Publications by authors named "Marcos Malumbres"

157 Publications

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.

JCI Insight 2021 Aug 23;6(16). Epub 2021 Aug 23.

Cell Division and Cancer group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.
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http://dx.doi.org/10.1172/jci.insight.146364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409993PMC
August 2021

Expanding the Differentiation Potential of Already-Established Pluripotent Stem Cells.

Methods Mol Biol 2021 Jun 15. Epub 2021 Jun 15.

Cell Division and Cancer group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Pluripotent stem cells (PSCs) have proven to be an essential tool in many research fields including basic cell biology, development, or human disease. In addition, we are only starting to see their potential in regenerative medicine. Manipulation and culture of PSCs, however, imposes limitations in the quality of these cells and their ability to differentiate into functional cells with physiological function. Here we propose a novel and simple technique based on the transient expression of a single microRNA molecule to expand the differentiation potency of a wide range of PSCs including induced PSCs (iPSCs) as well as embryonic stem cells (ESCs). This method requires no genetic modification of PSCs and achieves stable improvement of the differentiation potential of these cells through several cell passages both in vitro and in vivo.
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http://dx.doi.org/10.1007/7651_2021_408DOI Listing
June 2021

USP7 limits CDK1 activity throughout the cell cycle.

EMBO J 2021 Jun 15;40(11):e99692. Epub 2021 Apr 15.

Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.
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http://dx.doi.org/10.15252/embj.201899692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167359PMC
June 2021

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis.

EMBO Rep 2021 04 21;22(4):e51030. Epub 2021 Feb 21.

Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain.

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo-like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo-like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.
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http://dx.doi.org/10.15252/embr.202051030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025030PMC
April 2021

Effects of energy metabolism on the mechanical properties of breast cancer cells.

Commun Biol 2020 10 20;3(1):590. Epub 2020 Oct 20.

Bionanomechanics Lab, Instituto de Micro y Nanotecnología, IMN-CNM (CSIC), Isaac Newton 8 (PTM), E-28760, Tres Cantos, Madrid, Spain.

Tumorigenesis induces actin cortex remodeling, which makes cancerous cells softer. Cell deformability is largely determined by myosin-driven cortical tension and actin fiber architecture at the cell cortex. However, it is still unclear what the weight of each contribution is, and how these contributions change during cancer development. Moreover, little attention has been paid to the effect of energy metabolism on this phenomenon and its reprogramming in cancer. Here, we perform precise two-dimensional mechanical phenotyping based on power-law rheology to unveil the contributions of myosin II, actin fiber architecture and energy metabolism to the deformability of healthy (MCF-10A), noninvasive cancerous (MCF-7), and metastatic (MDA-MB-231) human breast epithelial cells. Contrary to the perception that the actin cortex is a passive structure that provides mechanical resistance to the cell, we find that this is only true when the actin cortex is activated by metabolic processes. The results show marked differences in the nature of the active processes that build up cell stiffness, namely that healthy cells use ATP-driven actin polymerization whereas metastatic cells use myosin II activity. Noninvasive cancerous cells exhibit an anomalous behavior, as their stiffness is not as affected by the lack of nutrients and ATP, suggesting that energy metabolism reprogramming is used to sustain active processes at the actin cortex.
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http://dx.doi.org/10.1038/s42003-020-01330-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576174PMC
October 2020

Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

EMBO J 2020 08 2;39(16):e104324. Epub 2020 Jul 2.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.
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http://dx.doi.org/10.15252/embj.2019104324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429746PMC
August 2020

Mammalian cell cycle cyclins.

Semin Cell Dev Biol 2020 11 22;107:28-35. Epub 2020 Apr 22.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO) Madrid, Spain. Electronic address:

Proper progression throughout the cell division cycle depends on the expression level of a family of proteins known as cyclins, and the subsequent activation of cyclin-dependent kinases (Cdks). Among the numerous members of the mammalian cyclin family, only a few of them, cyclins A, B, C, D and E, are known to display critical roles in the cell cycle. These functions will be reviewed here with a special focus on their relevance in different cell types in vivo and their implications in human disease.
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http://dx.doi.org/10.1016/j.semcdb.2020.03.009DOI Listing
November 2020

Mechanisms of Sensitivity and Resistance to CDK4/6 Inhibition.

Cancer Cell 2020 04;37(4):514-529

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain. Electronic address:

Inhibiting the cell-cycle kinases CDK4 and CDK6 results in significant therapeutic effect in patients with advanced hormone-positive breast cancer. The efficacy of this strategy is, however, limited by innate or acquired resistance mechanisms and its application to other tumor types is still uncertain. Here, through an integrative analysis of sensitivity and resistance mechanisms, we discuss the use of CDK4/6 inhibitors in combination with available targeted therapies, immunotherapy, or classical chemotherapy with the aim of improving future therapeutic uses of CDK4/6 inhibition in a variety of cancers.
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http://dx.doi.org/10.1016/j.ccell.2020.03.010DOI Listing
April 2020

CDK4/6 Inhibitors Impair Recovery from Cytotoxic Chemotherapy in Pancreatic Adenocarcinoma.

Cancer Cell 2020 03 27;37(3):340-353.e6. Epub 2020 Feb 27.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO) Madrid, Madrid 28029, Spain. Electronic address:

Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents.
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http://dx.doi.org/10.1016/j.ccell.2020.01.007DOI Listing
March 2020

Squamous differentiation requires G2/mitosis slippage to avoid apoptosis.

Cell Death Differ 2020 08 20;27(8):2451-2467. Epub 2020 Feb 20.

Cell Cycle, Stem Cell Fate and Cancer Laboratory, Institute for Research Marqués de Valdecilla (IDIVAL), 39011, Santander, Spain.

The cellular mechanisms controlling cell fate in self-renewal tissues remain unclear. Cell cycle failure often leads to an apoptosis anti-oncogenic response. We have inactivated Cdk1 or Polo-like-1 kinases, essential targets of the mitotic checkpoints, in the epithelia of skin and oral mucosa. Here, we show that inactivation of the mitotic kinases leading to polyploidy in vivo, produces a fully differentiated epithelium. Cells within the basal layer aberrantly differentiate and contain large or various nuclei. Freshly isolated KO cells were also differentiated and polyploid. However, sustained metaphase arrest downstream of the spindle anaphase checkpoint (SAC) due to abrogation of CDC20 (essential cofactor of anaphase-promoting complex), impaired squamous differentiation and resulted in apoptosis. Therefore, upon prolonged arrest keratinocytes need to slip beyond G2 or mitosis in order to initiate differentiation. The results altogether demonstrate that mitotic checkpoints drive squamous cell fate towards differentiation or apoptosis in response to genetic damage.
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http://dx.doi.org/10.1038/s41418-020-0515-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370216PMC
August 2020

Molecular Basis of the Mechanisms Controlling MASTL.

Mol Cell Proteomics 2020 02 18;19(2):326-343. Epub 2019 Dec 18.

The Novo Nordisk Foundation Center for Protein Research, Protein Structure & Function Programme, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.
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http://dx.doi.org/10.1074/mcp.RA119.001879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000116PMC
February 2020

A Chemical Screen Identifies Compounds Capable of Selecting for Haploidy in Mammalian Cells.

Cell Rep 2019 07;28(3):597-604.e4

Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain; Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 21 Stockholm, Sweden. Electronic address:

The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells.
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http://dx.doi.org/10.1016/j.celrep.2019.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656781PMC
July 2019

An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability.

Nucleic Acids Res 2019 Aug;47(14):7716-7717

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

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http://dx.doi.org/10.1093/nar/gkz587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698732PMC
August 2019

p38γ is essential for cell cycle progression and liver tumorigenesis.

Nature 2019 04 10;568(7753):557-560. Epub 2019 Apr 10.

Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.
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http://dx.doi.org/10.1038/s41586-019-1112-8DOI Listing
April 2019

Detection of novel fusion-transcripts by RNA-Seq in T-cell lymphoblastic lymphoma.

Sci Rep 2019 03 26;9(1):5179. Epub 2019 Mar 26.

Department of Cellular Biology and Immunology, Severo Ochoa Molecular Biology Center (CBMSO), CSIC-Madrid Autonomous University, Madrid, 28049, Spain.

Fusions transcripts have been proven to be strong drivers for neoplasia-associated mutations, although their incidence in T-cell lymphoblastic lymphoma needs to be determined yet. Using RNA-Seq we have selected 55 fusion transcripts identified by at least two of three detection methods in the same tumour. We confirmed the existence of 24 predicted novel fusions that had not been described in cancer or normal tissues yet, indicating the accuracy of the prediction. Of note, one of them involves the proto oncogene TAL1. Other confirmed fusions could explain the overexpression of driver genes such as COMMD3-BMI1, LMO1 or JAK3. Five fusions found exclusively in tumour samples could be considered pathogenic (NFYG-TAL1, RIC3-TCRBC2, SLC35A3-HIAT1, PICALM MLLT10 and MLLT10-PICALM). However, other fusions detected simultaneously in normal and tumour samples (JAK3-INSL3, KANSL1-ARL17A/B and TFG-ADGRG7) could be germ-line fusions genes involved in tumour-maintaining tasks. Notably, some fusions were confirmed in more tumour samples than predicted, indicating that the detection methods underestimated the real number of existing fusions. Our results highlight the potential of RNA-Seq to identify new cryptic fusions, which could be drivers or tumour-maintaining passenger genes. Such novel findings shed light on the searching for new T-LBL biomarkers in these haematological disorders.
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http://dx.doi.org/10.1038/s41598-019-41675-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435891PMC
March 2019

Aurora A controls CD8 T cell cytotoxic activity and antiviral response.

Sci Rep 2019 02 18;9(1):2211. Epub 2019 Feb 18.

Servicio de Inmunología, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Instituto Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain.

Aurora A is a serine/threonine kinase whose role in cell cycle progression and tumour generation has been widely studied. Recent work has revealed an unexpected function for Aurora A during CD4 T cell activation and, also, in graft versus host disease development. However, it remains unknown whether Aurora A is involved in CD8 T cell effector function and in cytotoxic T lymphocyte-mediated antiviral response. Here, we show that Aurora A chemical inhibition leads to an impairment of both the peptide-specific cytotoxicity and the degranulation activity of CD8 T cells. This finding was similarly proven for both mice and human CD8 CTL activity. As a result of Aurora A blockade, we detected a reduction in the expression induced by T cell activation of genes classically related to the effector function of cytotoxic T lymphocytes such as granzyme B or perforin1. Finally, we have found that Aurora A is necessary for CD8 T cell-mediated antiviral response, in an in vivo model of vaccinia virus infection. Thus, we can conclude that Aurora A activity is, indeed, needed for the proper effector function of cytotoxic T lymphocytes and for their activity against viral threats.
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http://dx.doi.org/10.1038/s41598-019-38647-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379542PMC
February 2019

Downregulation of specific FBXW7 isoforms with differential effects in T-cell lymphoblastic lymphoma.

Oncogene 2019 06 11;38(23):4620-4636. Epub 2019 Feb 11.

Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

FBXW7 is a driver gene in T-cell lymphoblastic neoplasia acting through proteasome degradation of key proto-oncogenes. FBXW7 encodes three isoforms, α, β and γ, which differ only in the N-terminus. In this work, massive sequencing revealed significant downregulation of FBXW7 in a panel of primary T-cell lymphoblastic lymphomas characterised by the absence of mutations in its sequence. We observed that decreased expression mainly affected the FBXW7β isoform and to a lesser extent FBXW7α and may be attributed to the combined effect of epigenetic changes, alteration of upstream factors and upregulation of miRNAs. Transient transfections with miRNA mimics in selected cell lines resulted in a significant decrease of total FBXW7 expression and its different isoforms separately, with the consequent increment of critical substrates and the stimulation of cell proliferation. Transient inhibition of endogenous miRNAs in a T-cell lymphoblastic-derived cell line (SUP-T1) was capable of reversing these proliferative effects. Finally, we show how FBXW7 isoforms display different roles within the cell. Simultaneous downregulation of the α and γ isoforms modulates the amount of CCNE1, whilst the β-isoform alone was found to have a prominent role in modulating the amount of c-MYC. Our data also revealed that downregulation of all isoforms is a sine qua non condition to induce a proliferative pattern in our cell model system. Taking these data into account, potential new treatments to reverse downregulation of all or a specific FBXW7 isoform may be an effective strategy to counteract the proliferative capacity of these tumour cells.
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http://dx.doi.org/10.1038/s41388-019-0746-1DOI Listing
June 2019

Two Interlinked Bistable Switches Govern Mitotic Control in Mammalian Cells.

Curr Biol 2018 12 15;28(23):3824-3832.e6. Epub 2018 Nov 15.

Genome Damage and Stability Centre, University of Sussex, Science Park Road, Brighton BN1 9RQ, UK. Electronic address:

Distinct protein phosphorylation levels in interphase and M phase require tight regulation of Cdk1 activity [1, 2]. A bistable switch, based on positive feedback in the Cdk1 activation loop, has been proposed to generate different thresholds for transitions between these cell-cycle states [3-5]. Recently, the activity of the major Cdk1-counteracting phosphatase, PP2A:B55, has also been found to be bistable due to Greatwall kinase-dependent regulation [6]. However, the interplay of the regulation of Cdk1 and PP2A:B55 in vivo remains unexplored. Here, we combine quantitative cell biology assays with mathematical modeling to explore the interplay of mitotic kinase activation and phosphatase inactivation in human cells. By measuring mitotic entry and exit thresholds using ATP-analog-sensitive Cdk1 mutants, we find evidence that the mitotic switch displays hysteresis and bistability, responding differentially to Cdk1 inhibition in the mitotic and interphase states. Cdk1 activation by Wee1/Cdc25 feedback loops and PP2A:B55 inactivation by Greatwall independently contributes to this hysteretic switch system. However, elimination of both Cdk1 and PP2A:B55 inactivation fully abrogates bistability, suggesting that hysteresis is an emergent property of mutual inhibition between the Cdk1 and PP2A:B55 feedback loops. Our model of the two interlinked feedback systems predicts an intermediate but hidden steady state between interphase and M phase. This could be verified experimentally by Cdk1 inhibition during mitotic entry, supporting the predictive value of our model. Furthermore, we demonstrate that dual inhibition of Wee1 and Gwl kinases causes loss of cell-cycle memory and synthetic lethality, which could be further exploited therapeutically.
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http://dx.doi.org/10.1016/j.cub.2018.09.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287978PMC
December 2018

Genetic Interactions between the Aurora Kinases Reveal New Requirements for AURKB and AURKC during Oocyte Meiosis.

Curr Biol 2018 11 25;28(21):3458-3468.e5. Epub 2018 Oct 25.

Department of Genetics, Rutgers University, 145 Bevier Road, Piscataway, NJ 08854, USA. Electronic address:

Errors in chromosome segregation during female meiosis I occur frequently, and aneuploid embryos account for 1/3 of all miscarriages in humans [1]. Unlike mitotic cells that require two Aurora kinase (AURK) homologs to help prevent aneuploidy (AURKA and AURKB), mammalian germ cells also require a third (AURKC) [2, 3]. AURKA is the spindle-pole-associated homolog, and AURKB/C are the chromosome-localized homologs. In mitosis, AURKB has essential roles as the catalytic subunit of the chromosomal passenger complex (CPC), regulating chromosome alignment, kinetochore-microtubule attachments, cohesion, the spindle assembly checkpoint, and cytokinesis [4, 5]. In mouse oocyte meiosis, AURKC takes over as the predominant CPC kinase [6], although the requirement for AURKB remains elusive [7]. In the absence of AURKC, AURKB compensates, making defining potential non-overlapping functions difficult [6, 8]. To investigate the role(s) of AURKB and AURKC in oocytes, we analyzed oocyte-specific Aurkb and Aurkc single- and double-knockout (KO) mice. Surprisingly, we find that double KO female mice are fertile. We demonstrate that, in the absence of AURKC, AURKA localizes to chromosomes in a CPC-dependent manner. These data suggest that AURKC prevents AURKA from localizing to chromosomes by competing for CPC binding. This competition is important for adequate spindle length to support meiosis I. We also describe a unique requirement for AURKB to negatively regulate AURKC to prevent aneuploidy. Together, our work reveals oocyte-specific roles for the AURKs in regulating each other's localization and activity. This inter-kinase regulation is critical to support wild-type levels of fecundity in female mice.
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http://dx.doi.org/10.1016/j.cub.2018.08.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234855PMC
November 2018

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets.

J Clin Invest 2018 12 29;128(12):5351-5367. Epub 2018 Oct 29.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.
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http://dx.doi.org/10.1172/JCI121876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264735PMC
December 2018

CDK4/6 Inhibitors: What Is the Best Cocktail?

Authors:
Marcos Malumbres

Clin Cancer Res 2019 01 16;25(1):6-8. Epub 2018 Aug 16.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

CDK4/6 inhibitors have shown great potential in the new armamentarium against cancer. However, their effect as single agents is limited, and the hopes are on new combinatory strategies. Recent data suggest that inhibiting mTOR may significantly cooperate with cell-cycle arrest in a variety of cancers..
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http://dx.doi.org/10.1158/1078-0432.CCR-18-2177DOI Listing
January 2019

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1.

Sci Rep 2018 08 8;8(1):11871. Epub 2018 Aug 8.

Instituto de Biología Molecular y Celular del Cáncer (IBMCC), Universidad de Salamanca-CSIC, Campus Miguel de Unamuno, 37007, Salamanca, Spain.

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.
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http://dx.doi.org/10.1038/s41598-018-30253-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082843PMC
August 2018

Plk1 overexpression induces chromosomal instability and suppresses tumor development.

Nat Commun 2018 08 1;9(1):3012. Epub 2018 Aug 1.

Division of Molecular Thoracic Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.

Polo-like kinase 1 (Plk1) is overexpressed in a wide spectrum of human tumors, being frequently considered as an oncogene and an attractive cancer target. However, its contribution to tumor development is unclear. Using a new inducible knock-in mouse model we report here that Plk1 overexpression results in abnormal chromosome segregation and cytokinesis, generating polyploid cells with reduced proliferative potential. Mechanistically, these cytokinesis defects correlate with defective loading of Cep55 and ESCRT complexes to the abscission bridge, in a Plk1 kinase-dependent manner. In vivo, Plk1 overexpression prevents the development of Kras-induced and Her2-induced mammary gland tumors, in the presence of increased rates of chromosome instability. In patients, Plk1 overexpression correlates with improved survival in specific breast cancer subtypes. Therefore, despite the therapeutic benefits of inhibiting Plk1 due to its essential role in tumor cell cycles, Plk1 overexpression has tumor-suppressive properties by perturbing mitotic progression and cytokinesis.
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http://dx.doi.org/10.1038/s41467-018-05429-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070485PMC
August 2018

CDK6 Antagonizes p53-Induced Responses during Tumorigenesis.

Cancer Discov 2018 07 13;8(7):884-897. Epub 2018 Jun 13.

Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria.

Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists , and The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in significantly more often than expected. CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031305PMC
July 2018

RNA-Seq reveals the existence of a CDKN1C-E2F1-TP53 axis that is altered in human T-cell lymphoblastic lymphomas.

BMC Cancer 2018 04 16;18(1):430. Epub 2018 Apr 16.

Department of Cellular Biology and Immunology, Severo Ochoa Molecular Biology Center (CBMSO), CSIC-Madrid Autonomous University, 28049, Madrid, Spain.

Background: Precursor T-cell lymphoblastic lymphomas (T-LBL) are rare aggressive hematological malignancies that mainly develop in children. As in other cancers, the loss of cell cycle control plays a prominent role in the pathogenesis in these malignancies that is primarily attributed to loss of CDKN2A (encoding protein p16INK4A). However, the impact of the deregulation of other genes such as CDKN1C, E2F1, and TP53 remains to be clarified. Interestingly, experiments in mouse models have proven that conditional T-cell specific deletion of Cdkn1c gene may induce a differentiation block at the DN3 to DN4 transition, and that the loss of this gene in the absence of Tp53 led to aggressive thymic lymphomas.

Results: In this manuscript, we demonstrated that the simultaneous deregulation of CDKN1C, E2F1, and TP53 genes by epigenetic mechanisms and/or the deregulation of specific microRNAs, together with additional impairing of TP53 function by the expression of dominant-negative isoforms are common features in primary human T-LBLs.

Conclusions: Previous experimental work in mice revealed that T-cell specific deletion of Cdkn1c accelerates lymphomagenesis in the absence of Tp53. If, as expected, the consequences of the deregulation of the CDKN1C-E2F1-TP53 axis were the same as those experimentally demonstrated in mouse models, the disruption of this axis might be useful to predict tumor aggressiveness, and to provide the basis towards the development of potential therapeutic strategiesin human T-LBL.
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http://dx.doi.org/10.1186/s12885-018-4304-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902834PMC
April 2018

An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability.

Nucleic Acids Res 2018 05;46(9):4546-4559

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity.
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http://dx.doi.org/10.1093/nar/gky218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961008PMC
May 2018

Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

Cell Death Differ 2018 05 11;25(5):828-840. Epub 2017 Dec 11.

Cell Division and Cancer group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.
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http://dx.doi.org/10.1038/s41418-017-0024-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943447PMC
May 2018

Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis.

Nat Med 2017 Aug 10;23(8):964-974. Epub 2017 Jul 10.

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Polo-like kinase 1 (PLK1), an essential regulator of cell division, is currently undergoing clinical evaluation as a target for cancer therapy. We report an unexpected function of Plk1 in sustaining cardiovascular homeostasis. Plk1 haploinsufficiency in mice did not induce obvious cell proliferation defects but did result in arterial structural alterations, which frequently led to aortic rupture and death. Specific ablation of Plk1 in vascular smooth muscle cells (VSMCs) led to reduced arterial elasticity, hypotension, and an impaired arterial response to angiotensin II in vivo. Mechanistically, we found that Plk1 regulated angiotensin II-dependent activation of RhoA and actomyosin dynamics in VSMCs in a mitosis-independent manner. This regulation depended on Plk1 kinase activity, and the administration of small-molecule Plk1 inhibitors to angiotensin II-treated mice led to reduced arterial fitness and an elevated risk of aneurysm and aortic rupture. We thus conclude that a partial reduction of Plk1 activity that does not block cell division can nevertheless impair aortic homeostasis. Our findings have potentially important implications for current approaches aimed at PLK1 inhibition for cancer therapy.
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http://dx.doi.org/10.1038/nm.4364DOI Listing
August 2017
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