Publications by authors named "Carmen Blanco-Aparicio"

62 Publications

Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC.

Cancers (Basel) 2021 Apr 29;13(9). Epub 2021 Apr 29.

Department of Clinical Medicine, Trinity Translational Medicine Institute, St. James's Hospital, Dublin, Ireland.

PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.
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http://dx.doi.org/10.3390/cancers13092139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125027PMC
April 2021

Omipalisib inspired macrocycles as dual PI3K/mTOR inhibitors.

Eur J Med Chem 2021 Feb 18;211:113109. Epub 2020 Dec 18.

Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), C/ Melchor Fernández Almagro 3, E-28029, Madrid, Spain. Electronic address:

Activation of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway occurs frequently in a wide range of human cancers and is a main driver of cell growth, proliferation, survival, and chemoresistance of cancer cells. Compounds targeting this pathway are under active development as anticancer therapeutics and some of them have reached advanced clinical trials or been approved by the FDA. Dual PI3K/mTOR inhibitors combine multiple therapeutic efficacies in a single molecule by inhibiting the pathway both upstream and downstream of AKT. Herein, we report our efforts on the exploration of novel small molecule macrocycles (MCXs) as dual PI3K/mTOR inhibitors. Macrocyclization is an attractive approach used in drug discovery, as the semi-rigid character of these structures could provide improved potency, selectivity and favorable pharmacokinetic properties. Importantly, this strategy allows access to new chemical space thus obtaining a better intellectual property position. A series of MCXs based on GSK-2126458, a known clinical PI3K/mTOR inhibitor is described. These molecules showed potent biochemical and cellular dual PI3K/mTOR inhibition, demonstrated strong antitumoral effects in human cancer cell lines, and displayed good drug-like properties. Among them, MCX 83 presented remarkable selectivity against a panel of 468 kinases, high in vitro metabolic stability, and favorable pharmacokinetic parameters without significant CYP450 and h-ERG binding inhibition. This profile qualified this compound as a suitable candidate for future in vivo PK-PD and efficacy studies in mouse cancer models.
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http://dx.doi.org/10.1016/j.ejmech.2020.113109DOI Listing
February 2021

Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases.

Nat Cell Biol 2020 10 28;22(10):1223-1238. Epub 2020 Sep 28.

Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF-MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF-MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.
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http://dx.doi.org/10.1038/s41556-020-0573-1DOI Listing
October 2020

Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas.

Proc Natl Acad Sci U S A 2020 09 10;117(39):24415-24426. Epub 2020 Sep 10.

Experimental Oncology, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain;

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.
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http://dx.doi.org/10.1073/pnas.2002520117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533841PMC
September 2020

Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells.

Cancers (Basel) 2020 Jul 4;12(7). Epub 2020 Jul 4.

Department of Cancer Biology, Instituto de Investigaciones Biomédicas "Alberto Sols" (IIBM), 28029 Madrid, Spain.

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC.
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http://dx.doi.org/10.3390/cancers12071790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407272PMC
July 2020

Pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one derivatives as CDK8 inhibitors.

Eur J Med Chem 2020 Sep 20;201:112443. Epub 2020 Jun 20.

Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), C/ Melchor Fernández Almagro 3, E-28029, Madrid, Spain. Electronic address:

CDK8 is a cyclin-dependent kinase that forms part of the mediator complex, and modulates the transcriptional output from distinct transcription factors involved in oncogenic control. Overexpression of CDK8 has been observed in various cancers, representing a potential target for developing novel CDK8 inhibitors in cancer therapeutics. In the course of our investigations to discover new CDK8 inhibitors, we designed and synthesized tricyclic pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one derivatives, by introduction of chemical complexity in the multi-kinase inhibitor Sorafenib taking into account the flexibility of the P-loop motif of CDK8 protein observed after analysis of structural information of co-crystallized CDK8 inhibitors. In vitro evaluation of the inhibitory activity of the prepared compounds against CDK8 led us to identify compound 2 as the most potent inhibitor of the series (IC = 8.25 nM). Co-crystal studies and the remarkable selectivity profile of compound 2 are presented. Compound 2 showed moderate reduction of phosphorylation of CDK8 substrate STAT1 in cells, in line with other reported Type II CDK8 inhibitors. We propose herein an alternative to find a potential therapeutic use for this chemical series.
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http://dx.doi.org/10.1016/j.ejmech.2020.112443DOI Listing
September 2020

The mTOR pathway is necessary for survival of mice with short telomeres.

Nat Commun 2020 03 3;11(1):1168. Epub 2020 Mar 3.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, E-28029, Madrid, Spain.

Telomerase deficiency leads to age-related diseases and shorter lifespans. Inhibition of the mechanistic target of rapamycin (mTOR) delays aging and age-related pathologies. Here, we show that telomerase deficient mice with short telomeres (G2-Terc) have an hyper-activated mTOR pathway with increased levels of phosphorylated ribosomal S6 protein in liver, skeletal muscle and heart, a target of mTORC1. Transcriptional profiling confirms mTOR activation in G2-Terc livers. Treatment of G2-Terc mice with rapamycin, an inhibitor of mTORC1, decreases survival, in contrast to lifespan extension in wild-type controls. Deletion of mTORC1 downstream S6 kinase 1 in G3-Terc mice also decreases longevity, in contrast to lifespan extension in single S6K1 female mice. These findings demonstrate that mTOR is important for survival in the context of short telomeres, and that its inhibition is deleterious in this setting. These results are of clinical interest in the case of human syndromes characterized by critically short telomeres.
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http://dx.doi.org/10.1038/s41467-020-14962-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054554PMC
March 2020

Preclinical evaluation of a novel triple-acting PIM/PI3K/mTOR inhibitor, IBL-302, in breast cancer.

Oncogene 2020 04 10;39(14):3028-3040. Epub 2020 Feb 10.

Medical Oncology Group, Department of Molecular Medicine, Royal College of Surgeons Ireland, Smurfit Building Beaumont Hospital, Beaumont, Dublin, Ireland.

The proviral integration of Moloney virus (PIM) family of protein kinases are overexpressed in many haematological and solid tumours. PIM kinase expression is elevated in PI3K inhibitor-treated breast cancer samples, suggesting a major resistance pathway for PI3K inhibitors in breast cancer, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K/AKT/mTOR signalling. We thus evaluated the preclinical activity of IBL-302, in a range of breast cancer models. Our results demonstrate in vitro efficacy of IBL-302 in a range of breast cancer cell lines, including lines with acquired resistance to trastuzumab and lapatinib. IBL-302 demonstrated single-agent, anti-tumour efficacy in suppression of pAKT, pmTOR and pBAD in the SKBR-3, BT-474 and HCC-1954 HER2+/PIK3CA-mutated cell lines. We have also shown the in vivo single-agent efficacy of IBL-302 in the subcutaneous BT-474 and HCC-1954 xenograft model in BALB/c nude mice. The combination of trastuzumab and IBL-302 significantly increased the anti-proliferative effect in HER2+ breast cancer cell line, and matched trastuzumab-resistant line, relative to testing either drug alone. We thus believe that the novel PIM and PI3K/mTOR inhibitor, IBL-302, represents an exciting new potential treatment option for breast cancer, and that it should be considered for clinical investigation.
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http://dx.doi.org/10.1038/s41388-020-1202-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118022PMC
April 2020

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status.

Nat Commun 2019 11 28;10(1):5428. Epub 2019 Nov 28.

Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Lab Block level 3, Box 231, Cambridge Biomedical Campus, Cambridge, CB2 0QQ, UK.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.
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http://dx.doi.org/10.1038/s41467-019-13315-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883072PMC
November 2019

Screening protocol for the identification of modulators by immunofluorescent cell-based assay.

Chem Biol Drug Des 2020 01 20;95(1):66-78. Epub 2019 Sep 20.

Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

High-throughput assays are a common strategy for the identification of compounds able to modulate a certain cellular activity. Here, we show an automatized analysis platform for the quantification of nuclear foci as inhibitory effect of compounds on a target protein labeled by fluorescent antibodies. Our experience led us to a fast analysis platform that combines cell-based assays, high-content screening, and confocal microscopy, with an automatic and user-friendly statistical analysis of plate-based assays including positive and negative controls, able to identify inhibitory effect of compounds tested together with the Z-prime and Window of individual plate-based assays to assess the reliability of the results. The platform integrates a web-based tool implemented in Pipeline Pilot and R, and allows computing the inhibition values of different parameters obtained from the high-content screening and confocal microscopy analysis. This facilitates the exploration of the results using the different parameters, providing information at different levels as the number of foci observed, the sum of intensity of foci, area of foci, etc, the detection and filtering of outliers over the assay plate, and finally providing a set of statistics of the parameters studied together with a set of plots that we believe significantly helps to the interpretation of the assay results.
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http://dx.doi.org/10.1111/cbdd.13616DOI Listing
January 2020

Multiple cancer pathways regulate telomere protection.

EMBO Mol Med 2019 07 13;11(7):e10292. Epub 2019 Jun 13.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain.

Telomeres are considered as universal anti-cancer targets, as telomere maintenance is essential to sustain indefinite cancer growth. Mutations in telomerase, the enzyme that maintains telomeres, are among the most frequently found in cancer. In addition, mutations in components of the telomere protective complex, or shelterin, are also found in familial and sporadic cancers. Most efforts to target telomeres have focused in telomerase inhibition; however, recent studies suggest that direct targeting of the shelterin complex could represent a more effective strategy. In particular, we recently showed that genetic deletion of the TRF1 essential shelterin protein impairs tumor growth in aggressive lung cancer and glioblastoma (GBM) mouse models by direct induction of telomere damage independently of telomere length. Here, we screen for TRF1 inhibitory drugs using a collection of FDA-approved drugs and drugs in clinical trials, which cover the majority of pathways included in the Reactome database. Among other targets, we find that inhibition of several kinases of the Ras pathway, including ERK and MEK, recapitulates the effects of Trf1 genetic deletion, including induction of telomeric DNA damage, telomere fragility, and inhibition of cancer stemness. We further show that both bRAF and ERK2 kinases phosphorylate TRF1 in vitro and that these modifications are essential for TRF1 location to telomeres in vivo. Finally, we use these new TRF1 regulatory pathways as the basis to discover novel drug combinations based on TRF1 inhibition, with the goal of effectively blocking potential resistance to individual drugs in patient-derived glioblastoma xenograft models.
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http://dx.doi.org/10.15252/emmm.201910292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609915PMC
July 2019

Discovery of novel triazolo[4,3-b]pyridazin-3-yl-quinoline derivatives as PIM inhibitors.

Eur J Med Chem 2019 Apr 19;168:87-109. Epub 2019 Feb 19.

Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), C/ Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Electronic address:

PIM kinase family (PIM-1, PIM-2 and PIM-3) is an appealing target for the discovery and development of selective inhibitors, useful in various disease conditions in which these proteins are highly expressed, such as cancer. The significant effort put, in the recent years, towards the development of small molecules exhibiting inhibitory activity against this protein family has ended up with several molecules entering clinical trials. As part of our ongoing exploration for potential drug candidates that exhibit affinity towards this protein family, we have generated a novel chemical series of triazolo[4,3-b]pyridazine based tricycles by applying a scaffold hopping strategy over our previously reported potent pan-PIM inhibitor ETP-47453 (compound 2). The structure-activity relationship studies presented herein demonstrate a rather selective PIM-1/PIM-3 biochemical profile for this novel series of tricycles, although pan-PIM and PIM-1 inhibitors have also been identified. Selected examples show significant inhibition of the phosphorylation of BAD protein in a cell-based assay. Moreover, optimized and highly selective compounds, such as 42, did not show significant hERG inhibition at 20 μM concentration, and proved its antiproliferative activity and utility in combination with particular antitumoral agents in several tumor cell lines.
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http://dx.doi.org/10.1016/j.ejmech.2019.02.022DOI Listing
April 2019

Lysosomal trapping of palbociclib and its functional implications.

Oncogene 2019 05 28;38(20):3886-3902. Epub 2019 Jan 28.

Spanish National Cancer Research Center (CNIO), Madrid, Spain.

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.
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http://dx.doi.org/10.1038/s41388-019-0695-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756094PMC
May 2019

Author Correction: STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis.

Nat Med 2018 Sep;24(9):1481

Brain Metastasis Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

In the version of this article originally published, the names of three authors were incorrect. The authors were listed as "Coral Fustero-Torres", "Elena Pineiro" and "Melchor Sánchez-Martínez". Their respective names are "Coral Fustero-Torre", "Elena Piñeiro-Yáñez" and "Melchor Sanchez-Martinez". The errors have been corrected in the print, HTML and PDF versions of this article.
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http://dx.doi.org/10.1038/s41591-018-0108-5DOI Listing
September 2018

STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis.

Nat Med 2018 07 11;24(7):1024-1035. Epub 2018 Jun 11.

Brain Metastasis Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.
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http://dx.doi.org/10.1038/s41591-018-0044-4DOI Listing
July 2018

Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies.

Clin Cancer Res 2018 08 27;24(15):3550-3559. Epub 2018 Mar 27.

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the /VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our and functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0103DOI Listing
August 2018

Inhibition of TRF1 Telomere Protein Impairs Tumor Initiation and Progression in Glioblastoma Mouse Models and Patient-Derived Xenografts.

Cancer Cell 2017 11;32(5):590-607.e4

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, 28029, Spain. Electronic address:

Glioblastoma multiforme (GBM) is a deadly and common brain tumor. Poor prognosis is linked to high proliferation and cell heterogeneity, including glioma stem cells (GSCs). Telomere genes are frequently mutated. The telomere binding protein TRF1 is essential for telomere protection, and for adult and pluripotent stem cells. Here, we find TRF1 upregulation in mouse and human GBM. Brain-specific Trf1 genetic deletion in GBM mouse models inhibited GBM initiation and progression, increasing survival. Trf1 deletion increased telomeric DNA damage and reduced proliferation and stemness. TRF1 chemical inhibitors mimicked these effects in human GBM cells and also blocked tumor sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Thus, targeting telomeres throughout TRF1 inhibition is an effective therapeutic strategy for GBM.
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http://dx.doi.org/10.1016/j.ccell.2017.10.006DOI Listing
November 2017

Modulation of telomere protection by the PI3K/AKT pathway.

Nat Commun 2017 11 2;8(1):1278. Epub 2017 Nov 2.

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain.

Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.
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http://dx.doi.org/10.1038/s41467-017-01329-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668434PMC
November 2017

Identification of novel PI3K inhibitors through a scaffold hopping strategy.

Bioorg Med Chem Lett 2017 11 30;27(21):4794-4799. Epub 2017 Sep 30.

Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Electronic address:

A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKT in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.
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http://dx.doi.org/10.1016/j.bmcl.2017.09.059DOI Listing
November 2017

Inflammation and stem markers association to PIM1/PIM2 kinase-induced tumors in breast and uterus.

Oncotarget 2017 Aug 22;8(35):58872-58886. Epub 2017 Jul 22.

Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas, Seville, Spain.

The PIM family of Ser/Thr kinase proteins has been implicated in tumorigenesis at different levels. PIM proteins are overexpressed in several tumor types and have been associated with chemoresistance. However, their role in hormone-dependent female tissues has not been explored, especially in the uterus, breast and ovary. We generated conditional transgenic mice with confined expression of human PIM1 or PIM2 genes in these tissues. We characterized the tumoral response to these genetic alterations corroborating their role as oncogenes since they induce hyperproliferation in all tissues and tumors in mammary gland and uterus. Furthermore, we observed a high degree of inflammatory infiltration in these tissues of transgenic mice accompanied by NFAT and mTOR activation and IL6 expression. Moreover, PIM1/2 were overexpressed in human breast, uterine and ovarian tumors, correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression provoke tissue alterations and a large IL6-dependent inflammatory response that may act synergistically during the process of tumorigenesis. The possible end-point is an increased percentage of cancer stem cells, which may be partly responsible for the therapy resistance found in tumors overexpressing PIM kinases.
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http://dx.doi.org/10.18632/oncotarget.19438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601700PMC
August 2017

The role of PIM1/PIM2 kinases in tumors of the male reproductive system.

Sci Rep 2016 11 30;6:38079. Epub 2016 Nov 30.

Instituto de Biomedicina de Sevilla, IBIS/Hospital Universitario Virgen del Rocío/Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, Avda. Manuel Siurot s/n 41013, Seville, Spain.

The PIM family of serine/threonine kinases has three highly conserved isoforms (PIM1, PIM2 and PIM3). PIM proteins are regulated through transcription and stability by JAK/STAT pathways and are overexpressed in hematological malignancies and solid tumors. The PIM kinases possess weak oncogenic abilities, but enhance other genes or chemical carcinogens to induce tumors. We generated conditional transgenic mice that overexpress PIM1 or PIM2 in male reproductive organs and analyzed their contribution to tumorigenesis. We found an increase in alterations of sexual organs and hyperplasia in the transgenic mice correlating with inflammation. We also found that PIM1/2 are overexpressed in a subset of human male germ cells and prostate tumors correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression is a common feature of male reproductive organs tumors, which provoke tissue alterations and a large inflammatory response that may act synergistically during the process of tumorigenesis. There is also a correlation with markers of cancer stem cells, which may contribute to the therapy resistance found in tumors overexpressing PIM kinases.
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http://dx.doi.org/10.1038/srep38079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128923PMC
November 2016

Tissue damage and senescence provide critical signals for cellular reprogramming in vivo.

Science 2016 11;354(6315)

Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid E28029, Spain.

Reprogramming of differentiated cells into pluripotent cells can occur in vivo, but the mechanisms involved remain to be elucidated. Senescence is a cellular response to damage, characterized by abundant production of cytokines and other secreted factors that, together with the recruitment of inflammatory cells, result in tissue remodeling. Here, we show that in vivo expression of the reprogramming factors OCT4, SOX2, KLF4, and cMYC (OSKM) in mice leads to senescence and reprogramming, both coexisting in close proximity. Genetic and pharmacological analyses indicate that OSKM-induced senescence requires the Ink4a/Arf locus and, through the production of the cytokine interleukin-6, creates a permissive tissue environment for in vivo reprogramming. Biological conditions linked to senescence, such as tissue injury or aging, favor in vivo reprogramming by OSKM. These observations may be relevant for tissue repair.
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http://dx.doi.org/10.1126/science.aaf4445DOI Listing
November 2016

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Carcinogenesis 2015 Jun;36 Suppl 1:S254-96

Superfund Research Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27560, USA.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
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http://dx.doi.org/10.1093/carcin/bgv039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480130PMC
June 2015

Disruptive chemicals, senescence and immortality.

Carcinogenesis 2015 Jun;36 Suppl 1:S19-37

Brunel Institute of Cancer Genetics and Pharmacogenomics, Health and Environment Theme, Institute of Environment, Health and Societies, Brunel University London, Kingston Lane, Uxbridge, UB8 3PH, UK.

Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated 'selection and succession' of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of 'driver mutations' enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes.
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http://dx.doi.org/10.1093/carcin/bgv029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565607PMC
June 2015

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage.

EMBO Mol Med 2015 Jul;7(7):930-49

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.
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http://dx.doi.org/10.15252/emmm.201404497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520658PMC
July 2015

A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes.

Breast Cancer Res 2014 Dec 9;16(6):482. Epub 2014 Dec 9.

IBB-Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal.

Introduction: The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells.

Methods: We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation.

Results: We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment.

Conclusions: The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.
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http://dx.doi.org/10.1186/s13058-014-0482-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303209PMC
December 2014

PIM kinases as potential therapeutic targets in a subset of peripheral T cell lymphoma cases.

PLoS One 2014 11;9(11):e112148. Epub 2014 Nov 11.

Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; Cancer Genomics Group, Marqués de Valdecilla Research Institute (IDIVAL) & Pathology Department, Hospital Universitario Marqués de Valdecilla, Santander, Spain.

Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL). The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs) and pharmacologically (mainly with the pan-PIM inhibitor (PIMi) ETP-39010) in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL) cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112148PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227704PMC
December 2015

Non-genotoxic activation of p53 through the RPL11-dependent ribosomal stress pathway.

Carcinogenesis 2014 Dec 24;35(12):2822-30. Epub 2014 Oct 24.

Tumour Suppression Group, Experimental Therapeutics Program and Confocal Microscopy Unit, Spanish National Cancer Research Centre (CNIO), Madrid, E28029, Spain

Nucleolar disruption has recently emerged as a relevant means to activate p53 through inhibition of HDM2 by ribosome-free RPL11. Most drugs that induce nucleolar disruption also possess important genotoxic activity, which can have lasting mutagenic effects. Therefore, it is of interest to identify compounds that selectively produce nucleolar disruption in the absence of DNA damage. Here, we have performed a high-throughput screening to search for nucleolar disruptors. We have identified an acridine derivative (PubChem CID-765471) previously known for its capacity to activate p53 independently of DNA damage, although the molecular mechanism underlying p53 activation had remained uncharacterized. We report that CID-765471 produces nucleolar disruption by inhibiting ribosomal DNA transcription in a process that includes the selective degradation of the RPA194 subunit of RNA polymerase I. Following nucleolar disruption, CID-765471 activates p53 through the RPL11/HDM2 pathway in the absence of detectable DNA damage. In a secondary screening of compounds approved for medical use, we identify two additional acridine derivatives, aminacrine and ethacridine, that operate in a similar manner as CID-765471. These findings provide the basis for non-genotoxic chemotherapeutic approaches that selectively target the nucleolus.
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http://dx.doi.org/10.1093/carcin/bgu220DOI Listing
December 2014

Genetic Modeling of PIM Proteins in Cancer: Proviral Tagging and Cooperation with Oncogenes, Tumor Suppressor Genes, and Carcinogens.

Front Oncol 2014 15;4:109. Epub 2014 May 15.

Biology Section, Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO) , Madrid , Spain.

The PIM proteins, which were initially discovered as proviral insertion sites in Moloney-murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anti-cancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim, and a third group of genes (including bmi1 and gfi1) as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate, and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all three isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis.
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http://dx.doi.org/10.3389/fonc.2014.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030178PMC
June 2014