Genetic studies of hereditary myeloproliferative disorders Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Jakub Zmajkovič aus Bratislava, Slowakei Basel, 2019 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Professor Dr. med Radek C. Skoda Professor Dr. Mihaela Zavolan Basel, den 11.12. 2018 Prof. Dr. Martin Spiess Dekan der Philosophisch- Naturwissenschaftlichen Fakultät Table of Contents ACKNOWLEDGMENTS ......................................................................................... 4 SUMMARY .............................................................................................................. 5 1 INTRODUCTION .............................................................................................. 7 1.1 Hematopoiesis ..................................................................................................................... 7 1.1.1 Embryonic hematopoiesis ................................................................................................. 8 1.1.2 Adult hematopoiesis .......................................................................................................... 9 1.2 Hematopoietic growth factors and their receptors ........................................................ 13 1.2.1 Hematopoietic growth factor-induced signaling .............................................................. 14 1.2.2 JAK-STAT signaling in hematopoiesis ............................................................................ 16 1.3 Erythropoiesis ................................................................................................................... 17 1.3.1 Erythropoiesis during fetal and embryonic development and postnatal life .................... 17 1.3.2 Transcriptional regulation of erythropoiesis .................................................................... 20 1.3.3 Erythropoietin (EPO) and erythropoietin receptor (EPOR) ............................................. 20 1.3.4 Erythropoietin regulation ................................................................................................. 23 1.3.5 Dysregulated EPO production and associated clinical syndromes ................................. 26 1.4 Thrombopoiesis ................................................................................................................ 27 1.4.1 Platelet biogenesis .......................................................................................................... 27 1.4.2 Transcriptional regulation of thrombopoiesis .................................................................. 29 1.4.3 Thrombopoietin (TPO) and thrombopoietin receptor (c-MPL) ........................................ 29 1.5 Hereditary myeloproliferative disorders ......................................................................... 31 1.5.1 Hereditary erythrocytosis ................................................................................................ 32 1.5.2 Hereditary thrombocytosis .............................................................................................. 33 2 RESULTS ....................................................................................................... 34 2.1 A Gain-of-Function Mutation in EPO in Familial Erythrocytosis .................................. 35 2.2 A mutation in the Kozak sequence of the THPO gene causes hereditary thrombocytosis .............................................................................................................................. 75 2.3 Murine models to study MPN ........................................................................................... 93 2.3.1 Lineage bias of HSPC population ................................................................................... 93 2.3.2 Tracking MPN initiation in situ ......................................................................................... 97 3 DISCUSSION ................................................................................................. 100 3.1 EPO gene mutation and erythrocytosis ..........................................................................100 3.2 THPO gene and thrombocytosis ..................................................................................... 105 3.3 Concluding remarks ......................................................................................................... 106 4 REFERENCES ............................................................................................... 108 Acknowledgments Acknowledgments I would like to sincerely thank everybody that contributed to this dissertation in any manner. Radek, thank you very much for giving me the opportunity to join your lab and work on several exciting projects. I have learned a lot in terms of scientific excellence and also human relationships. However, what I value the most is 1. a scientific rigor you use to assess your work, and you also demand from the people in the lab (I will never forget Sunday evenings with you aligning the axes in the figures) and 2. independence, I gained under your supervision. All of above will be invaluable in my future career and I am very grateful for it. The same gratitude goes to my colleagues and friends in the lab: Morgane, Ronny, Takafumi, Pontus, Jean, Basia, Lucia, Yukiko, Julian, Shivam and Nils for the warm welcome and the great time we spent together. You were the best lab mates one can imagine, and I hope to stay in touch with you also after we move to other places. Special thank you also goes to our good souls in the lab, Gabi, Hélène and Hui. Thank you very much for your everyday help running the lab smoothly and supporting us immensely! I am also thankful to Mihaela and Lukas – thank you for your support and guidance in the committee meetings. Many thanks go as well to the colleagues from the other hematology labs (Myeloid malignancies, Childhood leukemia, and Stem cells and hematopoiesis) for their support! It was great to share our lab space with you, as it led to unanticipated and fruitful discussions about science and everyday life. Furthermore, I would like to thank all the core facilities members for providing us with the expertise and framework to perform our experiments and Swiss Cancer League, Swiss National Foundation and the University of Basel for supporting my research. Nakoniec by som rád poďakoval mojim najbližším, ktorí počas môjho doktorandského štúdia stáli pri mne a bez ktorých by táto práca určite nevznikla. Ďakujem Katka, za tvoju každodennú starostlivosť o nás, vždy si mi bola a budeš mojou vedeckou a nevedeckou oporou. Ďakujem aj nášmu slniečku – Ninka, vďaka tebe sa mi vždy podarilo rýchlo zabudnúť na pracovné povinnosti a problémy, tvoj smiech je pre mňa balzam na dušu. Ďakujem mami a tati, za všetku vašu podporu od začiatku až doteraz, nikdy vám to nezabudnem. Ďakujem aj všetkým ostatným členom mojej rodiny a všetkým kamarátom, za vašu lásku, pomoc a podporu. 4 Genetic studies of hereditary myeloproliferative disorders | Jakub Zmajkovic Summary More than 300 billion blood cells are being replaced daily in a process called hematopoiesis. Hematopoiesis is orchestrated by hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs produce multipotent and lineage-restricted progenitors, that are responsible for the supply of mature blood cells. Production of blood cells is governed by hematopoietic growth factors that are required for the survival and proliferation of blood cells at all stages of development. Mutations in genes responsible for the regulation of this fine-tuned system cause aberrant proliferation of different blood compartments. Myeloproliferative neoplasms (MPN) are characterized by the abnormal expansion of erythroid, megakaryocytic, and myeloid lineages, that is caused either by somatic mutations or by germline mutations transmitted through Mendelian inheritance within the family. The main topic of my doctoral research was the investigation of two distinct pedigrees diagnosed with erythrocytosis and thrombocytosis, respectively. Erythrocytosis occurred in ten individuals of Norwegian family that presented elevated hemoglobin and erythropoietin (EPO) serum levels. We performed genome-wide linkage analysis using SNP arrays coupled with targeted sequencing and identified a heterozygous single base deletion (ΔG) in exon 2 of the EPO gene as the sole candidate gene mutation in affected family members. EPO stimulates the proliferation of erythrocyte progenitors and prevents their apoptosis in order to produce mature erythrocytes. Surprisingly, ΔG introduces a frame-shift that generates a novel, 51-residue long polypeptide, which would predict a loss of erythropoietin function, and is at odds with the erythrocytosis phenotype. To elucidate the mechanism by which the loss-of-function mutation causes gain-of- function phenotype, we utilized the CRISPR/Cas9 genome editing to introduce the ΔG mutation into Hep3B cells, a human hepatoma cell line that expresses EPO. We found that cells with ΔG mutation produce excessive amounts of biologically active EPO and reproduces the observation form the affected family members. On the molecular level, in addition to the known transcript originating from the physiologic promoter (P1), we identified novel transcripts that initiate in intron 1 of EPO from a putative alternative