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Stapled Peptide Targeting the CDK4/Cyclin D Interface Combined Stapled peptide targeting the CDK4/Cyclin D interface combined with Abemaciclib inhibits KRAS mutant lung cancer growth Céline Bouclier, Matthieu Simon, Guillaume Laconde, Morgan Pellerano, Sebastien Diot, Sylvie Lantuejoul, Benoit Busser, Laetitia Vanwonterghem, Julien Vollaire, Véronique Josserand, et al. To cite this version: Céline Bouclier, Matthieu Simon, Guillaume Laconde, Morgan Pellerano, Sebastien Diot, et al.. Sta- pled peptide targeting the CDK4/Cyclin D interface combined with Abemaciclib inhibits KRAS mu- tant lung cancer growth. Theranostics, Ivyspring International Publisher, 2020, 10 (5), pp.2008-2028. 10.7150/thno.40971. inserm-02440284 HAL Id: inserm-02440284 https://www.hal.inserm.fr/inserm-02440284 Submitted on 15 Jan 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Theranostics 2020, Vol. 10, Issue 5 2008 Ivyspring International Publisher Theranostics 2020; 10(5): 2008-2028. doi: 10.7150/thno.40971 Research Paper Stapled peptide targeting the CDK4/Cyclin D interface combined with Abemaciclib inhibits KRAS mutant lung cancer growth Celine Bouclier1,*, Matthieu Simon1,*, Guillaume Laconde1, Morgan Pellerano1, Sebastien Diot1, Sylvie Lantuejoul2, Benoit Busser2,3, Laetitia Vanwonterghem3, Julien Vollaire3, Véronique Josserand3, Baptiste Legrand1, Jean-Luc Coll3, Muriel Amblard1, Amandine Hurbin3,#, May C. Morris1,# 1. Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France 2. CHU Grenoble Alpes, Université Grenoble Alpes, Grenoble, France 3. Institut pour l’Avancée des Biosciences, INSERM U1209, CNRS UMR-5309, Université Grenoble Alpes, Grenoble, France * These authors contributed equally # These authors contributed equally Corresponding author: May C. Morris. Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, 34093 Montpellier, France. Phone: +34 (0) 4 11 75 96 24; Email: [email protected] © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.10.07; Accepted: 2019.11.19; Published: 2020.01.12 Abstract CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS-mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy. Methods: As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1. Results: We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib. Conclusion: The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer. Key words: CDK4; Stapled Peptide; Inhibitor; Lung cancer (NSCLC); KRAS mutation Introduction Lung cancer is the leading cause of 30%). Standard radiotherapy and chemotherapy were cancer-related death worldwide in men and women the only alternative, until the recent discovery of [1]. Non-small-cell lung carcinoma (NSCLC) accounts “driver” oncogenic mutations in a subset of for 85% of all lung cancer cases, and includes adenocarcinomas and the development of adenocarcinoma (ADC) representing half of lung corresponding targeted therapies, however mainly cancers and squamous cell carcinoma (SCC) (nearly http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 2009 limited to patients harbouring the targeted genetic virtue of their stable, well-defined and predictable aberration [2]. conformation, and therefore constitute particularly CDKs are heterodimeric protein kinases formed attractive and tractable biomolecules for development through association of a CDK catalytic subunit with a of targeted therapeutics [21, 22]. Especially, cyclin regulatory partner [3, 4]. CDK4 complexed with all-hydrocarbon stapled α-helical peptides have cyclin D isoforms, constitutes an established emerged as suitable pharmacological drug candidates pharmacological target in several human cancers, with a large number of studies demonstrating their associated with mutation of CDK4, amplification of therapeutic potential [23-27]. A leading example is the cyclin D or overexpression of p16INK4a, all of which stapled peptide developed by Aileron Therapeutics, lead to hyperactivation of this kinase. CDK4/cyclin D displaying an anti-tumor activity, and currently in activity coordinates exit from quiescence and growth phase I trials for solid tumor [28] and in phase II trials factor-stimulated entry into and progression through for lymphoma [29]. G1 through phosphorylation of the Retinoblastoma The primary interface between CDK and cyclin tumour suppressor proteins (pRb, p107, p130). Cyclin partners is mediated by the C helix of the CDK and D expression and its association with CDK4 are the α5 helix of the cyclin partner [30, 31]. Given its induced by mitogenic signals, notably via Ras critical implication in assembly of an active signaling pathway. Amplification of the cyclin D1 CDK/cyclin complex, we previously showed that it locus is observed in 5-30% of NSCLC and high levels constituted a molecular target and designed a peptide of cyclin D1 protein are found in 18-76% of invasive derived from the PSTAIRE helix of CDK2, which NSCLC [5] and correlate with a worse outcome [6]. efficiently and specifically inhibited CDK2/Cyclin A CDK4 overexpression in lung cancer may accelerate [32]. Targeting the primary interface between CDK4 tumour progression and leads to an overall shorter and cyclin D could therefore constitute an attractive survival time in lung cancer patients [7]. In particular, alternative to ATP-pocket binding compounds. In this KRAS-driven NSCLC is particularly dependent on study we have designed a stapled peptide derived CDK4 activity. Targeting this kinase in NSCLC has from the C helix of CDK4, characterized by a therefore been proposed as a therapeutic strategy in stabilized helical conformation that binds cyclin D1 KRAS-mutant lung cancer that is resistant to with high affinity, compared to a linear peptide conventional and targeted therapies [8]. derived from the same sequence. This stapled peptide A large number of CDK/cyclin inhibitors have penetrates readily into cultured cells, colocalizes with been identified from natural substances, in high CDK4 and cyclin D1 and inhibits the ability of CDK4 throughput screening assays, or through to phosphorylate Rb. Moreover it inhibits lung cancer structure-guided approaches [9-13]. The vast majority cell proliferation and efficiently prevents growth of of these inhibitors are ATP-competitive, and there are orthotopic NSCLC tumours in mice when combined currently more than 20 CDK inhibitors in clinical trials with the ATP-competitive inhibitor Abemaciclib. for anticancer therapeutics, including the FDA-approved ATP-competitive CDK4 inhibitors Materials and Methods Palbociclib (PD-0332991), Abemaciclib (LY2835219), Patients, tissue samples, and and Ribociclib (LEE011) [14-19]. To date, these immunohistochemistry compounds suffer from an overall lack of selectivity and toxicity, and have had a limited success when Lung adenocarcinoma and squamous cell used as single agents, but exhibit potent synergistic carcinoma samples were obtained form surgical effects in combination with cytotoxic drugs such as rsections and retrieved from the Biological Resource cisplatin or paclitaxel. Moreover they induce Center of Grenoble University hospital (CRB) emergence of resistant mutants [18, 20], highlighting (authorized by Ministry of Higher Education and the need to develop novel therapeutic approaches. Research - Accreditation number AC 2017-2949- BRIF Targeting protein/protein interactions (PPIs) BB-0033-00069). All tumours were classified according that are essential for enzyme function represents a to the 2015 WHO classification [1]. particularly attractive alternative to ATP-competitive Immunohistostainings were performed on 3 µm inhibitors. However, the modulation of PPIs with formalin-fixed paraffin-embedded tissue sections on selectivity and potency represents a major challenge, Benchmark Autostrainer (Ventana, Tucson, AZ). given the highly conserved structural features of Sections were incubated with cyclin D1 rabbit mAb protein kinases.
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