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Decoding the Metabolic Program of Deregulated MYCN Expression in Neuroblastoma

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Decoding the Metabolic Program of Deregulated MYCN Expression in Neuroblastoma Decoding the metabolic program of deregulated MYCN expression in neuroblastoma vorgelegt von M. Sc. Birte Arlt an der Fakult¨atIII - Prozesswissenschaften der Technischen Universit¨atBerlin zur Erlangung des akademischen Grades Doktorin der Naturwissenschaften - Dr. rer. nat. - genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Jens Kurreck Gutachter: Prof. Dr. Roland Lauster Gutachterin: PD Dr. Hedwig Deubzer Gutachter: Prof. Dr. Juri Rappsilber Gutachterin: Prof. Dr. Angelika Eggert Tag der wissenschaftlichen Aussprache: 10. M¨arz2020 Berlin 2020 Diese Arbeit wurde von August 2015 bis Januar 2020 an der Charit´eUniversit¨atsmedizin Berlin und dem Max-Delbr¨uckCenter f¨urMolekulare Medizin unter Leitung von PD Dr. Hedwig E. Deubzer und Dr. Stefan Kempa angefertigt. Acknowledgment I am deeply thankful to all the wonderful colleagues, friends and family members who went this inspiring and challenging, but also exhaustive journey together with me. During my PhD I had the pleasure to be part of two labs. I would like to acknowledge my two supervisors PD Dr. Hedwig Deubzer and Dr. Stefan Kempa for the opportunity to work in their labs, for their guidance throughout the project, for their willingness to frequently travel between the two institutes and for constructive scientific discussions. They gave me the freedom to develop not only my project but also myself during the last four years. A huge thank you goes to my former and present lab members for all your support, encouraging words, inspiration and for sharing tears of laughter and frustration. To the former and present lab members from Hedi's group: Jasmin, Sebastian, Marco, Sabine, Daniela, Rasmus, Maddalena, Annika and Constantin. Thank you for being such a fantastic team to work and travel with. Legendary will remain our hilarious conversa- tions, weird songs and nerdy games in the office. I would especially like to thank Jasmin W¨unschelfor her endless experimental support, for great talks at the cell culture bench while preparing incredibly high numbers of culture plates and for generating the most beautiful western blots. To the former and present lab members from Stefan's group: Guido, Christin, Martin, Nadine, Jenny, Julia, Fardad, Safak, Henning, Tobias and Ela. Thank you for providing an exceptional working atmosphere combining scientific passion with cooking and good drinks. Special thanks go to Christin for introducing me to the field of data analysis, supporting me with ideas for neat-looking figures and sharing her scientific expertise. Furthermore I would like to thank Guido for introducing me to the world of nucleotides and proteins and supporting me with Italian coffee. I would like to thank Prof. Angelika Eggert for contributing with experimental advice and introducing me to the scientific community. I thank Dr. Kathy Astrahantseff for her scientific input and support in writing scientific essays. Special thanks go to every group member from the pediatric oncology department - it was a great pleasure to work with you! I further like to thank Annika for introducing me to CRISPR and her help in generating the knockout clones. Furthermore I would like to acknowledge my cooperation partners Dr. Jana Wolf and Dr. Dennis G¨urgen for carrying out the mice experiments and Dr. Anja K¨uhlfor her expertise and support on the immunohistochemistry stainings. I would also like to thank all patients and their families for making precious neuroblastoma samples available for research purposes. I would like to acknowledge Prof. Roland Lauster for his scientific support as well as Prof. Juri Rappsilber and Prof. Jens Kurreck for being part of the scientific commission. Furthermore, I would like to thank my diligent thesis readers: Christin, Martin, Guido, Annika, Jasmin and Kerstin. Ich danke von Herzen meiner Familie und meinen Freunden. Liebe Familie, ihr seid immer f¨urmich da, unterst¨utztmich in jeder Lebenslage und habt mich stets ermutigt meine Interessen und Ziele zu verfolgen. Daf¨urbin ich euch unendlich dankbar! Tobi, du hast mich immer wieder aufgemuntert, mir Kraft gegeben und unersch¨utterlichesVerst¨andnisgezeigt. Danke, dass es dich gibt! Liebe Freunde, vielen Dank f¨ureure vielf¨altigeUnterst¨utzungin all den unterschiedlichen Lebenslagen! Ihr seid die Besten! Why do we do basic research? To learn about ourselves. Walter Gilbert Summary The oncogene MYCN is amplified in approximately 22% of all neuroblastomas and correlates with aggressive disease and unfavorable patient survival. Directly targeting MYCN remains a challenge due to its biochemical structure. We aimed for a deeper understanding of key metabolic pathways associated to amplified MYCN to discover druggable nodes that can be directly targeted for high-risk neuroblastoma therapy. We performed GC-MS and LC-MS based methodologies for quantitative analysis of metabolites and proteins within the central carbon metabolism (CCM). Pulsed stable isotope-resolved metabolomics experiments (pSIRM) enabled the monitoring of glucose- derived carbons through the CCM. In order to decipher MYCN-dependent changes in the metabolic network, we quantified the proteome of a representative cohort of primary neuroblastoma biopsies and neuroblastoma cell lines by performing shotgun proteomics. High-level MYCN expression strongly correlated with the expression of proteins within the de novo serine synthesis pathway and the one-carbon metabolism. We identified phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis, as a metabolic target of MYCN. High-level PHGDH expression correlated with amplified MYCN on transcriptional and translational level in vitro. We observed a recruitment of MYCN to two different regions within the PHGDH promoter site by applying ChIP qRT-PCR, indicating a MYCN-mediated transcriptional regulation of PHGDH in neuroblastoma cells. Further, we have shown by pSIRM experiments that glucose-derived carbon routing through de novo serine synthesis is enhanced in MYCN -amplified compared to MYCN non-amplified cells. Serine and glycine starvation of MYCN -amplified cells did not affect proliferation and nucleotide pools, arguing for an independence of these cells to exogenous serine and glycine supply. This starvation induced PHGDH protein expression and enhanced de novo serine synthesis in MYCN non-amplified cells. However, proliferation was arrested and nucleotide pools were diminished in these cells. We constructed PHGDH knockout clones by the CRISPR/Cas9 technology to genetically inhibit PHGDH expression in vitro. Proliferation of these clones was significantly decreased compared to the MYCN -amplified control cells. Pharmacological suppression of PHGDH activity with two small molecule inhibitors arrested proliferation in MYCN -amplified and MYCN non-amplified neuroblastoma cells in vitro. In-vivo PHGDH suppression in mice carrying a patient-derived MYCN -amplified neuroblastoma xenograft hindered initial tumor growth which relapsed in later stages. A combination treatment of PHGDH inhibition with the chemotherapeutic drug cisplatin even induced an antagonizing effect on chemotherapy efficacy in vivo. In addition, we performed pSIRM experiments in vitro and observed a rerouting of glucose-derived carbons within the central carbon metabolism by PHGDH inhibition which will be further investigated. Moreover, we found evidence that glutamine starvation disrupted the MYCN-mediated regulation of PHGDH in neuroblastoma cells. Antifolates targeting the one-carbon metabolism reduced proliferation more efficiently compared to PHGDH inhibitors in vitro. This observation was accompanied by a significant decrease in nucleotide levels in MYCN -amplified and MYCN non-amplified cells. The herein presented thesis provides novel insights in the regulation of the de novo serine synthesis pathway and the one-carbon metabolism in neuroblastoma. We identified PHGDH as a promising target in dependency of MYCN expression. However, PHGDH inhibition did not induce lethal effects in neuroblastoma cells in in vitro and in vivo experiments. Thus additional research is needed to further exploit the application of small compounds targeting de novo serine synthesis and the one-carbon metabolism for the development of novel therapeutic strategies. Zusammenfassung Das Neuroblastom ist eine b¨osartigeKrebserkrankung des sympathischen Nervensystems, die vor allem im fr¨uhenKindesalter auftritt. In circa 22 Prozent aller F¨alleliegt eine Am- plifizierung des Onkogens MYCN vor, welche oft mit einer aggressiven Tumorbiologie und niedrigen Uberlebensraten¨ assoziiert ist. Die Anwendung von niedermolekularen Inhibitoren als Therapeutikum ist aufgrund der biochemischen Struktur des Onkogens wenig geeignet. Aus diesem Grund untersucht die vorliegende Arbeit MYCN-abh¨angigeStoffwechselwege mit dem Ziel direkte Angriffspunkte f¨urneue Therapieformen zu identifizieren. Die Anwendung von Massenspektrometrie-basierter Methoden (GC-MS / LC-MS) er- m¨oglichtedie Quantifizierung von Metaboliten und Proteinen des zentralen Kohlenstoff- wechsels. Dieser ist ein wesentlicher Produzent notwendiger zellul¨arerBausteine wie Aminos¨aurenund Nukleotide, die zur Erhaltung der energetischen Hom¨oostaseund damit zum Zellwachstum beitragen. Zus¨atzlicherm¨oglichtenMarkierungsexperimente unter An- wendung von stabilen Isotopen (pSIRM) die quantitative Betrachtung der Stoffwechselak- tivit¨atsowie deren Regulation. Ein Vergleich der Proteinexpressionen zwischen etablierten Zellkulturmodellen des Neuroblastoms
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