Diversité et plasticité des tumeurs de l’enfant

Publications de l’équipe

Année de publication : 2020

Gabriele Manzella, Leonie D Schreck, Willemijn B Breunis, Jan Molenaar, Hans Merks, Frederic G Barr, Wenyue Sun, Michaela Römmele, Luduo Zhang, Joelle Tchinda, Quy A Ngo, Peter Bode, Olivier Delattre, Didier Surdez, Bharat Rekhi, Felix K Niggli, Beat W Schäfer, Marco Wachtel (2020 Sep 16)

Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity.

Nature communications : 4629 : DOI : 10.1038/s41467-020-18388-7 En savoir plus
Résumé

Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.

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Inge M Ambros, Gian-Paolo Tonini, Ulrike Pötschger, Nicole Gross, Véronique Mosseri, Klaus Beiske, Ana P Berbegall, Jean Bénard, Nick Bown, Huib Caron, Valérie Combaret, Jerome Couturier, Raffaella Defferrari, Olivier Delattre, Marta Jeison, Per Kogner, John Lunec, Barbara Marques, Tommy Martinsson, Katia Mazzocco, Rosa Noguera, Gudrun Schleiermacher, Alexander Valent, Nadine Van Roy, Eva Villamon, Dasa Janousek, Ingrid Pribill, Evgenia Glogova, Edward F Attiyeh, Michael D Hogarty, Tom F Monclair, Keith Holmes, Dominique Valteau-Couanet, Victoria Castel, Deborah A Tweddle, Julie R Park, Sue Cohn, Ruth Ladenstein, Maja Beck-Popovic, Bruno De Bernardi, Jean Michon, Andrew D J Pearson, Peter F Ambros (2020 Sep 9)

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable -Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

Journal of clinical oncology : official journal of the American Society of Clinical Oncology : 3685-3697 : DOI : 10.1200/JCO.18.02132 En savoir plus
Résumé

For localized, resectable neuroblastoma without amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome.

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Shu-Hong Lin, Joshua N Sampson, Thomas G P Grünewald, Didier Surdez, Stephanie Reynaud, Olivier Mirabeau, Eric Karlins, Rebeca Alba Rubio, Sakina Zaidi, Sandrine Grossetête-Lalami, Stelly Ballet, Eve Lapouble, Valérie Laurence, Jean Michon, Gaelle Pierron, Heinrich Kovar, Udo Kontny, Anna González-Neira, Javier Alonso, Ana Patino-Garcia, Nadège Corradini, Perrine Marec Bérard, Jeremy Miller, Neal D Freedman, Nathaniel Rothman, Brian D Carter, Casey L Dagnall, Laurie Burdett, Kristine Jones, Michelle Manning, Kathleen Wyatt, Weiyin Zhou, Meredith Yeager, David G Cox, Robert N Hoover, Javed Khan, Gregory T Armstrong, Wendy M Leisenring, Smita Bhatia, Leslie L Robison, Andreas E Kulozik, Jennifer Kriebel, Thomas Meitinger, Markus Metzler, Manuela Krumbholz, Wolfgang Hartmann, Konstantin Strauch, Thomas Kirchner, Uta Dirksen, Lisa Mirabello, Margaret A Tucker, Franck Tirode, Lindsay M Morton, Stephen J Chanock, Olivier Delattre, Mitchell J Machiela (2020 Sep 4)

Low-frequency variation near common germline susceptibility loci are associated with risk of Ewing sarcoma.

PloS one : e0237792 : DOI : 10.1371/journal.pone.0237792 En savoir plus
Résumé

Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor.

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Johanna Theruvath, Elena Sotillo, Christopher W Mount, Claus Moritz Graef, Alberto Delaidelli, Sabine Heitzeneder, Louai Labanieh, Shaurya Dhingra, Amaury Leruste, Robbie G Majzner, Peng Xu, Sabine Mueller, Derek W Yecies, Martina A Finetti, Daniel Williamson, Pascal D Johann, Marcel Kool, Stefan Pfister, Martin Hasselblatt, Michael C Frühwald, Olivier Delattre, Didier Surdez, Franck Bourdeaut, Stephanie Puget, Sakina Zaidi, Siddhartha S Mitra, Samuel Cheshier, Poul H Sorensen, Michelle Monje, Crystal L Mackall (2020 Apr 29)

Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors.

Nature medicine : DOI : 10.1038/s41591-020-0821-8 En savoir plus
Résumé

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. ), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.

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Caroline Louis-Brennetot, Olivier Delattre, Isabelle Janoueix-Lerosey (2020 Mar 23)

High CD44 expression is not a prognosis marker in patients with high-risk neuroblastoma.

EBioMedicine : 102702 : DOI : S2352-3964(20)30077-3 En savoir plus
Résumé

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Martin F Orth, Tilman L B Hölting, Marlene Dallmayer, Fabienne S Wehweck, Tanja Paul, Julian Musa, Michaela C Baldauf, Didier Surdez, Olivier Delattre, Maximilian M L Knott, Laura Romero-Pérez, Merve Kasan, Florencia Cidre-Aranaz, Julia S Gerke, Shunya Ohmura, Jing Li, Aruna Marchetto, Anton G Henssen, Özlem Özen, Shintaro Sugita, Tadashi Hasegawa, Takayuki Kanaseki, Stefanie Bertram, Uta Dirksen, Wolfgang Hartmann, Thomas Kirchner, Thomas G P Grünewald (2020 Mar 14)

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Cancers : DOI : E644 En savoir plus
Résumé

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for -positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including -positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by -positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

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S Melloul, J-F Mosnier, J Masliah-Planchon, C Lepage, K Le Malicot, J-M Gornet, J Edeline, D Dansette, P Texereau, O Delattre, P Laurent Puig, J Taieb, J-F Emile (2020 Feb 22)

Loss of SMARCB1 expression in colon carcinoma.

Cancer biomarkers : section A of Disease markers : 399-406 : DOI : 10.3233/CBM-190287 En savoir plus
Résumé

SMARCB1 is a tumor suppressor gene, which is part of SWI/SNF complex involved in transcriptional regulation. Recently, loss of SMARCB1 expression has been reported in gastrointestinal carcinomas. Our purpose was to evaluate the incidence and prognostic value of SMARCB1 loss in colon carcinoma (CC). Patients with stage III CC (n= 1695), and a second cohort of 23 patients with poorly differentiated CC were analyzed. Immunohistochemistry for SMARCB1 was performed on tissue microarrays, and cases with loss of expression were controlled on whole sections. Loss of SMARCB1 was compared with the clinico-pathological and molecular characteristics, and the prognostic value was evaluated. Loss of SMARCB1 was identified in 12 of 1695 (0.7%) patients with stage III CC. Whole section controls showed a complete loss in only one of these cases, corresponding to a medullary carcinoma. SMARCB1 loss was not associated with histological grade, tumor size nor survival. In the cohort of poorly differentiated CC, we detected 2/23 (8.7%) cases with loss of SMARCB1; one was rhabdoid while the other had medullary and mucinous histology. These 2 cases were deficient for MisMatched Repair (dMMR) and mutated for BRAF. SMARCB1 loss is rare in stage III CC, but appears more frequent in poorly differentiated CC.

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Marie-Ming Aynaud, Olivier Mirabeau, Nadege Gruel, Sandrine Grossetête, Valentina Boeva, Simon Durand, Didier Surdez, Olivier Saulnier, Sakina Zaïdi, Svetlana Gribkova, Aziz Fouché, Ulykbek Kairov, Virginie Raynal, Franck Tirode, Thomas G P Grünewald, Mylene Bohec, Sylvain Baulande, Isabelle Janoueix-Lerosey, Jean-Philippe Vert, Emmanuel Barillot, Olivier Delattre, Andrei Zinovyev (2020 Feb 13)

Transcriptional Programs Define Intratumoral Heterogeneity of Ewing Sarcoma at Single-Cell Resolution.

Cell reports : 1767-1779.e6 : DOI : 10.1016/j.celrep.2020.01.049 En savoir plus
Résumé

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.

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Manuel Rodrigues, Khadija Ait Rais, Flore Salviat, Nathalie Algret, Fatoumata Simaga, Raymond Barnhill, Sophie Gardrat, Vincent Servois, Pascale Mariani, Sophie Piperno-Neumann, Sergio Roman-Roman, Olivier Delattre, Nathalie Cassoux, Alexia Savignoni, Marc-Henri Stern, Gaëlle Pierron (2020 Jan 3)

Association of Partial Chromosome 3 Deletion in Uveal Melanomas With Metastasis-Free Survival.

JAMA ophthalmology : DOI : 10.1001/jamaophthalmol.2019.5403 En savoir plus
Résumé

Studies on uveal melanomas (UMs) have demonstrated the prognostic value of 8q gain and monosomy 3, but the prognosis of UMs with partial deletion of chromosome 3 remains to be defined.

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Johannes Ommer, Joanna L Selfe, Marco Wachtel, Eleanor M O'Brien, Dominik Laubscher, Michaela Roemmele, Stephanie Kasper, Olivier Delattre, Didier Surdez, Gemma Petts, Anna Kelsey, Janet Shipley, Beat W Schäfer (2020 Jan 1)

Aurora A Kinase Inhibition Destabilizes PAX3-FOXO1 and MYCN and Synergizes with Navitoclax to Induce Rhabdomyosarcoma Cell Death.

Cancer research : 832-842 : DOI : 10.1158/0008-5472.CAN-19-1479 En savoir plus
Résumé

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. SIGNIFICANCE: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

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Année de publication : 2019

Amaury Leruste, Jimena Tosello, Rodrigo Nalio Ramos, Arnault Tauziède-Espariat, Solène Brohard, Zhi-Yan Han, Kevin Beccaria, Mamy Andrianteranagna, Pamela Caudana, Jovan Nikolic, Céline Chauvin, Leticia Laura Niborski, Valeria Manriquez, Wilfrid Richer, Julien Masliah-Planchon, Sandrine Grossetête-Lalami, Mylene Bohec, Sonia Lameiras, Sylvain Baulande, Celio Pouponnot, Aurore Coulomb, Louise Galmiche, Didier Surdez, Nicolas Servant, Julie Helft, Christine Sedlik, Stéphanie Puget, Philippe Benaroch, Olivier Delattre, Joshua J Waterfall, Eliane Piaggio, Franck Bourdeaut (2019 Nov 12)

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors.

Cancer cell : 597-612.e8 : DOI : S1535-6108(19)30482-9 En savoir plus
Résumé

Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8 T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

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Amal M El-Naggar, Syam Prakash Somasekharan, Yemin Wang, Hongwei Cheng, Gian Luca Negri, Melvin Pan, Xue Qi Wang, Alberto Delaidelli, Bo Rafn, Jordan Cran, Fan Zhang, Haifeng Zhang, Shane Colborne, Martin Gleave, Anna Mandinova, Nancy Kedersha, Christopher S Hughes, Didier Surdez, Olivier Delattre, Yuzhuo Wang, David G Huntsman, Gregg B Morin, Poul H Sorensen (2019 Nov 1)

Class I HDAC inhibitors enhance YB-1 acetylation and oxidative stress to block sarcoma metastasis.

EMBO reports : e48375 : DOI : 10.15252/embr.201948375 En savoir plus
Résumé

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5′-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.

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Swati Srivastava, Nishanth Belugali Nataraj, Arunachalam Sekar, Soma Ghosh, Chamutal Bornstein, Diana Drago-Garcia, Lee Roth, Donatella Romaniello, Ilaria Marrocco, Eyal David, Yuval Gilad, Mattia Lauriola, Ron Rotkopf, Adi Kimchi, Yuya Haga, Yasuo Tsutsumi, Olivier Mirabeau, Didier Surdez, Andrei Zinovyev, Olivier Delattre, Heinrich Kovar, Ido Amit, Yosef Yarden (2019 Oct 3)

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

Cell reports : 104-117.e4 : DOI : S2211-1247(19)31150-7 En savoir plus
Résumé

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR’s transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

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Julian Musa, Florencia Cidre-Aranaz, Marie-Ming Aynaud, Martin F Orth, Maximilian M L Knott, Olivier Mirabeau, Gal Mazor, Mor Varon, Tilman L B Hölting, Sandrine Grossetête, Moritz Gartlgruber, Didier Surdez, Julia S Gerke, Shunya Ohmura, Aruna Marchetto, Marlene Dallmayer, Michaela C Baldauf, Stefanie Stein, Giuseppina Sannino, Jing Li, Laura Romero-Pérez, Frank Westermann, Wolfgang Hartmann, Uta Dirksen, Melissa Gymrek, Nathaniel D Anderson, Adam Shlien, Barak Rotblat, Thomas Kirchner, Olivier Delattre, Thomas G P Grünewald (2019 Sep 13)

Cooperation of cancer drivers with regulatory germline variants shapes clinical outcomes.

Nature communications : 4128 : DOI : 10.1038/s41467-019-12071-2 En savoir plus
Résumé

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine.

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Simon Durand, Cécile Pierre-Eugène, Olivier Mirabeau, Caroline Louis-Brennetot, Valérie Combaret, Léo Colmet-Daage, Orphée Blanchard, Angela Bellini, Estelle Daudigeos-Dubus, Virginie Raynal, Gudrun Schleiermacher, Sylvain Baulande, Olivier Delattre, Isabelle Janoueix-Lerosey (2019 Aug 28)

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations.

Oncotarget : 4937-4950 : DOI : 10.18632/oncotarget.27119 En savoir plus
Résumé

The gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

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