ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1254

Mechanisms of MedulloblastomaDissemination and Novel TargetedTherapies

SARA BOLIN

ISSN 1651-6206ISBN 978-91-554-9692-0urn:nbn:se:uu:diva-300907

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, DagHammarskjölds väg 20, Uppsala, Friday, 4 November 2016 at 09:15 for the degree of Doctorof Philosophy (Faculty of Medicine). The examination will be conducted in English. Facultyexaminer: Professor Martine F. Roussel (St. Jude Children's Research Hospital, Memphis, TN,USA).

AbstractBolin, S. 2016. Mechanisms of Medulloblastoma Dissemination and Novel TargetedTherapies. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty ofMedicine 1254. 49 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9692-0.

Medulloblastomas are the most frequent malignant childhood brain tumors, arising in theposterior fossa of children. The overall 5-year survival is 70%, although children often suffersevere long-term side effects from standard medical care. To improve progression-free survivaland quality of life for these children, finding new therapeutic targets in medulloblastoma isimperative.

Medulloblastoma is divided in to four molecular subgroups (WNT, SHH, Group 3 andGroup 4) based on key developmental pathways essential for the initiation and maintenanceof tumor development. The MYC family of proto-oncogenes regulates cell proliferation anddifferentiation in normal brain. Aberrant expression of MYC proteins occurs commonly inmedulloblastoma.

Our studies on Group 3 medulloblastoma identify the transcription factor SOX9 as a noveltarget for the E3 ubiquitin ligase FBW7, and show that increased stability of SOX9 confersan increased metastatic potential in medulloblastoma. Moreover, SOX9-positive cells drivedistant recurrences in medulloblastoma when combining two regulatable TetON/OFF systems.MYCN depletion leads to increased SOX9 expression in Group 3 medulloblastoma cells,and the recurring tumor cells are more migratory in vitro and in vivo. Segueing to treatmentof medulloblastoma, we show that BET bromodomain inhibition specifically targets MYC-amplified medulloblastoma cells by downregulating MYC and MYC-transcriptional targets, andthat combining BET bromodomain- and cyclin-dependent kinase- inhibition improves survivalin mice compared to single therapy. Combination treatment results in decreased MYC levelsand increased apoptosis, and RNA-seq confirms upregulation of apoptotic markers along withdownregulated MYC target genes in medulloblastoma cells.

This thesis addresses novel findings in transcription factor biology, recurrence and treatmentin Group 3 medulloblastoma, the most malignant subgroup of the disease.

Keywords: Medulloblastoma, Recurrence, MYC, SOX9, FBW7, Treatment, BETbromodomains, Cyclin-dependent kinases

Sara Bolin, Department of Immunology, Genetics and Pathology, Neuro-Oncology,Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden.

© Sara Bolin 2016

ISSN 1651-6206ISBN 978-91-554-9692-0urn:nbn:se:uu:diva-300907 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-300907)

Till mamma & pappa,

I have not failed. I've just found 10,000 ways that won't work.

- Thomas A. Edison

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Rahmanto AS*, Savov V*, Brunner AW**, Bolin S**, Weishaupt H**,

Čančer M, Rosén G, Spruck C, Taylor MD, Maljukova A, Cho YJ, Pfist-er S, Kool M, Korshunov A, Swartling FJ#, Sangfelt O# FBW7 suppres-sion leads to SOX9 stabilization and increased malignancy in medullo-blastoma. EMBO J. 2016 Sept 13.

II Savov V, Bolin S, Rosen G, Rahmanto AS, Fotaki G, Hill RM, Dubuc

A, Remke M, Čančer M, Ramaswamy V, Clifford SC, Sangfelt O, Tay-lor MD, Weishaupt H and Swartling FJ. Metastasis and tumor recur-rence from rare SOX9-positive cells in MYCN-driven medulloblastoma. Manuscript.

III Bandopadhayay P*, Bergthold G*, Nguyen B, Schubert S, Gholamin S,

Tang Y, Bolin S, Schumacher SE, Zeid R, Masoud S, Yu F, Vue N, Gibson WJ, Paolella BR, Mitra SS, Cheshier S, Qi J, Liu KW, Wechsler-Reya RJ, Weiss WA, Swartling FJ, Kieran MW, Bradner JE, Beroukhim R*, Cho YJ*. BET-bromodomain inhibition of MYC-amplified Medullo-blastoma. Clin Cancer Res. 2013 Dec 2.

IV Bolin S, Borgenvik A*, Persson C*, Sundström A, Qi, J, Cho J-Y, Brad-

ner, JE, Weishaupt H, Swartling FJ. Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma. Manuscript submit-ted.

*, **, # authors contributed equally to the work.

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11Medulloblastoma ...................................................................................... 11

Subgroups and molecular classification ............................................... 11Treatment strategies and brain druggability ........................................ 14Recurrence and metastatic dissemination ............................................ 16

The UPS system and its implications in cancer ........................................ 16SOX9 and its role in cancer ...................................................................... 18MYC proteins and their oncogenic roles .................................................. 18Super-Enhancers and BET bromodomains ............................................... 21Cyclin-Dependent Kinases as therapeutic targets .................................... 22Murine experimental models in cancer .................................................... 23

Tetracycline-controlled transgenic models .......................................... 24Murine models to study medulloblastoma ........................................... 25

Present investigations .................................................................................... 27Paper I. FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma ............................................................... 27Paper II. Metastasis and tumor recurrence from rare SOX9-positive cells in MYCN-driven medulloblastoma .................................................. 29Paper III. BET-bromodomain inhibition of MYC-amplified medulloblastoma ....................................................................................... 31Paper IV. Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma ................................................................. 32

Conclusions ................................................................................................... 34

Future perspectives ........................................................................................ 35

Populärvetenskaplig sammanfattning ........................................................... 38

Acknowledgements ....................................................................................... 39

References ..................................................................................................... 43

Abbreviations

APC APC ATM ATR bHLH BBB BET BRDs CDK CNS CPD CT CTNNB1 CUL1 DCN DOX DDX3X E1-3 EC ECM ESC EGF EGL ERK FBW7 FGF FZD GAB1 GEM GFAP GLI GLT-1 GSK3β GCP GNP HMG

Adenomatous polyposis coli Anaphase promoting complex Ataxia-telangiectasia-mutated Ataxia and rad3 related Basic helix-loop helix Blood brain barrier Bromodomain and extra-terminal Bromodomain-containing proteins Cyclin-dependent kinase Central nervous system Cdc4 phospho degron Chemotherapy Beta-catenin Cullin-1 Deep cerebellar nuclei Doxycycline DEAD-Box Helicase 3, X-Linked Ubiquitin proteins Endothelial cell Extracellular matrix Embryonic stem cells Epidermal growth factor External granular layer Extracellular signaling-regulated kinase F-box and WD repeat domain-containing 7 Fibroblast growth factor Frizzled GRB2 Associated Binding Protein Genetically engineered model Glial fibrillary acidic protein Glioma-Associated Oncogene Homolog Glutamate transporter-1 Glycogen synthase kinase 3 Granule cell progenitors Granule neuron precursor High mobility group

IGL L/CA LEF/TCF MAX MB MIZ-1 ML MYC NSC PTCH1 PL RCAS RL RTK S62 SCF SE SHH SKP1 SMO SOX SUFU T58 TERT Tet TP53 TRE TF tTA/rtTA TRKA t-va UPS URL WNT YAP1

Internal granular layer Large-cell/anaplastic Lymphoid enhancer-binding/Transcription factors MYC Associated Factor X Medulloblastoma Myc-Interacting Zinc Finger Protein 1 Molecular layer V-Myc Myelocytomatosis Viral Oncogene Homolog Neural stem cells Patched 1 Purkinje cell layer Replication competent ALV splice acceptor Rhombic lip Receptor tyrosine kinase Serine 62 SKP1, CUL1 and F-box protein Super-enhancer Sonic Hedgehog S-phase kinase associated protein 1 Smoothened SRY-related HMG-box protein Suppressor of Fused Homolog Threonine 58 Telomerase Reverse Transcriptase Tetracycline Tumor Protein P53 Tetracycline responsive promotor element Transcription factor Tetracycline transactivator/reverse Tropomyosin receptor kinase A Genetic locus for susceptibility to ASLV viruses Ubiquitin proteasome system Upper rhombic lip Wingless Yes Associated Protein 1

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Introduction

Medulloblastoma Medulloblastomas (MB) are the most common malignant pediatric brain tumors and constitute 25% of all intracranial neoplasms [1]. The overall incidence of medulloblastoma is 1.8 cases per 1 million population, com-pared to the drastically higher childhood incidence of 6 cases per 1 million children [1]. 77% of all patients affected by medulloblastoma are under the age of 19, with peak incidences in 3 and 7 year-old children [1]. Prognosis is determined by the age of the patient and the extent to which the tumor has spread. Current therapies of MB improve patient survival by approximately 70% and include surgical resection, radiation and chemotherapy [2, 3]. De-spite this, children often suffer from severe long-term side effects from the current standard care, which is why identification of novel therapeutic tar-gets in MB is important for improving progression-free survival and quality of life in these patients.

Subgroups and molecular classification Cancer is characterized by abrogated control of cell proliferation and devel-ops as a consequence of genetic mutations in oncogenes, tumor suppressor genes and/or genes associated with genome stability. Epigenetic changes, alterations in gene expression not caused by changes in the DNA sequence, also influence tumorigenesis. Differential response to treatment among pa-tients, caused by primarily tumor heterogeneity, poses a great challenge for the development of new therapeutics. The identification of intertumoral dif-ferences allows subgrouping of tumors and consequently enables develop-ment of subgroup-specific therapeutic strategies. Subgroup-specific molecu-lar features together with the cellular origin of tumors are imperative to de-velop strategies to therapeutically target diverse molecular subtypes [4, 5].

MB pathogenesis is initiated by early embryonic aberrations in one or more important developmental genes that predispose children to MB, and is thought to arise from stem cells or granule neuron progenitors (GNPs) of the developing cerebellum or the brain stem [6, 7]. Gene expression profiling has divided MB into four molecularly distinct subgroups based on key de-velopmental pathways involved in MB pathogenesis: Wingless (WNT), Son-ic hedgehog (SHH), Group 3 and Group 4 [8-12]. In 2016, the SHH group

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was further classified by the world health organization (WHO) according to the mutational status of the tumor suppressor gene, TP53 [1] (Figure 1). All medulloblastomas, irrespective of molecular subgroup, are histologically classified as WHO Grade IV tumors [13].

Figure 1. Classification of medulloblastoma tumors into three subgroups according to WHO [1]. Key differences in histopathology, genetics and cell of origin between the subgroups are shown in the figure.

WNT-activated medulloblastoma Wingless (WNT) proteins play a pivotal role in embryonic brain patterning [14] and the WNT-signalling pathway maintains pluripotency in murine embryonic stem cells [15] and determines neuronal precursor cell fates [16]. Cytosolic β-catenin is normally maintained at low levels by glycogen syn-thase kinase 3β (GSK3β) in cells, but when WNT proteins bind to its recep-tor family Frizzled (FZD) Dishevelled inhibits GSK3β activity and levels of β-catenin increase. High cytosolic levels of β-catenin trigger its translocation to the nucleus and binding of transcription factors (TFs) of the LEF/TCF family, promoting e.g. Cyclin D1 and MYC expression. In WNT-activated MB activating β-catenin (CTNNB1) mutations result in deregulated β-catenin levels, which promote gene transcription of MYC and Cyclin D1 [17]. Muta-tions are often found in areas encoding the β-catenin phosphorylation site, leading to increased levels of β-catenin due to evasion of protein degradation [18].

The majority of WNT-activated MBs displays classic (undifferentiated cells with a uniform cellular morphology) MB tumor histology and present in older children (7-14 yrs). Fortunately, these WNT-activated tumors have an exceptional prognosis, 90-100 % overall survival, following standard therapy [19]. 10-15 % of all MBs have mutated CTNNB1, and monosomy 6

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[10] which, together with nuclear β-catenin immunoreactivity, constitute the main WNT-activated MB characteristics [20]. Other mutated genes found in WNT-activated MB are DDX3X and TP53 [21]. Germline mutations in ade-nomatous polyposis coli (APC) confer an increased risk of WNT MB devel-opment associated with an increased risk of colorectal adenomas (Turcot syndrome) [22]. The cellular origin of WNT-activated MB has been assigned to progenitor cells in the embryonic dorsal brainstem and lower rhombic lip (not of GNP origin) and the tumors frequently display cerebellar midline localization [7]. SHH-activated medulloblastoma Sonic Hedgehog (SHH) is a morphogen crucial in developmental processes such as somite differentiation, limb patterning and neural tube induction and patterning. SHH is secreted by Purkinje neurons in the cerebellum [23, 24] and acts as a mitogen, stimulating GNP proliferation [24, 25]. The SHH transduction cascade is initiated by a transmembrane complex formed by Smoothened (SMO) and Patched (PTCH1). SHH binds to PTCH1, relieving SMO from inhibition leading to activation of the GLI family of transcription activators, resulting in expression of a number of target genes including MYC, MYCN, Blc-2, BMP, Twist1, Bmi1 and Nanog, many of which are important for self-renewal. A mutation in genes such as SMO, PTCH1 and GLI, leading to loss of control of the downstream gene activation, is com-mon in SHH-activated MB.

SHH-activated MB makes up about 30% of all MB cases. Recently, the WHO further divided the SHH-activated MB subgroup based on TP53 muta-tional status. Differences in risk stratification and treatment response are associated with TP53 mutation and accompanying gene mutations [26-28]. SHH-activated TP53-wildtype MB (SHH/TP53wt) is generally linked with an SHH pathway activation and has a desmoplastic/nodular histology (round, pale nodules). Infants and adults more frequently present with SHH/TP53wt MB, with SMO, PTCH1, SUFU genes and the TERT promoter commonly mutated [8, 29]. SHH-activated TP53-mutant MB (SHH/TP53mt) shows large cell/anaplastic (LC/A; large, angulated cells with marked nucle-ar atypia) morphology and usually present in children aged 4-17. SHH/TP53mt display a SHH pathway activation associated with amplifica-tion of MYCN and GLI2. MYCN and GLI2 encode for proteins which are downstream in the SHH pathway, compared to common SHH/TP53wt muta-tions such as PTCH1 and SMO.

SHH-activated MB most likely develops from GNPs of the external gran-ule layer (EGL) in the cerebellum [6, 30] and is YAP1 and GAB1 immuno-reactive. Gorlin syndrome is an inherited condition caused by heterozygous germline mutations in PTCH1 and associated with SHH-activated MB, basal cell carcinoma and developmental abnormalities [31].

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Non-WNT/non-SHH medulloblastoma The Non-WNT/non-SHH subgroup consists of Group 3 and Group 4 MB, and makes up about 60 % of all MB (Group 3: 20 %, Group 4: 40 %). Both infants and children present with Group 3 MB and metastatic disease, alt-hough metastasis occur more frequently in infants. Group 3 MB can, similar to SHH/TP53mt MB, spread to surrounding tissue and may disseminate through the cerebrospinal fluid (CSF). Group 4 MB can present in all age groups, including adults. Histologically, Group 3 and Group 4 tumors show classic or LC/A characteristics and can be distinguished from WNT/SHH MB by their positive cytoplasmic β-catenin and negative GAB1 and YAP1 immunoreactivity [20].

Transcriptional profiling of Group 3 and Group 4 does not reveal individ-ual signaling pathways involved in pathogenesis as seen in the previously mentioned subgroups of MB. However, Group 3 displays enrichment of MYC/retinal gene signatures and Group 4 exhibits enrichment of neuronal signature genes [10, 11]. Moreover, regulators of histone methylation are deregulated in Group 3 and Group 4. The H3K27 trimethyl mark (H3K27me3) is known to repress expression of genes involved in stem cell differentiation, and methylases (EZH2) and demethylases (KDM6A) of H3K27me3 are found upregulated or downregulated respectively in these subgroups, leading to maintained stem-like epigenetic states in the tumor cells [21]. MYC and MYCN amplifications are common (10-15 %), mutually exclusive and associated with a poor prognosis in MB [32-34]. MYC ampli-fication and isodicentric 17q are common in Group 3 MB [35]. Isodicentric 17q is also strongly associated with Group 4 MB, as well as recurrent muta-tions or amplifications in MYCN and CDK6 [8]. MYC amplification is asso-ciated with a high risk of metastatic disease and poor outcome for children with MB [36].

The cellular origins of Group 3 and Group 4 MB are largely unknown. Two studies show that Atoh1-positive GNPs or Prominin1-positive cerebel-lar stem cells (SC) give rise to Group 3 MB, however it was shown that cells either undergo de-differentiation or silence lineage markers upon MYC-induced transformation, suggesting a neural stem cell (NSC) origin [37, 38]. Less is known about the origin of Group 4. However, a recent study tracing subgroup-specific master TFs of Group 4 MB using zebra fish, suggested that immature deep cerebellar nuclei (DCN), or their early progenitors from the upper rhombic lip (URL), may be putative cells of origin [39].

Treatment strategies and brain druggability Standard medical care for MB greatly improves patient survival. However, for the majority of the patients, who are young children, the treatment causes pronounced damage to the developing brain. Therefore, treatment studies aiming to replace or reduce the use of standard radiotherapy and chemother-

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apy (CT) are of great importance to increase survival and improve the pro-spects of the affected children.

Surgical intervention aims to safely remove as much of the tumor as pos-sible. If a tumor is completely resected, patients are given a low dose of ra-diation before receiving CT. If the tumor cannot be completely resected or if the tumor has spread, patients will typically receive a high dose of radiation preceding CT. CT drugs targets dividing cells by interrupting the cell cycle, a highly regulated process of cell division. Tumor cells acquire a higher rate of proliferation compared to normal cells in the body, which allows CT drugs to effectively target fast dividing cancer cells. Importantly, CT drugs also affect normal, healthy, cells, which can give rise to severe side effects and secondary malignancies. There are several groups of CT drugs based on their mechanisms of action. The groups most commonly used for treating medulloblastoma are alkylating agents (cisplatin, cyclophosphamide & lo-mustine), topoisomerase inhibitors (etoposide), and mitotic inhibiors (vin-cristine) [40]. Craniospinal radiation, using high energy X-rays, is common-ly accompanied with a combination of CT drugs in patients [40].

Treatment for children under the age of three is mainly focused on com-plete resection together with a cocktail of chemotherapeutic agents [41]. Radiation therapy is never considered for patients below the age of 3, due to adverse effects on the immature nervous system. For recurrent medullo-blasoma, children may receive autologous stem cell transplantation followed by high doses of CT and/or radiation, aiming to kill tumor cells and to rein-troduce healthy hematopoietic stem cells to regenerate the bone marrow. There are several techniques that physicians use to increase visualization of the tumor, such as functional MRI (fMRI), image-guided stereotaxis and conformal radiation therapy, to reduce damage to the brain. Most clinical trials investigate the combinatorial effects of CT and/or radiation, however currently there are a few trials looking into vaccine immunotherapy (NCT01326104), TPI-287 a microtubule-stabilizing agent (NCT00867568) and a hedgehog signaling-inhibitor Sonidegib (NCT01125800) for treating recurring MB.

Maintenance of brain homeostasis is controlled by the blood brain barrier (BBB), which is a highly selective permeability barrier surrounding the brain. The BBB consists of an integral network of specialized endothelial cells (ECs) that generate the cerebral blood vessels. These ECs, together with astrocytes, pericytes and extracellular matrix (ECM), separate the cen-tral nervous system (CNS) from the peripheral blood circulation [42]. The main features of the BBB ECs are continuous intercellular tight junctions, a lack of fenestration and a low rate of transcytosis [42]. These features enable the BBB to tightly regulate transport to the brain through highly selective transporters, which readily transport nutrients and oxygen, while rejecting toxins. Thus, only molecules that conform to the characteristic stringency of the BBB are allowed to pass. However elegant the BBB preserves brain in-

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tegrity, it is also a feature that makes systemic treatment of CNS tumors challenging. A study demonstrated that WNT-activated MBs produced high amounts of Wnt inhibitor factor 1 (WIF1), which disrupted WNT signaling in ECs and resulted in a deregulated BBB and sensitivity to drugs that are normally BBB impenetrable [43]. These novel findings gear toward pharma-cological disruption of the BBB to increase drug penetration into brain tu-mors [43].

Recurrence and metastatic dissemination If treatment fails to eliminate all neoplastic cells from the tumor-bed or if tumor cells develop resistance to therapy, the surviving cells have the poten-tial to cause disease recurrence. The recurring tumor can be local or a dis-tant, metastatic, recurrence [44]. Tumor recurrence is often associated with treatment resistance and hence a more aggressive, uncontrollable, growth [28] due to additional mutations or selective pressure acquired as a result of the primary treatment. Metastatic recurrence implies a relapse at a different site from the primary tumor, often in another organ where tumor cells have disseminated to via the blood or in the case of MB – the CSF [44]. The stag-es of cancer are grouped based on the extent of tumor spread. M0 grading denotes no spread whereas M4, the highest grading, indicates advanced or metastatic cancer characterized by spread to other organs.

Recurrent and metastatic MB display an ability to maintain the molecular integrity of the primary tumor [44, 45] and stay within the CNS [46]. Plate-let-derived growth factor receptor α (PDGFRA), a tyrosine-kinase protein important in embryonic development, cell proliferation and chemotaxis, is highly expressed in metastatic MB tumors [47, 48]. Moreover, insulin-like growth factor-1 receptor (IGF1R) activity has been described in MB [49, 50] and has most recently been linked to MB progression [51]. Both PDGFRA and IGF1R could be promising targets for treating metastatic MB.

Apart from brain tumors themselves recurring in the CNS, about 10-20% of all other neoplasms will eventually result in metastatic lesions to the brain [52]. The dismal outcome upon recurrence and metastatic dissemination to the brain requires focused research to better define the role of oncogene drivers and their interacting partners in recurrence and development of treatment resistance [28].

The UPS system and its implications in cancer The ubiquitin proteasome system (UPS) regulates protein turnover and there-fore serves as a rate-limiting step in the activity of substrate proteins in-volved in many cellular processes. The UPS system utilizes ubiquitin ligases

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which covalently attach ubiquitin chains to substrate proteins, marking them for proteasomal degradation [53].

Ubiquitin tagging is a joint effort of three enzymes E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugation enzyme) and E3 (ubiquitin protein ligase) [54]. SCF complexes, consisting of SKP1 (S-phase kinase-associated protein 1), CUL1 (cullin-1) and F-box protein (E3 ubiquitin lig-ase), act as scaffolds for protein recognition and ubiquitylation (Figure 2).

Figure 2. Schematic illustration of SCF ubiquitin complex with E3 ubiquitin ligase FBW7 and its substrates being marked for proteosomal degradation. Modified from [55].

The E3 ubiquitin ligases enable substrate specificity by recognizing specific motifs (Cdc4 phospho-degrons, CPDs) on the substrate protein. An im-portant E3 ligase, F-box and WD repeat domain-containing 7 (FBW7), binds to several important regulators of cell growth and cell division including MYC, cyclin E and Notch1 and most recently we have identified Sex-determining region-Y (SRY) box 9 (SOX9) as a novel substrate for FBW7 [56]. Due to the proto-oncogenic nature of its targets, FBW7 is considered a tumor suppressor [53]. In order for FBW7 to recognize its substrate, the pro-teins need to be phosphorylated within their conserved CPD motif, which is mainly achieved through the activity of GSK3β.

FBW7 encodes three transcripts (FBW7 α, β & γ) each with their own iso-form-transcriptional control, and their expression seems to be somewhat tissue-specific. In mice Fbw7α is ubiquitously expressed whereas β and γ are expressed in brain and muscle respectively [53]. Disruption of FBW7 leads to the accumulation of the above mentioned oncoproteins and therefore re-sults in deregulated cell growth [57]. The role of FBW7 in cancer may be direct, where FBW7 is mutated or deleted, or indirect, where the CPD recog-nition motif on the substrates is mutated. Regardless of the mechanism, pro-teins ultimately circumvent ligase recognition and degradation [57].

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SOX9 and its role in cancer SOX (SRY-related HMG-box) proteins share a conserved high mobility group (HMG) DNA binding domain [58] and are important in key areas of stem cell biology, such as development, reprogramming and tissue homeo-stasis, as transcriptional regulators [59]. SOX proteins are divided into sub-groups (A-H) based on their HMG-sequence homology. All SOX proteins require complex formation to exert their gene regulatory functions. Whereas SOX B/C/F proteins only form heterologous complexes, the SOXE proteins have two modes of complex formation, utilizing both hetero- and homo-dimerizing properties [60].

SOX9 is a member of the SOXE family and is normally involved in sex-determination, chondrogenesis and neurogenesis. Focusing on its role in neurogenesis, SOX9 is expressed in the embryonic and adult CNS in radial glia, astrocytes and oligodendrocyte progenitors and its expression is re-quired to sustain multipotency in NSCs [61]. Studies on mice have shown that from embryonic day 11.5 (E11.5) SOX9 and SOX2 are coexpressed in most cells of the developing CNS and SOX9 was needed to transition SOX2-expressing progenitors to multipotent stem cells [61]. To further high-light the importance of SOX9 in multipotency, it has been described that SOX9, in collaboration with the transcription factor Slug, can revert differ-entiated epithelial cells back to epithelial SCs [62]. Furthermore, SOX9 and Slug have been shown to collaborate during embryonic development in guid-ing neural crest cell specification [63], which may suggest a preserved coop-erative role in SC maintenance.

Although found to be over-expressed in cancers of the skin, lung, prostate and brain [64], SOX9 does not have a clearly defined role in cancer yet. On its own, high SOX9 levels are, to date, not sufficient to cause neoplasia. However in combination with an oncogene or transforming agent, SOX9 contributes to tumor formation [64]. In a study on colorectal cancer, re-searchers showed that increased SOX9 levels, in collaboration with mutated K-RAS, correlated with advancing tumor stages [64]. Amplification of SOX9 in colorectal cancer, interaction of SOX9 and Slug in breast cancer, and most recently SOX9’s metastatic potential in MB delineate a role for SOX9 as a mediator of metastatic disease across cancers [56, 62, 65].

MYC proteins and their oncogenic roles MYC-family proteins are basic Helix-Loop-Helix (bHLH) TFs which form homo- or heterodimeric complexes. There are three members of the MYC gene family c-MYC (MYC), MYCN and MYCL. MYC proteins are essential during normal development, particularly MYC and MYCN which is demon-strated by the embryonic lethal response in mice lacking the functional genes

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[66, 67]. MYCN is highly expressed in developing hindbrain and forebrain and serves important functions in cerebellar stem cell and GNP replication [68]. Up-regulation of MYCN in GNPs is mediated by SHH signaling and MYCN levels regulate cell cycle progression in GNPs [69]. Moreover, MYC expression and activity is important in processes such as cell cycle progres-sion, apoptosis and regulation of transcriptional activation of a large number of genes [70]. Several important signaling pathways like WNT, TGF-β, NOTCH and SHH regulate MYC expression [69, 71].

MYC-family protein activity is dependent on dimerization with other bHLH proteins, such as MAX, and is controlled by a rapid turnover facilitat-ed by the UPS system. The MYC/MAX complex acts as a transcriptional activator when binding Enhancer Box sequences (E-Boxes), whereas re-cruitment of Miz1 results in a complex that represses gene transcription [72]. MYC-family proteins are stabilized by phosphorylation of the serine-62 (S62) residue by extracellular-regulated kinase 1, 2 (ERK) [73] or cyclin-dependent kinases (CDKs) [74]. MYC turnover is regulated by GSK3β phosphorylation at threonine 58 (T58) on MYC proteins, and dephosphory-lation of S62 by protein phosphatase 2A (PP2A) is a prerequisite for T58-tag recognition by FBW7 (Figure 3). FBW7, E1/2 and the SCF complex facili-tate proteosomal degradation of MYC.

Figure 3. Receptor-tyrosine kinase (RTK) pathway activation lead to MYC stabili-zation and subsequent proteosomal degradation. Dashed lines indicate phosphoryla-tion/dephosphorylation of essential substrate residues.

A mutation resulting in an amino acid substitution, from threonine to alanine on residue 58, was discovered in the transforming v-MYC viral gene [75] and in MYC in Burkitt's lymphoma patients [76]. The stabilized form of MYC-T58A escape degradation by the ubiquitin system, much like mutated SOX9 [56], thereby prolonging the otherwise short half-life (20-30 min) of the pro-tein [77]. Also, mutations in FBW7 and GSK3β genes lead to increased

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MYC protein stability, which facilitate uncontrolled induction of cellular proliferation and growth.

Amplification or overexpression (Figure 4) of the members of the MYC family of genes (mainly MYC and MYCN) are found in several MB subtypes [34]. MYC is the most frequently observed gene amplification across multi-ple cancer types [78] and Group 3 MB tumors are commonly associated with amplification of MYC [35]. Group 3 tumors without MYC amplification are often characterized by overexpression of MYC [11] or amplification of MYCN [35]. MYCL amplifications have been reported in a few SHH tumor cases but SHH and Group 4 tumors all have prevalent proportions of MYCN amplifications [35].

Figure 4. Illustration of gene and protein levels when the MYC gene is amplified or overexpressed. Gene amplification denotes increased number of copies of a gene; overexpression does not have to be associated with gene amplification it can also be due to an overly active promotor.

As previously mentioned, targeting tumors and the causative onco-genes/oncoproteins in the CNS is difficult due to physical barriers such as the BBB, however targeting TFs such as MYC proteins proves to be yet another obstacle. In general, it is difficult to target intracellular proteins that lack enzymatic activity, which is why targeting kinases and cell surface pro-teins have long been the primary goal for therapy development. Ideally, dis-rupting MYC-complexes, such as MYC/MAX, would result in abolished MYC-dependent transcriptional activation. Moreover, MYC/Miz1 complex-es have been shown to repress genes important for neuronal differentiation in MYC-dependent Group 3 MB, thus allowing tumor cells to retain a stem-like state, targeting this complex would also be of therapeutic interest [72]. Today, the understanding of TF biology and constantly developing technol-ogies enable highly specific binding of small molecular inhibitors to TFs. Still, direct targeting of MYC is difficult, because MYC lacks suitable bind-ing sites for drugs and is pleiotropic in nature. Therefore, interruption of MYC-dependent pathways and MYC regulatory units is the preferred way to target MYC proteins.

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Super-Enhancers and BET bromodomains Enhancer regions are distant or proximal sites in the DNA, in relation to the promoter region of a gene, where regulator complexes bind and activate gene-specific transcription. Super-enhancer regions are clusters of enhancer regions that act as scaffolds for a high number of TFs, epigenetic modula-tors, co-activators and normal transcription machinery [79, 80]. When com-pared to normal enhancers, super-enhancers (SE) are larger in size, more sensitive to changes in their milieu, have considerably more associated regu-lators and a more prominent ability to activate gene transcription [79].

SE regions can be found in the DNA of normal cells but have more nota-bly been identified in several cancers including multiple myeloma, GBM and small-cell lung cancer [81]. SE have been linked to key oncogenic drivers in cancer such as MYC and GLI [39, 81, 82]. Moreover, SOX9 was shown to regulate SE activity, thereby indirectly governing cell plasticity through SE [83]. Abundant master regulators and complexes like BRD4 (a bromo-domain-containing proteins belonging to the bromodomain and extra-terminal (BET) bromodomain family) and Mediator (multiprotein complex) play prominent roles in regulating super-enhancer activity and sensitivity to perturbations. As previously mentioned, SEs are sensitive to changes in lev-els of regulators and co-activators, most likely because of the cooperative and synergistic interaction of several regulators that causes greatly reduced activation upon disruption [81]. Disruption of SE activity (and thereby asso-ciated oncogenic driver-genes) by BRD-inhibition is being extensively in-vestigated as a therapeutic option in many malignancies [84-87].

BET bromodomains (BRD2, 3, 4, T) are important in several cellular pro-cesses, such as mitosis and transcriptional regulation [88]. BRD4 recognizes ε-N-lysine acetylation motifs on open chromatin and interact with Mediator [89] and the positive transcription elongation factor b (P-TEFb) [90] control-ling phosphorylation of RNA polymerase II that leads to transcriptional acti-vation [81].

There are several small molecule inhibitors targeting BET bromodomains, such as TEN-010 (NCT01987362) and OTX105 (NCT02259114), currently in clinical trials [91]. One of the first and most extensively studied (although not clinically eligible) BRD4 inhibitor was JQ1 [92]. JQ1 displaces BRD4 from chromatin through competitive binding of the acetyl-lysine recognition pocket [92]. Several studies have shown that BET inhibition by JQ1 results in downregulation of MYC transcription, [84-87] and lead to down-regulation of target genes. We have previously showed that BET bromo-domain inhibition effectively targets MYC-amplified Group 3 MB [87].

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Cyclin-Dependent Kinases as therapeutic targets CDKs and their corresponding cyclins are key regulatory units in cell cycle integrity and progression. The cell cycle tightly regulates the precise steps of genetic duplication, cellular division and proofreading. Briefly, the cell cycle consists of G1 (growth phase), S (DNA synthesis), G2 (premitotic growth) and M (mitotic) phases [93]. The cell cycle would be unable to maintain its high integrity without accumulation of the regulatory units, CDKs and the oscillating cyclins, at each checkpoint of the cell cycle.

Mitogenic signals are initiated by the expression of cyclin D, which acti-vates CKD4 and 6 in the early stages of the cell cycle. CDK2 is activated by cyclin E in the late stages of G1 and is then reactivated by cyclin A in late S phase to facilitate transition to G2 [94]. CDK1/cyclin B complexes trigger the G2/M transition and, importantly, phosphorylate the anaphase promoting complex (APC) ensuring completion of mitosis. The G1/S checkpoint (re-striction point) aims to safeguard the integrity of the cell’s DNA and cell growth before progressing further in the cell cycle. DNA-damage cell cycle-arrest is dependent on P53. DNA damage rapidly increases P53 activity by phosphor-activation by protein kinases ataxia-telangiectasia-mutated (ATM) and ataxia and rad3 related (ATR). P53 induces transcription of genes such as Bax, Mdm2 and p21 [95], and p21 prevents replication of damaged DNA by its inhibitory action on CDK2 complexes [96]. Deregulation of the cell cycle is often associated with several diseases, such as cancer.

Functional activation of CDK2 kinase activity is dependent on two con-formational changes triggered by cyclin binding. The first conformational change shifts a steric hindrance from the catalytic cleft, enabling substrate access and an activating phosphorylation of Thr160 [97] on CDK2. The se-cond change prepares the residues in the active site for ATP binding and subsequent phosphate transfer [98, 99]. Therefore, cyclin levels dictate the activation and activity of CDK2. CDK2/cyclin A complexes are involved in many processes such as DNA replication and inactivation of G1 TFs. Im-portantly, CDK2 is also involved in regulating protein stability of MYC proteins. Moreover, overexpression of MYC activates CDK2 and increases cyclin A/E gene expression leading to progression in the cell cycle [100, 101]. These interactions exemplify the intricate relationship between MYC and CDK2, and demonstrate a potential benefit from disrupting these feed-back-loops therapeutically since CDK/cyclin complexes also have been found frequently deregulated in cancers [102]. Cells that have deregulated levels of CDK/cyclin complexes or acquired other mitosis-promoting altera-tions may be more prone to increased mitogenic signaling and deregulated responses to anti-mitogenic signals, ultimately leading to uncontrolled cell proliferation [94].

CDKs and their cyclin complexes are good candidates for therapeutic tar-geting, due to their regulatory role in cell cycle progression. Similarly to

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many CT drugs, CDK inhibitors halt cell proliferation by disrupting progres-sion in the cell cycle. Allosteric, ATP-competitive and ATP-non-competitive CDK inhibitiors are used to target CDK protein activity. A CDK4/CDK6 inhibitor, Palbociclib, is currently evaluated in clinical trials for different solid tumors and has been approved for treatment of breast cancer [103]. Several studies have shown that Palbociclib targeted neoplastic cells in breast cancer [104, 105] and glioma [106, 107], and that treatment resulted in an increased sensitivity to radiotherapy in MB [108]. Another CDK-inhibitor, Milciclib, which targets CDK2 has shown great promise in a phase I clinical trial on solid tumors [109]. To evaluate CDK2 as a therapeutic target in MB we investigated the ATP-competitive CDK2-inhibitior Milciclib together with epigenetic inhibition in paper IV.

Murine experimental models in cancer To understand how tumors develop and how they respond to different treat-ments, models that allow researchers to study a complete biological system are needed. In vivo modeling enables a more robust approach to evaluate interactions in a biological system and disease models can more readily bridge discoveries to the clinic given the relatively high cross-species integ-rity. Genetically engineered models (GEM) are important tools to model human disease. Genes can be turned on or off in genetically altered experi-mental animals. A transgene, a genetically altered gene/a gene of interest, is often under the control of an endogenous tissue-specific promoter that drives the expression.

Xenograft/allograft models use immunocompromised or immunocompe-tent hosts respectively for transplantation of tissue from different species or from same species. Due to the high integrity of the immune system, cross-species transplantation in immunocompetent recipients is not possible. Using xenograft models, one can generate secondary tumors that recapitulate the original human tumor. A downside to xenograft models is the absence of a complete microenvironment in the immunocompromised mice, leading to potentially important roles of immune cells in the tumor microenvironment being overlooked.

To target specific somatic cell populations for genetic alteration postnatal-ly, viral vectors can be used as modes of delivery. A commonly used retrovi-rus, Replication Competent ALV Splice acceptor (RCAS) can be manipulat-ed to express any oncogene of interest. The RCAS virus is avian and propa-gated in chick cells and as such RCAS is not able to infect mammalian cells without expression of the receptor, t-va, which is commonly linked to tissue specific promoters [110-112].

The main experimental models used in this thesis are the transgenic te-tON/OFF models together with xenograft models. Our studies require pre-

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cise control over the timing of gene expression, already during embryogene-sis, something we have previously shown to be crucial in tumor development [110], which is why we use the tetON/OFF models for our spontaneous mu-rine MB studies. To further increase the clinical relevance, xenografts are used to study patient-derived MB cells for evaluating effects of drugs or delineating roles of transcription factors in human MB in complete biologi-cal systems.

Tetracycline-controlled transgenic models To control induction of gene expression, transcription is normally turned on and off in cells. This can also be done in transgenic models when transgenes are under control of a regulatable system. In tetracycline (tet) controlled transgenic models tetracycline trans-activating (tTA/rtTA) proteins are under the control of a tissue specific promoter, and activate a tetracycline respon-sive element (TRE) (repeats of bacterial tet-O sequence) (Figure 5) [113]. The system can be a tet-ON model - where administration of tetracycline induces gene expression, or a tet-OFF model - where administration of tetra-cycline impairs gene expression. A more stable tet analog called doxycycline (DOX) is usually used in these systems to regulate expression of transgenes.

Figure 5. Schematic representation of gene regulation using tetOFF/ON transgenic models. Tetracycline-controlled transactivator (tTA) and reverse tetracycline-controlled transactivator (rtTA) are under control of a tissue-specific promotor (TSP), activating/inactivating transgenes (Tg) of interest and commonly a reporter such as luciferase (Luc). DOX inactivates expression of the target gene in the tetOFF model and conversely activates expression of the target gene in the tetON model.

An example of a tet-OFF transgenic non-SHH MB model is the GLT1-tTA, TRE-MYCN-Luciferase (GTML) model [114], generated to drive human MYCN in hindbrain NSCs from the Glutamate transporter 1 (GLT1). This system utilize a bidirectional promoter to drive both MYCN and reporter-luciferase expression. Administration of DOX turns off MYCN expression leading to tumor regression [114]. An advantage of using tetracycline-controlled transgenic models, compared to conditional/non-conditional gene

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expression systems, is the reversibility of the system as well as the control of gene activation/inactivation at precise time points. However, the initial re-sponse may be somewhat slower in tetracycline controlled transgenic models compared to conditional gene expression systems, due to transcriptional and translational events.

Murine models to study medulloblastoma NSCs are multipotent, self-renewing cells, with the ability to give rise to cells in the neuronal, astrocytic and oligodendrocytic lineages. NSC differen-tiation is conducted by an interplay of spatially and temporally controlled gene expression of the SHH, BMP, WNT and FGF pathways. Generation of GNPs starts in the second germinal center, situated anterior of the rhombic lip (RL) structure. Cells migrate from the anterior part of the RL, creating the EGL with MATH1-positive, highly proliferative, granule cell progenitors (GCPs). In a fully developed cerebellum the EGL is transformed into an internal granule layer (IGL) bordered by a Purkinje cell layer and a molecu-lar layer consisting of interneurons and Purkinje cell arbors. The IGL consist of matured GCPs, granule neurons, which are the most abundant cell type in the entire brain. The pathways of important developmental signals are often deregulated in MB and can together with potential cells of origin be used to model the disease in mice. WNT models GEMMs used to model WNT MB include animals that drive mutated β-catenin (CTNNB1lox) together with p53-depletion, (TRP53flx/flx). These tumors are thought to arise from brain lipid-binding protein (BLBP)-positive brain stem precursors [7]. SHH models SHH-activated MB is the easiest subgroup to model, evident by the plethora of conditional/non-conditional knockout mouse models and genetically en-gineered cells used in studies [115]. Mutations in PTCH1, SUFU or SMO are, as mentioned previously, common genetic alterations found in SHH MB and these signatures are frequently used to model SHH MB [6, 30, 116-122]. PTCH loss causes EGL progenitors/cerebellar stem cells to initiate MB gen-esis [6, 116] whereas SMO mutations are thought to initiate EGL progeni-tors/rhombip lip progenitors [30, 123, 124] in MB genesis. SHH MB can be initiated by both GNPs and NSCs using GNP-specific Math1-cre/PtchC/C and NSC-specific hGFAP-cre/PtchC/C knockout mice [6]. Math1 regulates genes that promote neuronal differentiation, maintaining sensitivity of GNPs to SHH signaling [125]. This suggests that the origin of SHH MB could be either of GNP or NSC cellular origin. NSC are required to commit to the granule lineage before initiating MB, and they show a marked latency of 3-4

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weeks compared to the already committed GNP, which give rise to tumors at around 3 months [6]. Non-WNT/non-SHH models Group 3 and 4 are the most challenging subgroups to model in mice. Never-theless, four mouse models using MYC or stabilized MYCT58A together with mutated TRP53 [37, 38], MYCNT58A alone [110] or MYCN expression from GLT1-positive cerebellar cells [114, 115] recapitulate Group 3 or 4 MB. Expression of MYC together with mutant TRP53 in cerebellar stem cells and cerebellar progenitor cells gives rise to tumors that recapitulate the most aggressive form of human MB [37, 38]. MYCNT58A-transduced embryonic or postnatal NSCs isolated from the cerebellum generate MB that resemble human SHH MB or Group 4 MB when orthotopically transplanted [110]. The spontaneous tetOFF-model, GTML, drives expression of MYCN in GLT1-positive cerebellar cells and generates tumors that most closely re-semble human Group 3 tumors [115]. Group 3 tumors are the most malig-nant MBs and are associated with the poorest survival, which is why we focused studies in this thesis on events influencing tumorigenesis of this subgroup.

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Present investigations

Paper I. FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

Figure 6. SOX9 is a novel substrate of the ubiquitin ligase FBW7. Evasion of deg-radation by FBW7 leads to increased migratory and metastatic potential, and treat-ment resistance of SOX9-positive cells in MB.

Aim and Results The aim was to identify putative substrates for the ubiquitin ligase FBW7, with particular focus on the transcription factor SOX9, and to determine the role of such a substrate in tumorigenicity, metastatic potential and treatment resistance. Findings were correlated to previously published clinical MB cohorts (Northcott et al) to bridge preclinical findings to the clinic.

Paper I highlights regulatory pathways of the transcription factor SOX9 and its role in medulloblastoma. Here SOX9 was identified as a novel target of FBW7. Further, a key phosphodegron motif (T236/T240) on SOX9 was shown to be essential for the interaction with FBW7. Interaction between the two proteins leads to proteosomal degradation that helps maintain transcrip-tion factor homeostasis in cells. Conditional regulation of FBW7 together

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with overexpressed SOX9 showed an increased rate of tumor development in mice when FBW7 was not expressed. Similarly, high levels of FBW7 were correlated with increased survival in large patient cohorts of MB. In the same cohort, elevated levels of FBW7 displayed a reduced rate of translation of SOX9 RNA to protein. Moreover, overexpression of SOX9 in Group 3 MB cell lines led to increased cell migration in vitro and in vivo. Concomi-tantly, the MB patient cohort showed significant correlation between high levels of SOX9 protein and metastatic stage 3 (spinal metastasis). RNA seq analysis of group 3 MB revealed how FBW7-driven SOX9 stability affects a number of SOX9 substrates, previously identified as pro-metastatic targets. Further, SOX9-positive MB cells proved more resistant to cisplatin treat-ment, a standard therapy cytotoxic drug. Accordingly, the increased re-sistance was reversed upon SOX9 ubiquitinylation by FBW7. Additionally, we showed that inhibiting the PI3K/Akt/mTOR pathway led to FBW7-dependent SOX9 degradation and sensitization of MB cells to cisplatin treatment.

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Paper II. Metastasis and tumor recurrence from rare SOX9-positive cells in MYCN-driven medulloblastoma

Figure 7. Upon MYCN oncogene depletion, SOX9-positive cells can drive recur-rence in a Group 3 MB model. A combination of the TetON and TetOFF systems allows a small population of SOX9-positive cells to survive when the primary tumor completely regresses, these cells can later give rise to distant recurrences in MB.

Aim and Results The aim of the study was to further evaluate the role of SOX9 in MB devel-opment and metastasis. We wanted to investigate whether a small subpopu-lation of tumor cells could drive tumor recurrence after primary MYCN on-cogene depletion taking advantage of both a tet-ON and a tet-OFF system. We used three transgenes (Glt1-tTA; TRE-MYCN-Luc; SOX9-rtTA) to assess the potential of SOX9-positive tumor cells in the GTML model to drive tu-mor recurrence.

In paper II we focused on tumor recurrence in MB. Recurrence is sub-group-specific, Group 3 tumors form distant relapses whereas tumors in the SHH subgroup display local recurrence. Since SOX9-positive cells in MB tumors from paper I revealed increased migration and metastatic potential

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we hypothesized that SOX9-positive MB cells could be involved in metastat-ic recurrence. We compared SOX9 levels between paired primary and meta-static tumors of human Group 3 and Group 4 MB and found that SOX9 ex-pression was significantly increased in the metastatic tumors compared to the corresponding primary tumors. We have previously shown that SOX9 levels are higher in MYCN-depleted MB tumors [110]. To model recurrence using SOX9-positive MB cells, we utilized the inducible tet-OFF GTML model [114] (expressing the MYCN oncogene) together with an inducible tet-ON SOX9 model, allowing us to control MYCN expression in two cell populations in vivo (named the GTS model). GTML tumors originated from a GLT1-positive cell population. Administration of DOX depleted MYCN-positive GLT1-positive cells in the tet-OFF model and concurrently stimu-lated survival of rare SOX9-positive cells using the tet-ON model. These rare SOX9-positive cells were sufficient to cause relapse in the combined tet-OFF/ON model, also resulting in increased dissemination to the spine. Transcriptome analysis further showed that, in line with human MB, relaps-ing tumors displayed similar transcriptional profiles to the primary tumor. These rare SOX9-positive cells have not yet been extensively characterized, however with this new recurrence model system one can evaluate the contri-bution of specific cell populations in tumor recurrence in many cancer types.

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Paper III. BET-bromodomain inhibition of MYC-amplified medulloblastoma

Figure 8. BET bromodomain inhibition target MYC and MYC-associated transcrip-tional targets in MB. The BET bromodomain inhibitor, JQ1, selectively inhibits proliferation of MYC-amplified MB and prolongs survival in vivo.

Aim and Results

MYC-amplified MBs are highly aggressive tumors and associated with poor prognosis in children. Recent studies show that BET bromodomain inhibi-tion shut down MYC-associated transcriptional activity in diverse cancers [126]. JQ1, a BET bromodomain inhibitor, inhibits BET bromodomain-containing proteins, including BRD4. In this study we consider BET bro-modomain inhibition for the treatment of MYC-amplified MB.

We studied the effect of BET bromodomain inhibition on proliferation, the cell cycle, apoptosis and efficacy in established MYC/MYCN ampli-fied/overexpressed MB cell lines and in vivo models. Moreover, the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity was analyzed.

MYC-amplified MB cells treated with JQ1 showed decreased cell viability coupled with G1 cell cycle arrest and apoptosis. We demonstrated that MYC-associated transcriptional targets were inhibited and MYC/MYCN expression was downregulated following JQ1 treatment. In an orthotopic xenograft model we showed that JQ1 significantly prolonged survival of MYC-amplified MB.

Inhibition of BET bromodomains, using the compound JQ1 suppressed MYC expression and MYC-associated transcriptional activity in MYC-amplified MBs, and displayed an overall decrease in MB cell viability.

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Paper IV. Combined BET-bromodomain and CDK2 inhibition in MYC-driven medulloblastoma

Figure 9. Combined BET bromodomain and cyclin-dependent kinase inhibition upregulate apoptotic markers and downregulate MYC target genes, and significantly increases survival in vivo compared to single compound treatment in Group 3 MB.

Aim and Results

We have previously shown that epigenetic inhibition of BET bromodomains effectively targets BRD4, resulting in down regulation of MYC proteins. In paper IV we investigate the mechanisms involved in MYC/MYCN depletion in childhood brain tumors, and whether combination therapy targeting dif-ferent paths in MYC signaling is needed for complete tumor regression. CDKs regulate key events in MYC function and processing, and they are essential cell cycle regulators. Targeting CDK proteins, together with epige-netic targeting of BRD4, could potentially reduce proliferation and arrest fast dividing tumor cells more efficiently.

In Paper IV, suppression of MYC expression by targeting BRDs and MYC destabilization using CDK2 inhibition reduced MB proliferation and promoted cell death. MYC levels and proliferation of murine and human MB was effectively reduced by a combination of specific CDK2-inhibition by Milciclib and the BET bromodomain inhibitor JQ1. Importantly, a sustained combination treatment over 7-10 days was needed in order to effectively abolish tumor cell proliferation. Both treatment strategies induced increased apoptosis as seen by elevated cleaved caspase-3 and upregulation of apoptot-ic markers using RNA seq. In addition, JQ1 together with Milciclib reduced tumor growth in orthotopical MB transplants and prolonged survival com-pared to JQ1 or Milciclib alone. Targeted MYCN suppression (DOX) com-pletely depleted MYCN-driven MB cells in vivo. Immediate transcriptional changes from such MYCN blockade were found by RNA-Seq and showed

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similarities to changes that occurred after CDK2 suppression or when inhib-iting BET Bromodomains. Importantly, RNA-seq data show that combina-tion treatment of human Group 3 MB resulted in upregulated apoptotic pathways and downregulated MYC target pathways, effects that were exclu-sive to combination therapy. Our data suggest that dual inhibition of CDK2 and BET bromodomains could be a novel treatment approach in suppressing MB by targeting MYC proteins.

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Conclusions

This thesis addresses novel findings in transcription factor biology, recur-rence and treatment in Group 3 MB, the most malignant subgroup of the disease. Summarized conclusions of each study follow below: PAPER I We show for the first time that SOX9 is a substrate for the ubiquitin ligase FBW7 and that deregulation of FBW7’s ability to recognize SOX9 drastical-ly affected the role of SOX9 in MB progression and dissemination. PAPER II Small populations of SOX9/MYCN-expressing cells drive tumor recurrence in mice, after complete elimination of the primary tumor, using the GTS model. Notably, the tetON/OFF model provides an elegant way of combin-ing two regulatable models to study the impact of multiple genes in vivo, which could be utilized in other cancer types. PAPER III BET bromodomain inhibition shows specific targeting of MYC-amplified MB cells and prolongs survival in a mouse xenograft model. These findings provide an important step towards evaluating BET bromodomain inhibition as a treatment option for patients with MYC-amplified MB. PAPER IV Combined BET bromodomain inhibition and CDK2 inhibition downregu-lates MYC-transcriptional targets, induces apoptosis and prolongs survival compared to single agent therapy in a MYC-amplified MB model.

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Future perspectives

The heterogeneity among MB tumors has long been troublesome, causing diverse prognostic and therapeutic responses among patients. Distinct mo-lecular signatures associated with important signaling pathways have divided MB tumors into four, more homogeneous, subgroups with different histolo-gy, prognosis and drug response. Delineating molecular signatures and de-veloping subgroup-specific treatments has become increasingly important in MB. A number of in vivo models have helped identify cell of origin for some of the subgroups and to evaluate treatment targets. Still, MYC-amplified MBs are the most aggressive tumors and have a very poor prognosis. It is therefore important to continue to decipher the origin and progression of these malignant tumors.

The rapid increase in genomic, proteomic and methylomic data on MB tumors, together with researchers willingness to share these data sets within the community, presents a good scaffold for making important discoveries based on the dynamic between genomic alterations, protein functions and epigenomic changes. Ideally, joint efforts investigating subgroup-specific molecular signatures together with the contribution of the vasculature and immune system to the tumor environment would be of importance to find new weaknesses in MBs and exploit them therapeutically.

The work in this thesis has contributed to increased understanding of the UPS system and TF turnover by identifying SOX9 as a novel substrate of FBW7. We have also showed that SOX9 increases the rate of tumor devel-opment when it is not targeted by FBW7 in MB. Moreover, using a new recurrence model we showed that a small population of SOX9-positive cells drives metastatic recurrence in MB. We also show that MYC protein levels are negatively correlated to SOX9 protein levels, something that most likely enable the migratory capacity of SOX9-positive cells. IHC stainings and protein expression of the recurrent tumors, initiated by SOX9-positive cells, showed very low levels of SOX9 and high levels of MYCN, much like the primary tumor. To better understand the dynamics of the recurring tumors and as proof-of concept we need to employ fate-mapping of these rare SOX9-positive cells, to show that these rare cells indeed give rise to the re-lapsed tumor. We will add two transgenes to the GTS model creating a GTS/Sox9ERTCre/R26R-lsl-YFP model, allowing us to induce Cre expres-sion in the surviving SOX9 population after DOX treatment and to follow the progeny using their YFP expression. This system allows us to identify

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cells that are SOX9-positive in the tumor and more importantly, to identify if the recurring SOX9-negative tumor bulk originates from these cells. Another approach would be to isolate SOX9-positve cells from tumors, using a re-porter gene linked to SOX9, and transplanting these cells back to a host and follow tumor formation. Transplantation of these cells has recently been attempted, however challenges such as the low number of SOX9-positive cells in tumors, the probability of getting triple-positive transgenic mice and the time-consuming nature of this spontaneous model have made this ap-proach challenging.

To improve the impact of our finding we need to establish how these SOX9-positive cells trigger relapse and moreover investigate the possibility of targeting signaling pathways in these cells. As previously reported by others, we also found PDGFRA significantly upregulated in our recurrent tumors. Inhibiting this signaling pathway would be an interesting approach to evaluate the contribution of the PDGFRA expression in the recurring tu-mors and if it could be of any therapeutic benefit. Interestingly, we have seen in our study that SOX9 cannot induce tumors on its own and previously we have shown that DOX-treated GTML cells resulted in a small surviving frac-tion of cells with high SOX9 expression and low MYCN. Without the SOX9-driven tetON system in these cells, the SOX9-positive cells remain senescent or quiescent after oncogene depletion. With that in mind, we could utilize the negative correlation found between SOX9 and MYCN protein, and theoretically treat these tumors with SOX9 to reduce MYCN levels and make the tumor cells quiescent.

Therapeutically interesting targets will, following in vitro evaluation, be assessed in our recurrence model to see if tumor recurrence can be prevent-ed. Given the dismal prognosis for patients following relapse, this combined tet-ON/OFF model could provide an important platform to better understand recurrence and its therapeutic implications.

In the second part of the thesis studies were focused on therapeutic bene-fits of using BET bromodomain inhibitors or CDK inhibitors in Group 3 MB treatment. We showed that both BET bromodomain and CDK2 inhibition increased survival in mice and that a combination of both further increased survival as compared to single treatment. We also showed that combination treatment resulted in downregulated MYC-transcriptional targets and apop-tosis. It remains to be elucidated how tumor cells evade the effects of BET bromodomain and CDK2 inhibition, seeing that the treatment groups also faced complete mortality, albeit delayed. Multiple studies have shown bene-ficial effects of the CDK4/6 inhibitor Palbociclib in breast cancer and glioma models, and studies on MB have shown increased response to radiotherapy together with Palbociclib in vitro. In our model of MB we show that target-ing CDK2 using Milciclib is more efficient than Palbociclib in killing tumor cells in vitro, and that CDK2 inhibition significantly prolongs survival in a xenograft model. Having used metastatic MYC-amplified patient-derived

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MB cells in this study, it would be valuable to try the combination therapy in other primary MYC-amplified tumor cells to see if non-metastatic cells re-spond differently. Given the rapid nature of this engrafted model angiogene-sis could perhaps be limited to the boarder of the tumor, thus it would be interesting to evaluate the role of the tumor vasculature in facilitating drug delivery in these models. The recent realization that MB subtypes also differ in their BBB integrity opens up the possibility of disrupting the BBB to in-crease penetration of normally inaccessible compounds to the tumor.

Moreover, we will extend our evaluation of the role of CDK2 in MB tu-morigenesis, mainly by crossing tamoxifen-inducible CDK2 knock-out (KO) mice with GTML mice, and by using the CDK2 KO mice together with the RCAS system tva-mice and isolate normal cells from different brain regions and KO CDK2 in vitro. This approach will enable us to study loss of CDK2 in normal cells but also, using the RCAS system we can introduce stabilized MYCN or any gene of interest using retroviruses and transplant the cells back to evaluate the tumorigenic potential in vivo.

The scope of this thesis addresses novel findings in transcription factor biology, recurrence and treatment in Group 3 MB. The continuation of these studies requires insight into the underlying mechanisms of tumor recurrence and treatment resistance to help identify reliable targets for treatment. Im-proving progression-free survival and quality of life for children affected by MB, by identifying new drug targets in the most malignant subgroup of MB is crucial.

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Populärvetenskaplig sammanfattning

Thesis summary in Swedish Medulloblastom är den vanligaste form av elakartade barnhjärntumörer. Medulloblastom uppstår i lillhjärnan, en region som vanligtvis styr koordina-tion av rörelser och balans. Cancergenen MYC och dess signalering och mekanismer för proteinstabilisering under hjärntumörutveckling studeras i denna avhandling med betoning på att förbättra behandlingsstrategier för barn som drabbas av medulloblastom samt att för att bättre förstå hur tumörer kan generera återfall efter behandling. Dagens standardterapi för patienter är kirurgi, strålning samt cytostatikabehandling vilket botar 3 av 4 patienter. En lyckosam behandling, men den är dock hård och tar inte bara död på cancerceller utan även viktiga normala celler i hjärnan, celler som behövs för kognition och minne. De barn som överlever behandling får därför ofta livslånga neurologiska biverkningar. Nya behandlingsalternativ, som specifikt riktas mot snabbt delande tumörceller utan att tillföra stor ska-da på kroppens normala celler, behövs för att öka överlevnad samt för att maximera livskvalitén hos de barn som överlever behandling. Avhandlingen är en sammanfattning av studier på medulloblastom, korta sammanfattningar av varje delarbete följer nedan:

I. Vi identifierade transkriptions faktor SOX9 som ett substrat till ubikvitin-ligas FBW7, samt studerade dessa proteiners roll i medulloblastom och hur de bidrar till tumörers förmåga att sprida sig.

II. Vi visade att en begränsad grupp av SOX9-uttryckande celler i tumörer har förmåga att driva återfall av medulloblastom i möss.

III. Men en epigenetisk behandlingsstrategi visade vi att BET-bromodomän-inhibitorer minskade aktivering av onkgenen MYC i medulloblastom och ökade överlevnad i möss.

IV. En kombination av epigenetisk- och cyklin-beroende kinas- in-hibering resulterade i ökad överlevnad jämfört med enkelbehan-dling i en aggressiv medulloblastom-modell.

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Acknowledgements

This work was carried out at the Rudbeck Laboratory, Department of Immu-nology, Genetics and Pathology, Science for Life Laboratory, Uppsala Uni-versity, Uppsala, Sweden. We warmly acknowledge financial support for our research from the Swedish Research Council, the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Association for International Cancer Research, Åke Wiberg Foundation, Ragnar Söderberg Foundation, the Swedish Brain Fund, the Swedish Society of Medicine, Lars Hiertas Minne and Lions Cancer Research Fund. I would like to express my heartfelt gratitude to the following people who supported me throughout my PhD studies and helped me prepare this thesis:

To my supervisor Fredrik Swartling – Almost five years ago I was in-terviewing for this PhD position and I had very little experience with the brain tumor field, despite that you chose to give me the opportunity to join your group - thank you for seeing potential in me and for being a leader and mentor that others will have difficulties measuring up to. And thank you for providing a wonderful research atmosphere and for caring so much about your group. Last but not least, thanks for all the fun times at conferences and retreats (and let’s not forget Frankie-boy).

To my co-supervisor Karin Forsberg-Nilsson – thank you for your sup-port throughout my PhD studies, for kindly accepting to be my co-mentor and for your invaluable advice on what to wear to AACR meetings.

To my faculty opponent Professor Martine F. Roussel – thank you for taking the time to scrutinize my thesis work and for traveling all the way to Sweden.

Thanks to the past and present Swartling lab: Vasil – for all the fun in San Diego, Berlin and of course outside the lab, Sanna – for the many talks about work and life in the (old) office, Holger – for great memories in San Diego and Fyrväpplingen and for all your help with bioinformatics, Ga-briela – for your godsend presence in the lab and for your heroic efforts as a tour guide (Berlin – we’ve seen it all ;) ), Anders – for all the help with bio-informatics and for allowing me to be better at Prism than you (small but important personal win), Sonja – for the short time we were office buddies AND roomies, Matko – for all the fun in Berlin and New Orleans (Huuu-ricanes FTW!) and Anna B – for putting up with me both as a supervisor and colleague (kudos), for the many chats about life and all the gliringar ;) .

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Geraldine – for bringing your important clinical point of view to the group, Lotta – for your positive attitude and hard work in the anim. fac., Oliver – welcome to the group! You guys have really made these years amazing, thanks for all the fun group activities! Thanks to my previous project stu-dents, Anna, Camilla and Johan for your curiosity and enthusiasm.

To all past and presents collaborators and co-authors – thank you for great collaborations. Olle and Aldwin – thank you for the great collaboration on the Sox9/Fbw7 paper. Mimi – thank you for the JQ1 collaboration and for having me visit the lab at Dana Farber.

Thanks to all the people in the Neuro-Oncology group for interesting scientific discussions, all the in-house knowledge and our Wednesday fika. A special thanks to Marianne & Annika for all your help.

Thanks to all the people in my past and present office(s) Vasil, Gabriela, E-Jean, Demet, Vikki, Anna S, Anna B, Sanna, Ludmila, Satish, Sonja, Yuan, Anqi – I´m so thankful that I didn’t have to eat all the choco-lates/cookies by myself.

Thanks to all colleagues at Rudbeck and IGP for contributing to a great work environment. A special thanks to: Vasil, Emma Y, Verónica, Eric, Sofia, Johan B, Moa, Miguel, Frank, Isa, Ross, Ammar, Emma G, Viktor L, Chiara, Anja, Luuk, Johanna, Mohammad, Anna S, Matko, Holger, Gabriela, Anders, Anna B, Gabriel G, Tor, Victor G, Argyris, Diego, Lucy, Mr Tabata, Marco, Maike, Linda, Antonia, Svea, Tobias, Demet, Jelena, Tobias, Leonor, Doroteya & Kiki – for all the fun chats, parties and activities over the years.

Lucy & Andrew – you awesome native speakers! Thank you so much for proof-reading my thesis and for just being awesome.

Thanks to everyone who helped make my research visit to Stanford Uni-versity possible – Fredrik, Yoon-Jae Cho, IGP/Stanford Admin, Cancer-fonden and Sederholm’s fund. And thanks to all the amazing ppl in the Cheshier lab – Sam, Sid, Suzana, Gregors, Sherri, Joy and Matt for adopting me and for the fun evenings together. Julian – thank you for your friendship, it meant more to me than you know.

Christina M – thank you for all your encouraging words, all the help throughout the years and for your contagious happiness. Let’s arrange that alumni event soon, right? ;)

Thanks to the IGP PhD student council, the Medical faculty PhD stu-dent council (MDR) and the Postgraduate Research Education Commit-tee (KUF) – for many laughs, activities and valuable discussions regarding the research education at Uppsala University.

Thanks to the Biovis facility, NGI Uppsala at Uppsala Genome Center and Animal husbandry facility for all the invaluable resources/help throughout my PhD. Thanks to vaktmästeriet och intendenturen at Rud-beck for all your help.

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Magnus Å – thank you for taking me in as a project student, for teaching me a lot of in vivo techniques and for encouraging me to continue with re-search. Alexis F – thank you for all the talks in the lab and yes, we are still as beautiful as we were ‘back then’ ;) Christian R – I’m really happy we had the opportunity to roam the corridors at BMC together for a while, and even happier to still have you in my life today.

The Old Circle training team, and the Newer one – thank you guys for all the fun evenings in the gym. I have truly loved being yelled at and loved yelling back at you. Thanks for all the laughs, the “friendly” jargon and the post-workout drinks! You guys kick-ass!

Sofia, Emil, Miguel & Vasil – It’s been fun. Well, when I’ve been around. ;) Thank you for all the oyster shucking, champagne popping and piñata bashing over the years (in its literal sense of course). I have loved every one of our ridiculous conversations and look forward to many more (we still have a few words left before we’ve exhausted the etymology con-versation). Oh, and Sofia – yes, I will probably regret the hairstyle on the back of the cover in a few years. Thanks! Berglund’s crew – thank you for all the great escape opportunities over the years and of course for the awe-some Freimarkt trips. Transport – thanks for all the great times spent riding scooters and drinking sangria on the beach. Susanna – thanks for all the fun parties and for exploring Kas with me. Shaun – thank you for your persis-tent interest in perfectly centered succulents and for all the escapades and laughs we’ve shared. Anna E, BB, Andrew, Lunde – Thank you for all the fantastic talks, dinners, parties and beach volley evenings over the years (hopefully many more to come). Coach Do/T-rex – thank you for the innu-merable hours spent at the gym, for the constant encouragement and for your friendship. Maria, Anna, Tina och Malin - tack för att ni finns i mitt liv. Oavsett hur lång tid det går mellan gångerna känns det alltid som att vi är tillbaka till förfesterna innan Vallen när vi väl ses (fast vi sköter oss ju lite bättre nu för tiden. Hoppas jag. ;) ).

Jenny – thank you for being a shining star in my life, for being the best combo, for making sure I don’t work on national holidays and for our beauti-ful trip to Italy three years ago. I’m so happy you decided to abandon your lawyer-dreams for rocks. A solid career choice. Ali – thank you for your big heart, for all the game nights, for all the evenings spent deep-frying stuff and for letting that magic 8-ball lead a big-city boy to small-town Uppsala ;). Thanks for being the bestest of friends I could ever wish for, for always put-ting up with/helping with pranks, for all the support throughout the years and for letting me be a part of your lives. 10 years next August - What Would Chuck Norris Do? Min älskade familj – De senaste åren har innehållit allt mellan lycka och sorg – tack för att ni har funnits där och för att vi alltid har varandra. Far-mor och farfar – tack för att ni alltid funnits vid min sida genom min

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uppväxt och för alla fina minnen vi delar. Lina & Hussain, Ida & Mwisa – tack för allt stöd, för att ni tror på mig mer än vad jag själv gör, för allt överseende med min frånvaro, för all er kärlek och för alla mina fantastiska syskonbarn. Isaac, Joseph, Mollie, Gabriel och Benjamin – ni är de busi-gaste och bästa kidsen jag vet, och jag är så stolt att vara er moster. Mamma och pappa – tack för er överväldigande kärlek, för er omtanke och för att ni genom hela min uppväxt alltid insisterat att mitt bästa är gott nog. Jag hade inte klarat mig igenom dessa år utan er alla – jag älskar er!

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1254

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-300907

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016