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PQR309 Is a Novel Dual PI3K/Mtor Inhibitor with Preclinical Antitumor Activity in Lymphomas As a Single Agent and in Combination

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PQR309 Is a Novel Dual PI3K/Mtor Inhibitor with Preclinical Antitumor Activity in Lymphomas As a Single Agent and in Combination Published OnlineFirst October 24, 2017; DOI: 10.1158/1078-0432.CCR-17-1041 Cancer Therapy: Preclinical Clinical Cancer Research PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy Chiara Tarantelli1, Eugenio Gaudio1, Alberto J. Arribas1,IvoKwee1,2,3, Petra Hillmann4, Andrea Rinaldi1, Luciano Cascione1,5, Filippo Spriano1, Elena Bernasconi1, Francesca Guidetti1, Laura Carrassa6, Roberta Bordone Pittau5, Florent Beaufils4,7, Reto Ritschard8, Denise Rageot4,7, Alexander Sele7, Barbara Dossena9, Francesca Maria Rossi10, Antonella Zucchetto10, Monica Taborelli9, Valter Gattei10, Davide Rossi1,5, Anastasios Stathis5, Georg Stussi5, Massimo Broggini6, Matthias P. Wymann7, Andreas Wicki8, Emanuele Zucca5, Vladimir Cmiljanovic4, Doriano Fabbro4, and Francesco Bertoni1,5 Abstract Purpose: Activation of the PI3K/mTOR signaling pathway is nostat, ibrutinib, lenalidomide, ARV-825, marizomib, and recurrent in different lymphoma types, and pharmacologic inhi- rituximab. Sensitivity to PQR309 was associated with spe- bition of the PI3K/mTOR pathway has shown activity in lym- cific baseline gene-expression features, such as high expres- phoma patients. Here, we extensively characterized the in vitro and sion of transcripts coding for the BCR pathway. Combining in vivo activity and the mechanism of action of PQR309 (bimir- proteomics and RNA profiling, we identified the different alisib), a novel oral selective dual PI3K/mTOR inhibitor under contribution of PQR309-induced protein phosphorylation clinical evaluation, in preclinical lymphoma models. and gene expression changes to the drug mechanism of Experimental Design: This study included preclinical in vitro action. Gene-expression signatures induced by PQR309 and activity screening on a large panel of cell lines, both as single agent by other signaling inhibitors largely overlapped. PQR309 and in combination, validation experiments on in vivo models showed activity in cells with primary or secondary resis- and primary cells, proteomics and gene-expression profiling, and tance to idelalisib. comparison with other signaling inhibitors. Conclusions: On the basis of these results, PQR309 appeared Results: PQR309 had in vitro antilymphoma activity as as a novel and promising compound that is worth developing single agent and in combination with venetoclax, panobi- in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. Ó2017 AACR. Introduction proliferation, and survival (1, 2). Class IA PI3Ks include hetero- dimers of p110 catalytic subunit and p85 regulatory subunit PI3Ks are enzymes belonging to PI3K/AKT/mTOR signaling (1–3). The class IA catalytic subunit isoforms are encoded by the pathways with a central role in the regulation of cell metabolism, genes PIK3CA, PIK3CB, and PIK3CD (1–3): p110a (PI3Ka), p110b (PI3Kb), and p110d (PI3Kd), respectively. These isoforms fi 1 can associate with any of ve regulatory isoforms, p85a and its Universita della Svizzera Italiana (USI), Institute of Oncology Research (IOR), PIK3R1 PIK3R2 Bellinzona, Switzerland. 2Dalle Molle Institute for Artificial Intelligence (IDSIA), splicing variants p55a and p50a ( ), p85b ( ), and PIK3R3 Manno, Switzerland. 3Swiss Institute of Bioinformatics (SIB), Lausanne, Switzer- p55g ( ), generally called p85 type regulatory subunits land. 4PIQUR Therapeutics AG, Basel, Switzerland. 5Oncology Institute of (1–3). Class IB PI3Ks are heterodimers of a p110g (PI3Kg) Southern Switzerland (IOSI), Bellinzona, Switzerland. 6IRCCS-Istituto di catalytic subunit (PIK3CG) with regulatory isoforms p101 7 Ricerche Farmacologiche Mario Negri, Milan, Italy. Department of Biomedicine, (PIK3R5) or p87 (PIK3R6). While PI3Ka and PI3Kb are ubiqui- University of Basel, Basel, Switzerland. 8Department of Oncology, University 9 tously expressed, PI3Kd, and PI3Kg are largely restricted to leu- Hospital Basel, Basel, Switzerland. Cytogenetics Laboratory, Ente Ospedaliero – Cantonale, Bellinzona, Switzerland. 10Clinical and Experimental Onco-Hematol- kocytes (1 3). ogy Unit, IRCCS-Centro di Riferimento Oncologico, Aviano, Italy. PI3K/AKT/mTOR signaling pathway activation is common in lymphomas. Examples are mutations or gains of PIK3CA in Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). diffuse large B-cell lymphoma (DLBCL), mantle cell lymphomas (MCL), and chronic lymphocytic leukemias (CLLs; refs. 4, 5), C. Tarantelli and E. Gaudio contributed equally to this article. inactivation or low expression of PTEN in DLBCL, follicular Corresponding Author: Francesco Bertoni, Institute of Oncology Research, via lymphomas (FL), MCL and CLL (5–8). Vela 6, Bellinzona 6500, Switzerland. Phone: 41-91-820-0367; Fax: 41-91-820- The approval by regulatory authorities of the mTOR inhibitor 0397; E-mail: [email protected] temsirolimus for MCL (9) and of the PI3Kd inhibitor idelalisib for doi: 10.1158/1078-0432.CCR-17-1041 FL and CLL (10, 11) strongly encouraged further development of Ó2017 American Association for Cancer Research. compounds targeting PI3K complex and/or mTOR (12–15). Dual 120 Clin Cancer Res; 24(1) January 1, 2018 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 24, 2017; DOI: 10.1158/1078-0432.CCR-17-1041 Novel Dual PI3K/mTOR Inhibitor for Lymphomas lyzed with a BX61 microscope (Olympus Italia, Milan, Italy) Translational Relevance using the Cytovision Imaging System (Applied Imaging). Targeting PI3K and mTOR is an important therapeutic opportunity to improve the outcome of individuals affected Compounds by lymphomas. PQR309 is a novel brain-penetrant dual PQR309 and apitolisib (GDC0980) were provided by Piqur PI3K/mTOR inhibitor with in vitro and in vivo antilymphoma Therapeutics, venetoclax, ibrutinib, idelalisib, duvelisib, activity as single agent and in combination, and importantly, AZD1208, lenalidomide, panobinostat were purchased from can overcome both primary and acquired resistance to idela- Selleckchem, ARV-825 and metformin from MedChem, mar- lisib, the most frequently used PI3K inhibitor in the clinical izomib from AdooQ and rituximab from Roche. setting. The presented highly overlapping changes in the lymphoma transcriptome observed by a direct comparison Primary cells of the gene expression changes induced by PQR309 and other Peripheral blood was obtained from patients diagnosed different BCR inhibitors may have implications in the design with CLL according to standard criteria. All patients signed of novel combination schemes. informed consent (BASEC 2016-00511, CE 3051). CLL cells were isolated by Ficoll density-gradient centrifugation and CLL cells (5 Â 105 cells/mL) were cultured for 48 hours, and the percentage of viable and apoptotic CLL cells [Annexin V positive/7-Aminoactinomycin D (7AAD) negative PI3K/mTOR inhibitors with strong activity toward all p110 iso- and Annexin V positive/7AAD positive] was determined by forms and mTOR combine multiple therapeutic efficacies in a double staining the cells with Annexin V–FITC/7AAD. single molecule (3). Pan PI3K inhibitors are expected to reduce the risk of drug resistance that might occur in case of treatment In vitro antitumor activity with compounds targeting a single p110 isoform (16) and, The antiproliferative activity in 384-well plates (21), the IC together with mTOR inhibition, could prevent feedback loop of 50 values estimate (22) and drug combinations screening (23) were AKT activation following mTOR inhibition (17). PQR309 (bimir- performed as previously described. Combinations were defined: alisib) is a novel orally bioavailable selective dual PI3K/mTOR synergistic if the Chou-Talalay Combination Index (CI) was <0.9; inhibitor (18, 19), and, differently from most PI3K and mTOR additive if CI between 0.9 and 1.1; antagonistic if CI > 1.1 (24). inhibitors (20), able to cross the brain blood barrier (18). Here, Apoptosis and cell cycle were evaluated as previously reported we present the preclinical characterization of PQR309 as single (22). Apoptosis induction was defined in the presence of a 2-fold agent and in combination with several other agents in pre-clinical or higher increase in comparison with DMSO-treated cells. models of lymphomas. Proteomic and/or genomic approaches were also used with the aim to uncover the mechanism of action of In vivo experiments the compound in parallel with other targeted signaling inhibitors. Three in vivo experiments were performed. The first two explored PQR309 as single agent and in combination with Materials and Methods ibrutinib in the RI-1 cell line or venetoclax in the SU-DHL-6 cell Cell lines line. For these, NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice A total of 49 human established lymphoma cell lines were used: were subcutaneously inoculated with the RI-1 cell line (1 Â 107 seven (RI-1, HBL-1, TMD8, U2932, SU-DHL-2, OCI-LY-3, cells in 0.1 ml of PBS, 1:1 Matrigel) or with the SU-DHL-6 cell line OCI-LY-10) from activated B-cell–like DLBCL (ABC DLBCL), (5 Â 106 cells in 100 mL of PBS, 1:1 Matrigel). Mice maintenance 18 (Pfeiffer, OCI-LY-1, OCI-LY-2, OCI-LY-7, OCI-LY-8, OCI-LY- and animal experiments were performed under the Guide of 18, OCI-LY-19, KARPAS422, SU-DHL-4, SU-DHL-6, SU-DHL-7, animal care and use (NCR 2011) and under the Chinese National SU-DHL-10, FARAGE, VAL, WSU-DLCL2, TOLEDO, RCK8, Standard (GB14925-2010; Crown Bioscience Inc.). For the third DOHH2) from germinal
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