Vol day 21. D, Representation of percentage of tumors, which increases more than 1.5-fold, from 1 to 1.5-fold, or less than 1-fold. E, Ki67 and cleaved caspase-3 immunostaining in tumor sections after the corresponding treatment. F, Quantification of E,percentageofKi67 positive cells. G, Quantification of E, cleaved caspase-3 area per field, comparing LY2835219 and A769662þLY2935219. H, Western blot analysis of phosphorylation of RB, p70S6K, and ACC proteins in tumor samples. I, Quantification of H, phospho-RB normalized to total RB. J, Quantification of H, phospho-p70S6K normalized to total p70S6K. K, Quantification of H, phospho-ACC normalized to total ACC. , P < 0.05; , P < 0.01; , P < 0.0001.

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Discussion feedback exists between proteasomal activity and autophagic We show here for the first time that CDK4 regulates lysosomal flux (49). Thus, the impairment of autophagic flux at the lyso- function and mTORC1 activity in cancer cells. We demonstrate somal degradation step can result in proteasomal activation and that CDK4, through phosphorylation of FLCN, facilitates the vice versa. migration of mTOR to the lysosomes in order to be activated. Despite the known requirement for lysosomes in cell-cycle The abrogation of mTOR activation by depletion or chemical progression (50, 51), little is known about the relationship inhibition of CDK4 consequently resulted in a substantial between lysosomes and senescence. Importantly, in eukaryotes, increase in the numbers of lysosomes and autophagosomes. autophagy impairment via lysosomal dysfunction has been These prove that CDK4 is necessary for the dissociation of described to be an important characteristic of oxidative FLCN from the lysosomes, and for the subsequent recruitment stress-induced senescence (52). Our findings suggest that the and activation of mTORC1. However, this increase was not ultimate fate of cells that lack CDK4 is the activation of correlated with greater lysosomal activity, which suggested that senescence due to lysosomal dysfunction. The absence of CDK4 has an mTOR-independent role in this process. mTORC1 senescence when cells are treated with rapamycin further activation is initiated at the lysosome and requires the presence demonstrates that mTORC1 inactivation is a consequence of of AAs in the cytosol and in the lysosomal lumen (21). Impor- lysosomal impairment due to CDK4 inhibition or depletion. tantly, we prove that CDK4 inhibition or depletion leads to the However, CDK4 inhibitor-induced lysosomal dysfunction was accumulation of intra-lysosomal undigested material, which not sufficient to prompt cell death in the tumors of the treated probably results in impaired AA recycling and blunted mice. Instead, cancer cells were arrested but were still alive, mTORC1 activation. We show here an additional mechanism which explained that the tumor burden was not decreased but in which CDK4 favors mTORC1 activation by promoting the only stabilized (Fig. 6). We reasoned that further forcing the digestionofproteinsinthelysosome,inturnprovidingmet- autophagic flux would create additional stress that could kill abolic intermediates that sustain cell growth and survival via these tumor cells. Autophagic induction in conditions where downstream effectors. This pathway would be particularly the lysosomal function is impaired abrogates the restoration of crucial during prolonged starvation conditions, such as the metabolic intermediates in the cytoplasm and the synthesis of typical environment of some tumors. macromolecules. Altogether, this might create a situation in Cells initially respond to nutrient deprivation by inactivat- which cells collapse and enter into apoptosis. With this aim, ing their energy-consuming processes, such as protein or lipid we used the AMPK activator A769662, which is known to biosynthesis, and by activating catabolism. At the same time, increase autophagy, in combination with the CDK4 inhibitor. other mechanisms are activated to recycle molecules to provide Indeed, cotreatment resulted in increased cancer cell death and the cell with enough substrates and metabolic intermediates therefore tumor regression (Fig. 7). This is a major finding that to survive—an important function of autophagy. In the long represents a paradigm switch regarding the use of CDK4 inhi- term, autophagy reactivates the mTORC1 pathway by replen- bitors for the treatment of cancer. Strikingly, and consistent ishing the lysosomes with digested proteins and AAs (40). with our findings, CDK4 inhibitors are not efficient as a single CDK4 inhibition or depletion could therefore mimic a star- drug in the clinical practice and are often used in combination vation signal. This hypothesis is in agreement with previous with other drugs (53–55). studies showing that CDK4/6 inhibitors induce autop- We show here that the effects of CDK4 in MDA-MB-231 cells are hagy (41, 42). We also observed an increase in the number likely to be independent of E2F1, the transcription factor mod- of autophagosomes upon CDK4 inhibition. However, when ulated by CDK4 during the cell cycle. It has been reported, analyzing the ultimate fate of the autophagosomes, we found however, that E2F1 regulates lysosomal positioning and activates that they accumulate due to the impairment of lysosomal mTORC1 by promoting its recruitment to the lysosomal sur- degradation upon CDK4 inhibition or depletion. Indeed, face (56, 57). These suggest a dual and complementary role for others have demonstrated that the inhibition of lysosomal CDK4 in the regulation of the mTORC1 pathway: first, through activity causes decreased fusion with autophagosomes and regulation of the lysosomal function; and second, through FLCN vice-versa (43–45). In this study, we show that inhibiting or phosphorylation. depleting CDK4, in addition to inducing autophagy, likely It was previously described that CDK4/6 inhibitors display through mTORC1 inactivation, impairs the autophagic flux at antitumor activity only in RB-positive cells (58). In addition, it the lysosomal degradation step. This observation may explain was unclear whether CDK4/6 inhibition had an effect on the increased susceptibility of CDK4 KO cells to autophagic TNBCs. Our findings are consistent with other studies showing stimuli. We also found that CDK4 inhibition or depletion that CDK4/6 inhibitors can still have some effects on RB- increased the expression of many lysosomal genes. It is well negative cells (59) and that CDK4/6 inhibitors do have anti- known that mTORC1 negatively regulates lysosomal biogen- tumor effects in TNBCs. Indeed, depending on the cell type, esis (46). This could be secondary to mTORC1 inhibition, or CDK4/6 inhibition triggers either a quiescence or senescence due to a compensatory mechanism to create new lysosomes, as response, not necessarily via the canonical RB-E2F pathway the existing ones are dysfunctional. (reviewed in refs. 53, 60). CDK4/6 inhibitors have been shown to accumulate into lyso- Overall, this study demonstrates a new role for CDK4 in the somes, a phenomenon called lysosomal trapping (47). However, regulation of lysosomal function, which ultimately leads to the use of CDK4 KO cells in our work demonstrates that CDK4 senescence in cancer cells and mTORC1 inactivation. In addition, rather stimulates lysosomal function and that the lysosomal we highlight the importance of lysosomes in cancer and we trapping of the drug is secondary to the lysosomal impairment propose that CDK4/6 inhibitors could be used in combination induced by CDK4 inhibition. In addition, CDK4/6 inhibition has with other drugs to target lysosomal function as a novel anticancer been found to result in proteasomal activation (48). A negative- strategy.

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Disclosure of Potential Conflicts of Interest Acknowledgments P. Romero is an Editor-in-Chief at the Journal for Immunotherapy of Cancer, TheauthorsacknowledgeallthemembersoftheFajaslaboratoryfor reports receiving a commercial research grant from Roche pRED – Zurich, has support and discussions. The authors thank Jean Daraspe (University of received speakers bureau honoraria from Roche, and has an unpaid consultant/ Lausanne, Switzerland) for technical assistance and the Electron Microscopy advisory board relationship with Immatics Biotechnologies. No potential Facility at the University of Lausanne for the use of electron microscopes. The conflicts of interest were disclosed by the other authors. authors thank Patrice Waridel and Manfredo Quadroni (from the Protein Analysis Facility, Center for Integrative Genomics, Faculty of Biology and Authors' Contributions Medicine, University of Lausanne, Switzerland) for their help with mass spectrometry analysis. This work from Prof. Fajas lab is supported by the Conception and design: L. Martínez-Carreres, J. Puyal, M. Orpinell, A. Giralt, Swiss National Foundation (31003A-159586). The work on autophagy of P. Romero, I.C. Lopez-Mejia, L. Fajas Julien Puyal is supported by the Swiss National Foundation (310030- Development of methodology: L. Martínez-Carreres, M. Orpinell, J. Castillo- 163064 and 310030_182332). The work of I.C. Lopez-Mejia is supported Armengol, A. Pabois by the Swiss National Science Foundation (Ambizione PZ00P3_168077). Acquisition of data (provided animals, acquired and managed patients, The authors thank Prof. Kei Sakamoto, Prof. Christian Widmann, Prof. Alejo provided facilities, etc.): L. Martínez-Carreres, J. Puyal, L.C. Leal-Esteban, Efeyan, and Prof. Mariano Barbacid for kindly providing some material used M. Orpinell, J. Castillo-Armengol, A. Giralt, O. Dergai, V. Barquissau, in this work. A. Nasrallah, A. Pabois, L. Zhang, P. Romero, I.C. Lopez-Mejia Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Martínez-Carreres, J. Puyal, L.C. Leal-Esteban, The costs of publication of this article were defrayed in part by the M. Orpinell, J. Castillo-Armengol, A. Giralt, L. Zhang, I.C. Lopez-Mejia, L. Fajas payment of page charges. This article must therefore be hereby marked Writing, review, and/or revision of the manuscript: L. Martínez-Carreres, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate J. Puyal, M. Orpinell, A. Giralt, V. Barquissau, A. Nasrallah, L. Zhang, this fact. P. Romero, I.C. Lopez-Mejia, L. Fajas Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Martínez-Carreres, M. Orpinell Received February 28, 2019; revised June 28, 2019; accepted August 1, 2019; Study supervision: M. Orpinell, L. Fajas published first August 8, 2019.

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CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival

Laia Martínez-Carreres, Julien Puyal, Lucía C. Leal-Esteban, et al.

Cancer Res 2019;79:5245-5259. Published OnlineFirst August 8, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0708

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