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      CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells

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          Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.


          To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.


          The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.


          LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

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          Most cited references 50

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          Comprehensive Molecular Characterization of Human Colon and Rectal Cancer

          Summary To characterize somatic alterations in colorectal carcinoma (CRC), we conducted genome-scale analysis of 276 samples, analyzing exome sequence, DNA copy number, promoter methylation, mRNA and microRNA expression. A subset (97) underwent low-depth-of-coverage whole-genome sequencing. 16% of CRC have hypermutation, three quarters of which have the expected high microsatellite instability (MSI), usually with hypermethylation and MLH1 silencing, but one quarter has somatic mismatch repair gene mutations. Excluding hypermutated cancers, colon and rectum cancers have remarkably similar patterns of genomic alteration. Twenty-four genes are significantly mutated. In addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9, and FAM123B/WTX. Recurrent copy number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive CRC and important role for MYC-directed transcriptional activation and repression.
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            Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.

            Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.
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              Identification and expansion of human colon-cancer-initiating cells.

              Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.

                Author and article information

                BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )
                May 2018
                7 April 2017
                : 67
                : 5
                : 903-917
                [1 ]Department of Biology, University of Rome “Tor Vergata” , Rome, Italy
                [2 ]Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità , Rome, Italy
                [3 ]Department of Research, Advanced Diagnostics and Technological Innovation, Regina Elena National Cancer Institute , Rome, Italy
                [4 ]Institute of General Pathology, Catholic University and A. Gemelli Polyclinic , Rome, Italy
                [5 ]Department of Science, University “Roma Tre” , Rome, Italy
                [6 ]Department of Molecular Medicine, University “La Sapienza” , Rome, Italy
                [7 ]SAFU, Department of Research, Advanced Diagnostics, and Technological Innovation, Translational Research Area, Regina Elena National Cancer Institute , Rome Italy
                [8 ]Department of Pathology, Regina Elena National Cancer Institute , Rome, Italy
                [9 ]Biostatistical Unit, Regina Elena National Cancer Institute , Rome, Italy
                [10 ]Department of Surgical Oncological and Stomatological Sciences, University of Palermo , Palermo, Italy
                [11 ]Genetics and Rare Diseases Research Division, Ospedale Pediatrico “Bambino Gesù” , Rome, Italy
                [12 ]Department of Experimental Medicine, University “La Sapienza” , Rome, Italy
                [13 ]Division of Medical Oncology 2, Regina Elena National Cancer Institute , Rome, Italy
                Author notes
                [Correspondence to ] Dr Ilio Vitale, Department of Biology, University of Rome “Tor Vergata”, via della Ricerca Scientifica 1, Rome 00133, Italy; iliovit@ Professor Ruggero De Maria, Catholic University and Gemelli Polyclinic, Largo Francesco Vito 1, Rome 00168, Italy; ruggero.demaria@

                GM, MS and AS contributed equally. IV and RDM are senior coauthors.

                Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to

                This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:

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