Published OnlineFirst March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3519 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia Anouchka P. Laurent1,2, Aurelie Siret1, Cathy Ignacimouttou1, Kunjal Panchal3,M’Boyba Diop4, Silvia Jenni5, Yi-Chien Tsai5, Damien Roos-Weil1, Zakia Aid1, Nais Prade6, Stephanie Lagarde6, Damien Plassard7, Gaelle Pierron8, Estelle Daudigeos4, Yann Lecluse4, Nathalie Droin1, Beat C. Bornhauser5, Laurence C. Cheung3,9, John D. Crispino10, Muriel Gaudry1, Olivier A. Bernard1, Elizabeth Macintyre11, Carole Barin Bonnigal12, Rishi S. Kotecha3,9,13, Birgit Geoerger4, Paola Ballerini14, Jean-Pierre Bourquin5, Eric Delabesse6, Thomas Mercher1,15, and Sebastien Malinge1,3 ABSTRACT ◥ Purpose: Children with Down syndrome (constitutive trisomy RAS/MAPK pathway activation in our cohort of DS-ALL, as 21) that develop acute lymphoblastic leukemia (DS-ALL) have a well as in other pediatric B-ALL presenting somatic gain of the 3-fold increased likelihood of treatment-related mortality coupled chromosome 21 (B-ALLþ21). In murine and human B-cell with a higher cumulative incidence of relapse, compared with other precursors, activated KRASG12D functionally cooperates with children with B-cell acute lymphoblastic leukemia (B-ALL). This trisomy 21 to deregulate transcriptional networks that promote highlights the lack of suitable treatment for Down syndrome increased proliferation and self renewal, as well as B-cell differ- children with B-ALL. entiation blockade. Moreover, we revealed that inhibition of Experimental Design: To facilitate the translation of new ther- RAS/MAPK pathway activation using the MEK1/2 inhibitor apeutic agents into clinical trials, we built the first preclinical cohort trametinib decreased leukemia burden in several PDX models of patient-derived xenograft (PDX) models of DS-ALL, compre- of B-ALLþ21, and enhanced survival of DS-ALL PDX in com- hensively characterized at the genetic and transcriptomic levels, and bination with conventional chemotherapy agents such as vin- have proven its suitability for preclinical studies by assessing the cristine. efficacy of drug combination between the MEK inhibitor trametinib Conclusions: Altogether, using novel and suitable PDX mod- and conventional chemotherapy agents. els, this study indicates that RAS/MAPK pathway inhibition Results: Whole-exome and RNA-sequencing experiments represents a promising strategy to improve the outcome of Down revealed a high incidence of somatic alterations leading to syndrome children with B-cell precursor leukemia. mechanisms driving leukemia in DS-ALL, so that novel and more Introduction targeted treatments can be developed for these children. B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most At the genetic level, apart from the constitutive trisomy 21, common type of childhood malignancy. Children with Down syn- DS-ALL presents a normal karyotype in more than 40% of cases, drome face a 27-fold increased risk to develop B-ALL (known as DS- and a lower incidence of ETV6-RUNX1 or high hyperdiploid (HeH) ALL; ref. 1), associated with a worse outcome and a significantly lower subtypes (2, 4). Half of DS-ALL samples overexpress cytokine event-free survival (EFS; 64% vs. 81%) and overall survival rate (OS; receptor-like factor 2 (CRLF2) either through microdeletions 74% vs. 89%) compared with other children (2). Treatment intensi- on chromosome X (P2RY8–CRLF2 fusion) or translocations to fication is limited in DS-ALL due to a higher therapy-related morbidity the IGH locus (5–7). JAK2-activating mutations, affecting the (TRM; 7% vs. 2%), ultimately leading to a higher rate of relapses (26% Arginine R683 residue located in the pseudokinase domain, are vs. 15%; refs. 2, 3). This emphasizes the need to better understand the also more frequently found in DS-ALL than in any other subgroups 1INSERM U1170, Gustave Roussy Institute, Universite Paris Saclay, Villejuif, Haematology, Oncology and Bone Marrow Transplantation, Perth Children's France. 2Universite Paris Diderot, Paris, France. 3Telethon Kids Cancer Centre, Hospital, Perth, Australia. 14Laboratoire d’Hematologie, Hopital^ Trousseau, Telethon Kids Institute, University of Western Australia, Perth, Australia. APHP, Paris-Sorbonne, Paris, France. 15Equipe Labellisee Ligue Nationale Contre 4Gustave Roussy Institute Cancer Campus, Department of Pediatric and Ado- le Cancer, Paris, France. lescent Oncology, INSERM U1015, Equipe Labellisee Ligue Nationale Contre le Note: Supplementary data for this article are available at Clinical Cancer Cancer, Universite Paris-Saclay, Villejuif, France. 5Department of Pediatric Research Online (http://clincancerres.aacrjournals.org/). Oncology, Children's Research Centre, University Children's Hospital Zurich, Zurich, Switzerland. 6Centre of Research on Cancer of Toulouse (CRCT), CHU Corresponding Author: Sebastien Malinge, Telethon Kids Institute, 15 Hospital Toulouse, Universite Toulouse III, Toulouse, France. 7IGBMC, Plateforme Geno- Avenue, Perth Children's Hospital, Nedlands, WA 6009, Australia. Phone: 61-8- mEast, UMR7104 CNRS, Ilkirch, France. 8Service de Gen etique, Institut Curie, 6319-1351; E-mail: [email protected] Paris, France. 9School of Pharmacy and Biomedical Sciences, Curtin University, – Bentley, Australia. 10Division of Hematology/Oncology, Northwestern Univer- Clin Cancer Res 2020;26:3307 18 11 sity, Chicago, Illinois. Hematology, Universite de Paris, Institut Necker-Enfants doi: 10.1158/1078-0432.CCR-19-3519 Malades and Assistance Publique–Hopitaux de Paris, Paris, France. 12Centre Hospitalier Universitaire de Tours, Tours, France. 13Department of Clinical Ó2020 American Association for Cancer Research. AACRJournals.org | 3307 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 27, 2020; DOI: 10.1158/1078-0432.CCR-19-3519 Laurent et al. my 21 and the somatic alterations found in DS-ALL remain largely Translational Relevance unknown. B-cell precursor acute lymphoblastic leukemia (B-ALL) is the In this study, we showed that trisomy 21 functionally cooperates most common type of childhood malignancies, accounting for with constitutive activation of RAS/MAPK pathway in murine more than 20% of all pediatric cancers worldwide. Although and human B-cell precursors, indicating that disrupting this mech- survival rates have significantly improved, many children with anism may have a therapeutic impact for Down syndrome children B-ALL continue to have a poor prognosis, and suffer from with B-cell leukemia. We built a comprehensive and preclinical treatment-related toxicity and relapse, which are clinical features cohort of patient-derived xenograft (PDX) models, and show exemplified in Down syndrome children with acute lymphoblastic efficacy of MEK1/2 inhibition on human B-ALL samples harboring leukemia (known as DS-ALL). Among the most frequent somatic NRAS, KRAS, JAK2,andCBL mutations. Altogether, these results alterations seen in childhood B-ALL, gain of chromosome 21 (þ21) indicated that the RAS/MAPK inhibitor trametinib is therapeuti- and RAS/MAPK alterations are found in 30% to 40% and 50%, cally exploitable to improve the outcome of Down syndrome respectively. In this study, using DS-ALL as a paradigm, we children with B-ALL when combined with conventional chemo- showed that both events functionally cooperate and that treatment therapy agents. with the MEK inhibitor trametinib improved the survival in patient-derived xenograft models of childhood B-ALLþ21 with RAS-activating mutations. The comprehensive repository of Materials and Methods preclinical models developed here is of general relevance to inves- Human samples and sequencing tigate new targeted therapies in childhood leukemia. Human peripheral blood (PB) and bone marrow (BM) samples were collected from Trousseau, Necker, Tours and Toulouse hospitals (France), and Perth Children's Hospital (Australia). Nine samples were provided under the MAPPYACTS protocol (clinical trial.gov: of pediatric B-ALL (8, 9), suggesting an oncogenic cooperation NCT02613962). All human samples were obtained with the written between cytokine signaling activation and constitutive trisomy 21. or signed consent of the patient or parents/guardians. This study Other genetic alterations affecting NRAS, KRAS, IKZF1, PAX5,or conforms to the provisions of the Declaration of Helsinki, was CDKN2A/B genes are commonly found in DS- and non-DS B- approved by independent ethics committees, and complied to insti- ALL (10, 11). tutional, local, and national regulations. The development of DS-ALL models, from the transgenic Ts1Rhr Cells were subjected to Ficoll gradient and then either used fresh or mice [trisomic for the Down Syndrome Critical Region (DSCR); frozen in FBS with 10% DMSO. DNA and RNA were extracted using ref. 12], has highlighted the complexity of DS-ALL leukemogenesis RNA/DNA/Protein Purification Plus Kit (Norgen). Whole-exome in vivo (13). Indeed, four additional alterations affecting CRLF2, JAK2, sequencing (WES) libraries were prepared using SureSelect All Exon IKZF1, and PAX5 genes, added to trisomy 21, were required to drive a V5 Clinical Research Exome V1 or CREV2 Kits (Agilent). Sequencing B-ALL phenotype in this model. Moreover, the lack of cellular models was performed on a HiSeq2000 (Illumina) with a median