Center for Molecular Medicine Cologne

Fischer, Matthias - A 03

The role of MAP2K7 inactivation in malignant transformation of neuroblastoma

Introduction

The molecular mechanisms driving malignant transformation of neuroblastoma are still incompletely understood.

Here, we aim to determine the role of MAP2K7 inactivation in neuroblastoma tumorigenesis, a gene that has not been recognized in the context of this malignancy before.

Our study will likely expand our knowledge on the complex genetic etiology and the molecular pathogenesis of high-risk neuroblastoma, which is prerequisite for developing novel therapeutic strategies against this deadly disease.

Neuroblastoma is a malignant pediatric tumor of the sympathetic nervous system with a broad spectrum of clinical courses, ranging from spontaneous differentiation or regression to fatal progression. We previously discovered that high-risk neuroblastoma is characterized by activation of telomere maintenance mechanisms, which are associated with genomic alterations of MYCN, TERT, or ATRX. In a substantial fraction of high-risk tumors, however, the genes driving malignant transformation have remained unknown.

We recently identified inactivating mutations in MAP2K7, encoding for a kinase of the JNK pathway, in a subset of primary neuroblastomas. Here, we aim to examine the mechanistic relevance of MAP2K7 inactivation in malignant transformation of neuroblastoma. To this end, we will determine whether genomic inactivation of MAP2K7 drives neuroblastoma tumorigenesis in genetically engineered mouse models. We will also generate MAP2K7 knock-out neuroblastoma cell lines to assess the phenotypic consequences of its inactivation in established neuroblastoma models. Finally, we will use both MAP2K7-mutated model systems and primary tumors to gain insights into the pathways by which MAP2K7 inactivation may promote neuroblastoma development.

Our Aims

  1. Does MAP2K7 inactivation drive malignant transformation of neuroblastic tumors
  2. Which are the phenotypic consequences of MAP2K7 inactivation in neuroblastoma cell line models?
  3. Which downstream pathways mediate the transforming effects upon MAP2K7 inactivation?

Previous Work

In our previous work, we have extensively characterized genomic and transcriptomic characteristics of the distinct neuroblastoma subtypes to gain insights into the molecular pathogenesis of this malignancy, to establish biomarkers for risk assessment, and to identify novel therapeutic targets. We discovered that high-risk neuroblastomas are recurrently affected by genomic rearrangements of the TERT (telomerase reverse transcriptase) locus (Peifer et al., Nature 2015).

The rearrangements invariably led to massive induction of TERT expression and telomerase activity, and occurred in mutually exclusive fashion with MYCN amplification and ATRX mutations. We also found that telomerase activity was strongly up-regulated in MYCN-amplified tumors, while the ALT pathway was activated in neuroblastomas bearing ATRX mutations. In contrast to high-risk tumors, telomere maintenance mechanisms were completely absent in low-risk neuroblastomas. Based on these observations, we hypothesized that activation of telomere maintenance mechanisms is a key feature of high-risk neuroblastoma, while low-risk tumors are defined by the lack of such alterations.

Telomere maintenance is essential for cancer cells to overcome replicative senescence by maintaining telomere length above a critical threshold, thus providing the capacity to divide infinitely. Our data suggest that low-risk neuroblastoma presumably lack immortal proliferation capacity, which may account for their propensity to undergo spontaneous regression or terminal differentiation.

To test our hypothesis, we determined alterations associated with telomerase activation and ALT in a cohort of 208 primary neuroblastomas (Ackermann et al., Science 2018).

We also wondered how mutations in genes of the RAS or p53 pathway, which occurred in 18% of the cases, affect the clinical phenotype. We observed that telomere maintenance mechanisms were strongly associated with poor survival, whereas patients whose tumors lacked such mechanisms had excellent outcome.

In addition, we noticed that the prognostic significance of RAS and p53 pathway mutations was strictly dependent on the presence of telomere maintenance mechanisms. Patients whose tumors were telomere maintenance-positive had dramatically inferior outcome when RAS/p53 pathway mutations were present, as compared to patients without such alterations. By contrast, patients whose tumors lacked telomere maintenance mechanisms had excellent outcome, and spontaneous regression or differentiation occurred both in the presence and absence of RAS/p53 pathway mutations.

Together, our data suggest a clear pathogenetic hierarchy of genetic alterations in neuroblastoma development, with telomere maintenance being the key characteristic of high-risk disease. Our studies thus provide a novel, clinically relevant classification of neuroblastoma based on a mechanistic understanding of the underlying tumor biology (Figure 1).

1. Ackermann S, Cartolano M, Hero B, Welte A, Kahlert Y, Roderwieser A, Bartenhagen C, Walter E, Gecht J, Kerschke L, Volland R, Menon R, Heuckmann JM, Gartlgruber M, Hartlieb S, Henrich KO, Okonechnikov K, Altmüller J, Nürnberg P, Lefever S, de Wilde B, Sand F, Ikram F, Rosswog C, Fischer J, Theissen J, Hertwig F, Singhi AD, Simon T, Vogel W, Perner S, Krug B, Schmid M, Rahmann S, Achter V, Lang U, Vokuhl C, Ortmann M, Büttner R, Eggert A, Speleman F, O’Sullivan RJ, Thomas RK, Berthold F, Vandesompele J, Schramm A, Westermann F, Schulte JH, Peifer M, Fischer M. (2018). A mechanistic classification of clinical phenotypes in neuroblastoma. Science362(6419), 1165-1170.

2. Roderwieser A, Sand F, Walter E, Fischer J, Bartenhagen C, Ackermann S, Otte F, Welte A, Gecht J, Kahlert Y, Lieberz D, Hertwig F, Reinhardt C, Simon T, Peifer M, Ortmann M, Büttner R, Hero B, O’Sullivan RJ, Berthold F, Fischer M. Telomerase is a prognostic marker of poor outcome and a therapeutic target in neuroblastoma. JCO Precision Oncology, in revision.

  • Beck L, Witt R, Nesper-Brock M, Milde T, Hettmer S, Frühwald MC, Rössig C, Fischer M, Reinhardt D, Taylor LA, Riedel C, Witt O, van Tilburg CM (2021). A study of regulatory challenges of paediatric oncology phase I/II trial submissions and guidance of protocol development. Clin Pharmacol Ther 110(4): 1025-1037. doi: 10.1002/cpt.2319.
  • Berthold F, Rosswog C, Christiansen H, Frühwald M, Hemstedt N, Klingebiel T, Fröhlich B, Schilling FH, Schmid I, Simon T, Hero B, Fischer M, Ernst A (2021). Clinical and molecular characterization of patients with stage 4(M) neuroblastoma aged less than 18 months without MYCN amplification. Pediatr Blood Cancer 68(8): e29038. doi: 10.1002/pbc.29038.
  • Berthold F, Ernst A, Ackermann S, Bartenhagen C, Christiansen H, Hero B, Rosswog C, von Schweinitz D, Klingebiel T, Schmid I, Simon T, and Fischer M (2021). Genetic Alterations and Resectability Predict Outcome in Patients with Neuroblastoma Assigned to High-Risk Solely by MYCN Amplification. Cancers (Basel)13. doi:10.3390/cancers13174360.
                                      • Chen J, Nelson C, Wong M, Tee AE, Liu PY, La T, Fletcher JI, Kamili A, Mayoh C, Bartenhagen C, Trahair TN, Xu N, Jayatilleke N, Wong M, Peng H, Atmadibrata B, Cheung BB, Lan Q, Bryan TM, Mestdagh P, Vandesompele J, Combaret V, Boeva V, Wang JY, Janoueix-Lerosey I, Cowley MJ, MacKenzie KL, Dolnikov A, Li J, Polly P, Marshall GM, Reddel RR, Norris MD, Haber M, Fischer M, Zhang XD, Pickett HA, and Liu T (2021). Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy. Clin Cancer Res27, 1438-1451. doi:10.1158/1078-0432.CCR-20-3044.
                                      • Fischer M, Moreno L, Ziegler DS, Marshall LV, Zwaan CM, Irwin MS, Casanova M, Sabado C, Wulff B, Stegert M, Wang L, Hurtado FK, Branle F, Geoerger B, Schulte JH (2021). Ceritinib in paediatric patients with anaplastic lymphoma kinase (ALK)-positive malignancies: a phase 1, open-label, dose-escalation study. Lancet Oncol 22(12), 1764-1776. doi: 10.1016/S1470-2045(21)00536-2.
                                      • Hartlieb SA, Sieverling L, Nadler-Holly M, Ziehm M, Toprak UH, Herrmann C, Ishaque N, Okonechnikov K, Gartlgruber M, Park YG, Wecht EM, Savelyeva L, Henrich KO, Rosswog C, Fischer M, Hero B, Jones DTW, Pfaff E, Witt O, Pfister SM, Volckmann R, Koster J, Kiesel K, Rippe K, Taschner-Mandl S, Ambros P, Brors B, Selbach M, Feuerbach L, and Westermann F (2021). Alternative lengthening of telomeres in childhood neuroblastoma from genome to proteome. Nat Commun12, 1269. doi:10.1038/s41467-021-21247-8.
                                                                                                                                                                        • Mitra S, Muralidharan SV, Marco MD, Juvvuna PK, Kosalai ST, Reischl S, Jachimowicz D, Subhash S, Raimondi I, Kurian L, Huarte M, Kogner P, Fischer M, Johnsen JI, Mondal T, and Kanduri C (2021). Subcellular Distribution of p53 by the p53-Responsive lncRNA NBAT1 Determines Chemotherapeutic Response in Neuroblastoma. Cancer Res81, 1457-1471. doi:10.1158/0008-5472.CAN-19-3499.
                                                                                                                                                                                                              • Raieli S, Di Renzo D, Lampis S, Amadesi C, Montemurro L, Pession A, Hrelia P, Fischer M, and Tonelli R (2021). MYCN Drives a Tumor Immunosuppressive Environment Which Impacts Survival in Neuroblastoma. Front Oncol11, 625207. doi:10.3389/fonc.2021.625207.
                                                                                                                                                                                                                      • Rosswog C, Bartenhagen C, Welte A, Kahlert Y, Hemstedt N, Lorenz W, Cartolano M, Ackermann S, Perner S, Vogel W, Altmuller J, Nurnberg P, Hertwig F, Gohring G, Lilienweiss E, Stutz AM, Korbel JO, Thomas RK, Peifer M, and Fischer M (2021). Chromothripsis followed by circular recombination drives oncogene amplification in human cancer. Nat Genet53, 1673-1685. doi:10.1038/s41588-021-00951-7.
                                                                                                                                                                                                                                        • Schmelz K, Toedling J, Huska M, Cwikla MC, Kruetzfeldt LM, Proba J, Ambros PF, Ambros IM, Boral S, Lodrini M, Chen CY, Burkert M, Guergen D, Szymansky A, Astrahantseff K, Kuenkele A, Haase K, Fischer M, Deubzer HE, Hertwig F, Hundsdoerfer P, Henssen AG, Schwarz RF, Schulte JH, and Eggert A (2021). Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions. Nat Commun12, 6804. doi:10.1038/s41467-021-26870-z.
                                                                                                                                                                                                                                                        • Seier JA, Reinhardt J, Saraf K, Ng SS, Layer JP, Corvino D, Althoff K, Giordano FA, Schramm A, Fischer M, and Holzel M (2021). Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma. J Immunother Cancer9. doi:10.1136/jitc-2020-001335.
                                                                                                                                                                                                                                                        • Mirzayi C, Renson A, Genomic Standards C, Massive A, Quality Control S, Zohra F, Elsafoury S, Geistlinger L, Kasselman LJ, Eckenrode K, van de Wijgert J, Loughman A, Marques FZ, MacIntyre DA, Arumugam M, Azhar R, Beghini F, Bergstrom K, Bhatt A, Bisanz JE, Braun J, Bravo HC, Buck GA, Bushman F, Casero D, Clarke G, Collado MC, Cotter PD, Cryan JF, Demmer RT, Devkota S, Elinav E, Escobar JS, Fettweis J, Finn RD, Fodor AA, Forslund S, Franke A, Furlanello C, Gilbert J, Grice E, Haibe-Kains B, Handley S, Herd P, Holmes S, Jacobs JP, Karstens L, Knight R, Knights D, Koren O, Kwon DS, Langille M, Lindsay B, McGovern D, McHardy AC, McWeeney S, Mueller NT, Nezi L, Olm M, Palm N, Pasolli E, Raes J, Redinbo MR, Ruhlemann M, Balfour Sartor R, Schloss PD, Schriml L, Segal E, Shardell M, Sharpton T, Smirnova E, Sokol H, Sonnenburg JL, Srinivasan S, Thingholm LB, Turnbaugh PJ, Upadhyay V, Walls RL, Wilmes P, Yamada T, Zeller G, Zhang M, Zhao N, Zhao L, Bao W, Culhane A, Devanarayan V, Dopazo J, Fan X, Fischer M, Jones W, Kusko R, Mason CE, Mercer TR, Sansone SA, Scherer A, Shi L, Thakkar S, Tong W, Wolfinger R, Hunter C, Segata N, Huttenhower C, Dowd JB, Jones HE, and Waldron L (2021). Reporting guidelines for human microbiome research: the STORMS checklist. Nat Med27, 1885-1892. doi:10.1038/s41591-021-01552-x.
                                                                                                                                                                                                                                                        • van Tilburg CM, Pfaff E, Pajtler KW, Langenberg KPS, Fiesel P, Jones BC, Balasubramanian GP, Stark S, Johann PD, Blattner-Johnson M, Schramm K, Dikow N, Hirsch S, Sutter C, Grund K, von Stackelberg A, Kulozik AE, Lissat A, Borkhardt A, Meisel R, Reinhardt D, Klusmann JH, Fleischhack G, Tippelt S, von Schweinitz D, Schmid I, Kramm CM, Bueren AO, Calaminus G, Vorwerk P, Graf N, Westermann F, Fischer M, Eggert A, Burkhardt B, Wößmann W, Nathrath M, Hecker-Nolting S, Frühwald MC, Schneider DT, Brecht IB, Ketteler P, Fulda S, Koscielniak E, Meister MT, Scheer M, Hettmer S, Schwab M, Tremmel R, Øra I, Hutter C, Gerber NU, Lohi O, Kazanowska B, Kattamis A, Filippidou M, Goemans B, Zwaan CM, Milde T, Jäger N, Wolf S, Reuss D, Sahm F, von Deimling A, Dirksen U, Freitag A, Witt R, Lichter P, Kopp-Schneider A, Jones DTW, Molenaar JJ, Capper D, Pfister SM, Witt O (2021). The pediatric precision oncology INFORM registry: clinical outcome and benefit for patients with very high-evidence targets. Cancer Discov 11(11), 2764-2779. doi: 10.1158/2159-8290.CD-21-0094. 
                                                                                                                                                                                                                                                        • Berthold F, Faldum A, Ernst A, Boos J, Dilloo D, Eggert A, Fischer M, Fruhwald M, Henze G, Klingebiel T, Kratz C, Kremens B, Krug B, Leuschner I, Schmidt M, Schmidt R, Schumacher-Kuckelkorn R, von Schweinitz D, Schilling FH, Theissen J, Volland R, Hero B, and Simon T (2020). Extended induction chemotherapy does not improve the outcome for high-risk neuroblastoma patients: results of the randomized open-label GPOH trial NB2004-HR. Ann Oncol31, 422-429. doi:10.1016/j.annonc.2019.11.011.
                                                                                                                                                                                                                                                        • Bhargava R, Fischer M, and O'Sullivan RJ (2020). Genome rearrangements associated with aberrant telomere maintenance. Curr Opin Genet Dev60, 31-40. doi:10.1016/j.gde.2020.02.005.
                                                                                                                                                                                                                                                                          • Cartolano M, Abedpour N, Achter V, Yang TP, Ackermann S, Fischer M, and Peifer M (2020). CaMuS: simultaneous fitting and de novo imputation of cancer mutational signature. Sci Rep10, 19316. doi:10.1038/s41598-020-75753-8.
                                                                                                                                                                                                                                                                          • Helmsauer K, Valieva M, Ali S, Chamorro Gonzalez R, Schöpflin R, Röefzaad C, Bei Y, Dorado Garcia H, Rodriguez-Fos E, Puiggròs M, Kasack K, Haase K, Keskeny C, Chen C, Kuschel LP, Euskirchen P, Heinrich V, Robson MI, Rosswog C, Toedling J, Szymansky A, Hertwig F, Fischer M, Torrents D, Eggert A, Schulte JH, Mundlos S, Henssen AG, Koche RP (2020). Enhancer hijacking determines intra- and extrachromosomal circular MYCN amplicon architecture in neuroblastoma. Nat Commun 11(1): 5823. doi: 10.1038/s41467-020-19452-y.
                                                                                                                                                                                                                                                                                                                                                                                                              • Koche RP, Rodriguez-Fos E, Helmsauer K, Burkert M, MacArthur IC, Maag J, Chamorro R, Munoz-Perez N, Puiggros M, Dorado Garcia H, Bei Y, Roefzaad C, Bardinet V, Szymansky A, Winkler A, Thole T, Timme N, Kasack K, Fuchs S, Klironomos F, Thiessen N, Blanc E, Schmelz K, Kunkele A, Hundsdorfer P, Rosswog C, Theissen J, Beule D, Deubzer H, Sauer S, Toedling J, Fischer M, Hertwig F, Schwarz RF, Eggert A, Torrents D, Schulte JH, and Henssen AG (2020). Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma. Nat Genet52, 29-34. doi:10.1038/s41588-019-0547-z.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Moreno L, Barone G, DuBois SG, Molenaar J, Fischer M, Schulte J, Eggert A, Schleiermacher G, Speleman F, Chesler L, Geoerger B, Hogarty MD, Irwin MS, Bird N, Blanchard GB, Buckland S, Caron H, Davis S, De Wilde B, Deubzer HE, Dolman E, Eilers M, George RE, George S, Jaroslav S, Maris JM, Marshall L, Merchant M, Mortimer P, Owens C, Philpott A, Poon E, Shay JW, Tonelli R, Valteau-Couanet D, Vassal G, Park JR, and Pearson ADJ (2020). Accelerating drug development for neuroblastoma: Summary of the Second Neuroblastoma Drug Development Strategy forum from Innovative Therapies for Children with Cancer and International Society of Paediatric Oncology Europe Neuroblastoma. Eur J Cancer136, 52-68. doi:10.1016/j.ejca.2020.05.010.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Mus LM, Lambertz I, Claeys S, Kumps C, Van Loocke W, Van Neste C, Umapathy G, Vaapil M, Bartenhagen C, Laureys G, De Wever O, Bexell D, Fischer M, Hallberg B, Schulte J, De Wilde B, Durinck K, Denecker G, De Preter K, and Speleman F (2020). The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness. Sci Rep10, 218. doi:10.1038/s41598-019-57076-5.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Peitz C, Sprüssel A, Linke RB, Astrahanseff K, Grimaldi M, Schmelz K, Toedling J, Schulte JH, Fischer M, Messerschmidt C, Beule D, Keilholz U, Eggert A, Deubzer HE, Lodrini M (2020). Multiplexed quantification of four neuroblastoma DNA targets in a single droplet digital PCR reaction. J Mol Diagn 22(11): 1309-1323. doi: 10.1016/j.jmoldx.2020.07.006.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Tong L, Wu PY, Phan JH, Hassazadeh HR, SEQC Consortium (including Fischer M), Tong W, Wang MD (2020). Impact of RNA-seq data analysis algorithms on gene expression estimation and downstream prediction. Sci Rep 10(1): 17925. doi: 10.1038/s41598-020-74567-y.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Prof. Dr. Matthias Fischer CMMC Cologne
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Prof. Dr. Matthias Fischer

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dept. of Exp. Pediatric Oncology - Clinic of Pediatric and Adolescent Medicine

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                CMMC - PI - A 03

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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                +49 221 478 32400

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dept. of Exp. Pediatric Oncology - Clinic of Pediatric and Adolescent Medicine

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Kerpener Str. 62

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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Group Members

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dr. Sandra Ackermann
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dr. Christoph Bartenhagen
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dr. Boris Decarolis
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dr. Janina Fischer-Mertens
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Anna-Maria Hellmann
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Dr. Carolina Rosswog
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Yvonne Kahlert
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Lisa Werr
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Felix Otto, cand. med.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Judith Pinnen
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Luca Pesch, cand. med.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Tancia Ngoy Manzambi Garcia, cand. med.