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CHK1-Targeted Therapy to Deplete DNA Replication-Stressed, P53

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CHK1-Targeted Therapy to Deplete DNA Replication-Stressed, P53 Gut Online First, published on April 7, 2017 as 10.1136/gutjnl-2016-312623 GI cancer ORIGINAL ARTICLE CHK1-targeted therapy to deplete DNA replication- Gut: first published as 10.1136/gutjnl-2016-312623 on 7 April 2017. Downloaded from stressed, p53-deficient, hyperdiploid colorectal cancer stem cells Gwenola Manic,1 Michele Signore,2 Antonella Sistigu,3 Giorgio Russo,3,4 Francesca Corradi,1 Silvia Siteni,3,5 Martina Musella,3,6 Sara Vitale,4 Maria Laura De Angelis,2 Matteo Pallocca,7 Carla Azzurra Amoreo,8 Francesca Sperati,9 Simone Di Franco,10 Sabina Barresi,11 Eleonora Policicchio,2,12 Gabriele De Luca,2 Francesca De Nicola,7 Marcella Mottolese,8 Ann Zeuner,2 Maurizio Fanciulli,7 Giorgio Stassi,10 Marcello Maugeri-Saccà,13 Marta Baiocchi,2 Marco Tartaglia,11 Ilio Vitale,1,3 Ruggero De Maria4 ▸ Additional material is ABSTRACT published online only. To view Objective Cancer stem cells (CSCs) are responsible for Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ tumour formation and spreading, and their targeting is gutjnl-2016-312623). required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer What is already known on this subject? (CRC), particularly for tumours carrying RAS-activating ▸ fi Cancer stem cells (CSCs), the subpopulation of For numbered af liations see mutations. The aim of this study was to identify novel end of article. cells driving tumour initiation and spreading, CSC-targeting strategies. are associated with cancer relapse, therapeutic Correspondence to Design To discover potential therapeutics to be resistance and poor patient prognosis thereby Dr. Ilio Vitale, Department of clinically investigated as single agent, we performed a limiting the efficacy of antineoplastic regimens. Biology, University of Rome screening with a panel of FDA-approved or ▸ Patient-derived cancer models enriched for “Tor Vergata”, via della Ricerca Scientifica 1, Rome investigational drugs on primary CRC cells enriched for CSCs are successfully employed for discovering 00133, Italy; [email protected] CSCs (CRC-SCs) isolated from 27 patients. Candidate novel cancer drugs, predicting the clinical Professor Ruggero De Maria, predictive biomarkers of efficacy were identified by fi ef cacy of novel antineoplastic regimens and http://gut.bmj.com/ Catholic University and Gemelli integrating genomic, reverse-phase protein microarray rapidly translating these novelties into the Polyclinic, Largo Francesco Vito (RPPA) and cytogenetic analyses, and validated by 1, Rome 00168, Italy; clinical setting. [email protected] immunostainings. DNA replication stress (RS) was ▸ We and others previously showed that the increased by employing DNA replication-perturbing or therapeutic resistance of CSCs is due to their GM, MS and AS contributed polyploidising agents. high efficiency in activating the DNA damage equally. Results The drug-library screening led to the IV and RDM are senior response (DDR) in the presence of DNA lesions, fi on September 24, 2021 by guest. Protected copyright. coauthors. identi cation of LY2606368 as a potent anti-CSC but further investigations are required to agent acting in vitro and in vivo in tumour cells from a elucidate the status and molecular mechanisms Received 15 July 2016 considerable number of patients (∼36%). By inhibiting of DDR in CSCs and how these insights can be Revised 3 February 2017 checkpoint kinase (CHK)1, LY2606368 affected DNA exploited for cancer therapy. Accepted 28 February 2017 replication in most CRC-SCs, including RAS-mutated ▸ Previous evidence indicated that the inhibition ones, forcing them into premature, lethal mitoses. of the ataxia telangiectasia mutated serine/ Parallel genomic, RPPA and cytogenetic analyses threonine kinase (ATM)-checkpoint kinase indicated that CRC-SCs sensitive to LY2606368 (CHK)2 or ataxia telangiectasia mutated and displayed signs of ongoing RS response, including the Rad3 related serine/threonine kinase phosphorylation of RPA32 and ataxia telangiectasia (ATR)-CHK1 axis of DDR, alone or in mutated serine/threonine kinase (ATM). This was combination with DNA-damaging associated with mutation(s) in TP53 and hyperdiploidy, chemotherapeutics, could kill cancer cells and made these CRC-SCs exquisitely dependent on displaying genomic instability, but the effect on CHK1 function. Accordingly, experimental increase of CSC survival needs further investigations. RS sensitised resistant CRC-SCs to LY2606368. Conclusions LY2606368 selectively eliminates What are the new findings? replication-stressed, p53-deficient and hyperdiploid ▸ We identified LY2606368 as a potent in vitro To cite: Manic G, CRC-SCs independently of RAS mutational status. and in vivo anti-CSC agent able to kill a Signore M, Sistigu A, et al. fi Gut Published Online First: These results provide a strong rationale for signi cant fraction (approximately a third) of [please include Day Month biomarker-driven clinical trials with LY2606368 our large panel of primary colorectal cancer Year] doi:10.1136/gutjnl- in patients with CRC. (CRC) cells enriched for CSCs (CRC-SCs). 2016-312623 Manic G, et al. Gut 2017;0:1–15. doi:10.1136/gutjnl-2016-312623 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BSG) under licence. GI cancer subsets of immature, undifferentiated cells known as cancer stem Significance of this study cells (CSCs).61011CSCs have been isolated from multiple malig- nancies, including CRC, by specific cell-surface markers or Gut: first published as 10.1136/gutjnl-2016-312623 on 7 April 2017. Downloaded from serum-free in vitro culture enrichment.12 13 This stem-like popu- lation is at the apex of the hierarchical organisation of the cancer ▸ The mechanism of CRC-SC killing by LY2606368 involves the system, where it constitutes a perpetual pool generating non- specific inhibition of CHK1, which alters the DNA replication tumorigenic progeny with variable degree of differentiation and and intra-S checkpoint of CRC-SCs, in turn resulting in the plasticity, and in dynamic crosstalk with the tumour microenvir- generation of an intolerable DNA damage burden and the 10 onment. CSCs are responsible for tumour initiation, and are consequent cell demise via replication catastrophe. involved in cancer progression, recurrence, metastasis and thera- ▸ The fraction of CRC-SCs displaying signs of DNA replication 10 14 15 peutic failure. Underscoring their clinical importance, spe- stress, such as phosphorylation of RPA32 or ATM, deficiency cific CSC-related gene-expression signatures have been associated in p53 and increased number of chromosomes 61617 to inferior clinical outcomes, and CRC patients with stem (hyperdiploidy) was shown to be highly dependent on CHK1 cell-like tumours have been documented to display lower 5-year activity and thus targetable with CHK1 inhibitors. disease-free survival rates than those having differentiated ▸ Boosting replication stress or increasing the number of 18 tumours. The therapeutic targeting of CSCs is thus advocated chromosomes by employing DNA replication-perturbing or as essential for the development of effective drugs. mitosis-perturbing agents was proven to be a strategy to Multicellular spheroids (best known as tumourspheres) are a sensitise formerly resistant CRC-SCs to LY2606368. cancer model employed to efficiently purify, enrich and propa- 13 How might it impact on clinical practice in the gate patient-derived CSCs, hence representing a powerful tool foreseeable future? for discovering anticancer drugs and companion predictive bio- ▸ Our results, together with the recent evidence on tolerable markers. Considering the enormous potential of CSC-based safety profiles and preliminary antineoplastic activity of assays as hypothesis-generating models for early clinical trials, LY2606368 as monotherapy in a phase I, non-randomised, we decided to screen freshly generated patient-derived CRC open-label, dose-escalation trial in patients affected by tumourspheres enriched for CSCs (hereafter referred to as advanced or metastatic solid tumours who underwent at CRC-SCs) with a panel of >300 drugs already approved or in least three previous lines of treatment, support the further clinical development. clinical development of LY2606368. ▸ The selective targeting of CHK1 by LY2606368 can be MATERIALS AND METHODS rapidly translated into the clinical settings for the eradication Cell lines, culture conditions and chemicals fi Unless otherwise indicated, media, supplements and plasticware of tumours with replication-stressed, p53-de cient and fi hyperdiploid CRC-SCs, which can be easily identified by p53 were purchased from Gibco-Thermo Fisher Scienti c(Waltham, sequencing combined with immunohistochemical analysis for Massachusetts, USA) or Corning-Life Sciences (Corning, New York, replication stress markers (phosphorylation of ATM and USA). CSC isolation and culture from samples of human patients http://gut.bmj.com/ RPA32) and ploidy status (measurement of nuclear area). with CRC (obtained in accordance with the standards of the institu- ▸ fi tional Ethics Committee on human experimentation, authorisation Our ndings also guide for the further development of 19 therapeutic regimens based on the induction of replication CE5ISS-09/282) were performed as previously reported and stress in patients with low DNA replication-stressed CRC described in online supplementary information. All CRC-SCs were (identified as reported above) so as to broaden the validated for their capability
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