ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1389

New Molecular Approaches toGlioblastoma Therapy

SATHISHKUMAR BASKARAN

ISSN 1651-6206ISBN 978-91-513-0126-6urn:nbn:se:uu:diva-329745

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen,Rudbeck Laboratory, Dag Hammarskjöld Road 20, Uppsala, Friday, 8 December 2017 at09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will beconducted in English. Faculty examiner: Professor Silvia Marino (Queen Mary University ofLondon, UK).

AbstractBaskaran, S. 2017. New Molecular Approaches to Glioblastoma Therapy. DigitalComprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1389.48 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0126-6.

Glioblastoma (GBM) is the most common high-grade brain tumor diagnosed in patients who aremore than 50 years of age. The standard of care treatment is surgery, followed by radiotherapyand chemotherapy. The median life expectancy of patients is only between 12 to 15 monthsafter receiving current treatment regimes. Hence, identification of new therapeutic compoundsand gene targets are highly warranted. This thesis describes four interlinked studies to attain thisgoal. In study 1, we explored drug combination effects in a material of 41 patient-derived GBMcell (GC) cultures. Synergies between three compounds, pterostilbene, gefitinib, and sertraline,resulted in effective killing of GC and can be predicted by biomarkers. In study 2, we performeda large-scale screening of FDA approved compounds (n=1544) in a larger panel of GCs (n=106).By combining the large-scale drug response data with GCs genomics data, we built a novelcomputational model to predict the sensitivity of each compound for a given GC. A notablefinding was that GCs respond very differently to proteasome inhibitors in both in-vitro and in-vivo. In study 3, we explored new gene targets by RNAi (n=1112) in a panel of GC cells. Wefound that loss of transcription factor ZBTB16/PLZF inhibits GC cell viability, proliferation,migration, and invasion. These effects were due to downregulation of c-MYC and Cyclin B1after the treatment. In study 4, we tested the genomic stability of three GCs upon multiplepassaging. Using molecular and mathematical analyses, we showed that the GCs undergo bothsystematic adaptations and sequential clonal takeovers. Such changes tend to affect a broadspectrum of pathways. Therefore, a systematic analysis of cell culture stability will be essentialto make use of primary cells for translational oncology.

Taken together, these studies deepen our knowledge of the weak points of GBM and provideseveral targets and biomarkers for further investigation. The work in this thesis can potentiallyfacilitate the development of targeted therapies and result in more accurate tools for patientdiagnostics and stratification.

Keywords: Glioblastoma, GBM, ZBTB16, PLZF, Heterogeneity, Proteasome inhibitors,Bortezomib, Pterostilbene, drug combinations

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

© Sathishkumar Baskaran 2017

ISSN 1651-6206ISBN 978-91-513-0126-6urn:nbn:se:uu:diva-329745 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-329745)

Dedicated -To my maternal grandparents

Diagnose the disease, detect its root cause,

discern its cure and then act aptly. -Thiruvalluvar

-

List of Articles

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

I. Schmidt, L., Baskaran, S., Johansson, P., Padhan, N., Matuszewski,

D. J., Green, L. C., Elfineh, L., Wee, S., Häggblad, M., Martens, U., Westermark, B., Forsberg-Nilsson, K., Uhrbom, L., Claesson-Welsh, L., Andäng, M., Sintorn, IM., Lundgren, B., Lönnstedt, I., Krona, C., Nelander, S. (2016). Case-specific potentiation of glio-blastoma drugs by pterostilbene. OncoTarget, 7(45), 73200–73215.

II. Johansson, P., Schmidt, L., Baskaran, S., Kundu, S., Gallant, C., Kling, T., Awe, O., Elfineh, L., Almstedt, E., Häggblad, M., Mar-tens, U., Lundgren, B., Lönnstedt, I., Frigault, M., Hurt, E., Jörnsten, R., Krona, C., Nelander, S. Targeting tumor heterogeneity: multi-omic modeling of glioblastoma drug response using an open-access library of patient-derived cells. Manuscript.

III. Baskaran, S., Johansson, P., Hansson, C., Spyrou, A., Kalushkova,

A., Ramachandran, M., Párraga, AA., Nordling, T., Elfineh, L., Martens, U., Häggblad, M., Kundu, S., Forsberg-Nilsson, K., Lundgren, B., Krona, C., Nelander, S. Loss of transcription fac-tor ZBTB16 induces cell death in patient-derived GBM cell lines. Manuscript.

IV. Baskaran, S*., Mayrhofer, M*., Kultima, H., Elfineh, L., Cavelier,

L., Isaksson, A†., Nelander, S†. Primary glioblastoma cells for pre-cision medicine: a quantitative portrait of genomic (in)stability dur-ing the first 30 passages. Manuscript in revision at Neuro-Oncology.

*, † Authors contributed equally to the work

Reprints were made with permission from the respective publishers.

Related Work by the Author

1. Schmidt, L., Kling, T., Monsefi, N., Olsson, M., Hansson, C., Baskaran, S., Lundgren, B., Martens, U., Häggblad, M., Wester-mark, B., Forsberg-Nilsson, K., Uhrbom, L., Karlsson-Lindahl, L., Gerlee, P., Nelander, S. (2013). Comparative drug pair screening across multiple glioblastoma cell lines reveals novel drug-drug in-teractions. Neuro-Oncology, 15(11), 1469–1478.

2. Xie. Y*., Bergström, T*., Jiang, Y*., Johansson, P*., Marinescu, V.

D., Lindberg, N., Segerman, A., Niklasson, M., Baskaran, S., Sreedharan, S., Everlien, I., Kastemar, M., Hermansson, A., Elfineh, L., Libard, S., Holland, E.C.H., Göran Alafuzoff, I., Westermark, B†., Nelander, S†., Forsberg-Nilsson, K†., Uhrbom, L†. (2015) The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes. EBioMedicine, 2(10), 1351–1363.

3. Ramachandran, M*., Yu, D*., Dyczynski, M., Baskaran, S., Zhang,

L., Lulla, A., Lulla, V., Saul, S., Nelander, S., Dimberg, A., Merits, A., Leja Jarblad, J†., Essand, M†. (2017) Safe and effective treatment of experimental neuroblastoma and glioblastoma using systemically delivered triple microrna-detargeted oncolytic semliki forest virus. Clinical Cancer Research, 23(6):1519-1530.

Table of Contents

Introduction ................................................................................................. 13 CNS cell types and functions .................................................................... 13Glioma and Astrocytomas ........................................................................ 13Glioblastoma (GBM) ................................................................................ 15Genomic characterization of GBM ........................................................... 16Important GBM signaling pathways ......................................................... 19GBM disease models ................................................................................ 21Clinical trials ............................................................................................ 23GBM therapeutic challenges .................................................................... 25ZBTB16/PLZF/ZNF145 ............................................................................ 27

Aims .............................................................................................................. 29

Present Investigations ................................................................................. 30Article I: Case-specific potentiation of glioblastoma drugs by pterostilbene ............................................................................................. 30Article II: Targeting tumor heterogeneity: multi-omic modeling of glioblastoma drug response using an open-access library of patient-derived cells .............................................................................................. 31Article III: Loss of transcription factor ZBTB16 induces cell death in patient-derived GBM cell lines ................................................................. 32Article IV: Primary glioblastoma cells for precision medicine: a quantitative portrait of genomic (in)stability during the first 30 passages .................................................................................................... 33

Discussion & Future Perspectives .............................................................. 34

Popular Science in Tamil ............................................................................ 37

Acknowledgements ...................................................................................... 38

References .................................................................................................... 40

Abbreviations

ADC Antibody-Drug Conjugate ALL Acute Lymphoblastic Leukemia APML Acute Promyelocytic Leukemia BBB Blood Brain Barrier BG O6 BenzylGuanine CAR Chimeric Antigen Receptor CBTRUS Central Brain Tumor Registry of the United States CDK Cyclin Dependent Kinase CDKN2B Cyclin Dependent Kinase Inhibitor 2B CL Classical CNS Central Nervous System CSF CerebroSpinal Fluid EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor FGF Fibroblast Growth Factor FISH Fluorescent In-situ Hybridization G Gefitinib G-CIMP Glioma-CpG Island Methylator Phenotype GBM Glioblastoma GC Patient-derived GBM cell cultures GEMM Genetically Engineered Mouse Models H3F3A H3 Histone, Family 3A HDAC Histone Deacetylase HGCC Human Glioma Cell Culture HSC Hematopoietic Stem Cell IDH Isocitrate Dehydrogenase KD Knock Down KLK4 Kallikrein-Related Peptidase 4 mAb Monoclonal AntiBody MDR1 Multi Drug Resistance Protein 1

MGMT O6 MethylGuanine DNA MethylTransferase MRP1 Multi drug Resistance related Protein 1 MS Mesenchymal mTOR Mammalian Target of Rapamycin mTORC1 Mammalian Target of Rapamycin Complex 1 NF1 Neurofibromin 1 NL Neural NOS Not Otherwise Specified NSC Neural Stem Cells NSCLC Non-Small-Cell Lung Carcinoma OS Overall survival P Pterostilbene PC Prostate Cancer PDGFRA Platelet-Derived Growth Factor Receptor Alpha PDX Patient-Derived Xenograft PI3K Phosphoinositide 3-kinase

PIK3CA Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic Subunit Alpha

PIP2 PhosphatidylInositol (4,5)-bisPhosphate PIP3 PhosphatidylInositol (4,5)-trisPhosphate PLZF Promyelocytic Leukemia Zinc Finger PN Proneural pRB Retinoblastoma protein PTEN Phosphatase and TENsin Homolog RARα Retinoic Receptor Alpha RER Renin/prorenin Receptor RTK Receptor Tyrosine Kinase S Sertraline TCGA Tumor Cancer Genome Atlas TMZ Temozolomide TTF Tumor Treating Field TXNIP Thioredoxin Interacting Protein VEGF Vascular Endothelial Growth Factor WHO World Health Organization ZBTB16 Zinc Finger And BTB Domain Containing 16

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Introduction

CNS cell types and functions The brain is predominantly composed of two cell types: neurons and glial cells (macroglia and microglia). Neurons are specialized cells that consist of dendrites, neuron cell body, and axons/synapses. Functionally, their role is to code (send electric signals through axons) and decode signals (receive electric signals via dendrites) transmitted to and from different organs. The glial cells, on the other hand, serve to structurally and functionally support the neurons and other functions of the central nervous system (CNS). The macroglial cells are classified into three cell types: astrocytes, oligodendro-cytes, and ependymal cells1. Astrocytes play a variety of functions ranging from backing the endothelial cells that form the blood-brain barrier (BBB), to uptake and discharging of neural transmitters2,3. Oligodendrocytes insu-late neuronal axons by forming myelin sheaths that facilitate the transmis-sion of electrical impulses through axons4. Ependymal cells are present in the cerebrospinal fluid (CSF) filled ventricles of the brain and they are in-volved in production and circulation of CSF in CNS5. Microglia cells are another important cell type present in CNS. They are the immune defense system of the CNS, which protects neurons against pathogens and helps to maintain brain homeostasis6.

Glioma and Astrocytomas Glioma is the commonly used name for a group of tumors that develops in the brain and spinal cord. Historically, the name glioma originates in the fact that the tumor cells of a glioma are histologically similar to neuroglia cells. According to the histological classification, the gliomas are subdivided into the main groups of astrocytoma, ependymoma, oligodendroglioma, brain stem glioma and oligoastrocytoma7. Astrocytoma is the most frequent type of glioma and it is most commonly diagnosed in adults and older age pa-tients8. In 2016, the World Health Organization (WHO) provided an updated CNS classification system, in which histological observations were com-plemented with genetic information9. The intent is to enable a more precise diagnosis of the tumor, which may have important clinical uses, such as designing of an appropriate treatment. In the revised WHO classification,

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astrocytoma still be histologically graded from I to IV and further sub-classified based on particular acquired genetic lesions9. In concise outline, grade I astrocytoma is well differentiated with a low cell proliferative index; a grade I tumor is often cured after a surgical resection of the tumor. Grade II astrocytoma is a moderately differentiated tumor cells. The more diffuse growth of grade II tumors increases the risk of tumor relapse after surgical resection. Grade II astrocytoma might also give rise to grade IV astrocytoma (secondary GBM). Grade III or Anaplastic astrocytoma is poorly differenti-ated tumor cells with high proliferative and invasive capacity. It is treated by tumor resection followed by radiotherapy and chemotherapy and has worse prognosis than other two low-grade tumors. Finally, Grade IV astrocytoma, referred as glioblastoma, is a highly invasive and mix of heterogeneous tu-mor cell population is widely diagnosed in adults10. In the new classification system a new variant of glioblastoma is included, termed epithelioid glio-blastoma, characterized by big epithelioid cells9.

Figure 1: Summary of 2016 updated WHO glioma classification system. The figure describes phathogentic landmarks of different types of glioma. Reprinted with per-mission11

The main highlight of new WHO classification system is, the genetic status of isocitrate dehydrogenase (IDH) 1/2 is used to genetically stratify astrocy-tomas9. The function of wild-type IDH 1/2 protein is to produce 2-oxoglutarate from isocitrate, which is an important step in the TCA cycle. But the mutant IDH1/2 produces an abnormal ‘oncometabolite’, 2-hydroxy glutarate instead of 2-oxoglutarate12. The most frequently detected somatic mutation is missense mutation of arginine into histidine at the 132nd and 172nd amino acid positions of IDH1 and IDH2 respectively13. In clinical practice, methods such as immunohistochemistry (to detect the mutant form of protein) and sequencing of IDH1/2 codons are used to determine the mu-tation status of IDH1/2. As a convention, if IDH1/2 status is not known, that

Histology Diffuse astrocytic and oligodendroglial gliomasWHO grade II or grade III

Diffuse astrocytic gliomas/glioblastomasWHO grade IV

Nuclear ATRXretained

Nuclear ATRX lost

Nuclear ATRX retained*

Nuclear ATRXretained

Nuclear ATRX lost

Nuclear ATRX retained or lost

Midlinelocation

Midlinelocation

IDH status

ATRX status

1p/19q status

H3-K27M status

Integrateddiagnosis

IDH-mutant

1p/19q- codeleted

1p/19q-non-codeleted

IDH-mutant IDH-mutantIDH-wild-type IDH-wild-typeIDH-wild-type

Oligodendroglioma, IDH-mutant and 1p/19q-codeleted, WHO grade II or III

H3-K27M-mutant, WHO grade IV

Astrocytoma, IDH-mutant, WHO grade II or III

Astrocytoma, IDH-wild-type, WHO grade II or III ‡

Glioblastoma, IDH-wild-type, WHO grade IV

Glioblastoma, IDH-mutant, WHO grade IV

H3-K27M-mutant

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particular tumor case will fall under NOS (not otherwise specified) category9,11 .

Based on genetic marks the majority of Grade II and Grade III astrocytomas belong to the IDH-mutant subclass. By contrast, more than 90% of Grade IV astrocytomas (glioblastoma) are IDH-wildtype (patients age >55 years) and remaining 10% or less of cases are diagnosed as IDH-mutant. There are chances that this glioblastoma IDH-mutant cases are relapsed from prior Grade II or III astrocytoma (secondary glioblastoma, patients age <55 years)9,11.

Glioblastoma (GBM) Glioblastoma (GBM) (primary GBM) is a highly malignant type of astrocy-toma, in which multiple hallmark phenotypes of cancer are evident, and which exhibits poor patient prognosis8,10,14,15. It is most commonly diagnosed in older adults at the age of more that 50 years16. Recent statistics from the central brain tumor registry of the United States (CBTRUS) hold out of all brain and CNS tumors, 15.1% are GBM, making it the second largest group after non-malignant meningioma (35.9%)17.

Figure 2: Snapshot of gliomagenesis. The list of important oncogenes and tumor suppressor genes involved in the process of primary and secondary GBM. Modified from18

The current treatment of GBM includes maximal surgical resection of the tumor followed by radiation and chemotherapy. The chemotherapy uses, temozolomide (TMZ), is an orally administered DNA alkylating/methylating agent that targets proliferating tumor cells. However, patients median sur-vival time is limited to between 12 to 15 months after implementing stand-ard-of-care treatment, and the survival benefit of the TMZ component of the treatment protocol is estimated to a mere 3 months19. Key reasons for the

16

poor prognosis of treated patients include invasive growth beyond the surgi-cal resection margin, presence of GBM stem-like cells after treatments that capable of regenerating the tumor, and resistance to TMZ treatment that mediated by overexpression of O6 methylguanine DNA methyltransferase (MGMT) gene20.

Recent clinical trials have evaluated new strategies to overcome TMZ re-sistance and to protect hematopoietic stem cells (HSC) from the treatment side effects. A direct inhibitor of MGMT, O6 benzylguanine (BG), was ad-ministrated along with TMZ, improving TMZ treatment sensitivity21,22. In another clinical trial, an autologous transplantation of genetically engineered hematopoietic stem cell (HSC) (P140K-modified HSC) decreased the toxici-ty to HSC during the combination treatment of BG and TMZ. Initial results using this strategy suggests a means to increase the tolerance to BG+TMZ combination therapy, resulting in an estimated overall survival (OS) rate to 20 months23,24.

In a second development, supplementing adjuvant TMZ treatment with tu-mor treating fields (TTFs) has been approved for GBM in a randomized phase III clinical trial (695 patients). TTFs are a method of applying low-intensity electric fields (200MHZ, 1-3 V/cm) at the site of tumor location. It is applied via a non-invasive, battery operated, head wearable device, with the possible mechanisms of interfering with mitotic division, leading to slow cell division or cell death25. Initial reported results obtained with this meth-od indicates a better OS rate of 20.5 months compared to 15.6 months with treatment by concomitant TMZ alone 26. This treatment indicates that further survival gains are possible for GBM patients in principle; it is however also associated with side effects, including scalp irritation and headache27–31. In order to improve OS period further, identification of new therapeutic gene targets and compounds against GBM are highly warranted.

Genomic characterization of GBM Genetically, GBM is a heterogeneous disease characterized by acquired somatic mutations in multiple signaling pathways. In a pilot effort during 2008, the cancer genome atlas consortium (TCGA) analyzed a set of GBM surgical samples (n=206) to chart the molecular changes (DNA, RNA, DNA methylation status) of GBM, using a range of genome-wide technologies. Analyses of the emerging data revealed a spectrum of genomic and tran-scriptional aberrations including inactivating mutation of neurofibromin 1 (NF1) gene, different types of activating mutations in EGFR gene (including EGFRvIII mutant). Together with this finding, activation of RTK/RAS/PI3K

17

pathway and inactivation of TP53 and RB pathways appear to be essential for the occurrence of GBM32 (Figure 3).

Figure 3: Major mutated pathways in GBM. (A) The activation of RTK pathways through different RTK receptors such as EGFR, ERBB2, PDGFRA, MET. (B), (C) The inactivation of TP53 and RB tumor suppressor protein pathways by different mechanisms. Reprinted with permission32

Integrated gene expression analysis of TCGA data revealed four molecular classes of GBM defined by 840 gene signatures, 210 genes form each sub-class: proneural (PN), classical (CL), mesenchymal (MS) and neural (NL) (Figure 4). To a significant degree, the subtypes correlate with genetic changes. The proneural subtype trends to have amplification of the locus encoding the platelet-derived growth factor receptor alpha (PDGFRA) gene, as well as mutations in IDH1 and TP53 genes; classical subtype is associated with a epidermal growth factor receptor (EGFR) gene profile and loss of NF1 gene, while expression of YKL40 and CD44 gene (mesenchyme and astrocytic markers) corresponds to mesenchymal subtype signatures. The neural subtype, however, does not possess any unique mutation like other subtypes but gene expression profile is quite similar to non-tumorous brain33. Of the four subtypes, patients with proneural subtype has better prognosis while mesenchymal subtype has worst prognosis 33,34. There are

a

b

EGFR ERBB2 PDGFRA MET

RASNF1

AKT

FOXO

PTEN

MDM4

TP53

MDM2

RB1

CDK4 CDK6CCND2

RTK/RAS/PI(3)Ksignalling altered

in 88%

p53signallingalteredin 87%

RBsignallingalteredin 78%

CDKN2CCDKN2BCDKN2A(P16/INK4A)

Homozygous deletion,mutation in 52%

Homozygousdeletion in 47%

Homozygousdeletion in 2%

Amplificationin 2%

Amplificationin 1%

Amplificationin 18%

G1/S progression

Activated oncogenes

ApoptosisSenescence

CDKN2A(ARF)

Mutation, homozygousdeletion in 35%

Homozygous deletion,mutation in 49%

Amplification in 7%

Amplification in 14%

Homozygous deletion,mutation in 11%

Amplification in 2%

Mutation in 1%

Mutation in 2% Mutation in 15%

Mutation, homozygousdeletion in 36%

Amplificationin 4%

Amplificationin 13%

Mutationin 8%

Mutation, amplificationin 45%

PI(3)KMutation, homozygousdeletion in 18%

Proliferationsurvival

translation

c

18

some signs that chemotherapy and radiation benefits patient with mesen-chymal and classical subtype, which is not observed in proneural subtype patients, who have similar survival period even with or without treatment33.

In addition to GBM subtypes based on gene expression data, TCGA DNA methylation data has revealed new insights of GBM biology. The TCGA GBM cases (n=272) displayed three clusters based on difference in methyla-tion status. Cluster 1 consists of samples having hyper methylation on CpG island (G-CIMP+ 8.8%). Cluster 2 and 3 is classified based on DNA meth-ylation status and no methylation on CpG island (G-CIMP-) respectively. By visualizing G-CIMP+ and G-CIMP- groups under the lights of gene expres-sion based subtypes, 87.5% of the G-CIMP+ cases fall under PN subtype which accounts for 30% of overall PN GBM cases. The pairwise gene ex-pression comparison of PN G-CIMP+ cases with PN G- CIMP-, CL, MS, and NL subtypes displayed significantly poor correlation. By this way indi-viduality of PN G-CIMP+ as a new subtype is verified. The stratification of survival period of patients showed, PN G-CIMP+ patients lived longer (me-dian survival = 150 weeks) than PN G-CIMP- (median survival = 42 weeks) 35. It is worth noting that PN G-CIMP+ patients are diagnosed at younger age than PN GCIMP- (p<0.0001) 35. To some degree, it appears possible to model GBM subtypes in the mouse. For instance, overexpression of PDGFA in Nestin/tv-a mouse background is sufficient for producing glioma, with a gene signature similar to human PN GBM. Also, a molecular subtype shift of PN GBM cell line into MS or CL has been observed when knocking down the NF1 gene36.

The histone 3 Family 3A (H3F3A) gene encodes the H3.3 protein, a key nuclear protein involved in epigenetic regulation of gene expression. Muta-tion in H3F3A gene has been reported in different types of cancer including adult and pediatric glioma, chronoblastoma and bone giant cell tumor37–39. In GBM, mutation of H3F3A is associated to observable changes in the epige-netic profile. Clustering of global methylation level in 210 adult and pediat-ric GBM patients displayed six new epigenetics based subtypes driven by IDH1 mutation, H3F3A K27 mutation (K27), H3F3A G34 mutation (G34), mutation in Receptor Tyrosine Kinase I (RTK I), mutation in RTK II and mutation in mesenchymal subtype genes (Figure 4).

The G34 subclass exhibits hypo methylated chromosomal ends suggesting a possible mechanism for tumor cells to extend the length of telomeres, there-by escaping replicative senascance39.

Taken together, the different types of classification will enable the identifi-cation of molecular differences between patients, which in turn could facili-tate the design of targeted treatment strategies40.

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Figure 4: Molecular subtypes of GBM based on transcript and methylation level with its hallmark signatures. Reprinted with permission39

Important GBM signaling pathways The p53, pRB, PI3K-Akt, and receptor tyrosine kinase (RTK) pathways are critical regulators of cell homeostasis, and are found to be affected by muta-tions in GBM33. TP53, which encodes p53 protein, is a well-known tumor suppressor gene41. The non-mutant form of p53 can inhibit tumor cell prolif-eration through many mechanisms: induction of cell cycle arrest by activat-ing p21 protein, induction of apoptosis, and activation of DNA repair path-ways42. In GBM, one or several of these crucial mechanisms are disrupted due to TP53 mutation. Clinically, the mutation rate of TP53 has been shown to be 54% and 32% in PN and MS subtype respectively33. Retinoblastoma protein (pRB) is another crucial tumor suppressor protein commonly mutated in GBM patients43. During G1 phase of the cell cycle,

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pRB is phosphorylated by the cyclin D-CDK4/6 complex leading to inacti-vation of pRB and release, activation of E2F-1 bound to it. E2F-1 belonging to E2F family plays a major role in transition of a cell from G1 phase to S phase. In GBM, downregulation of pRb disrupts cell cycle regulation and increases G1 to S phase transition through E2F1 and several other pathways. pRb in GBM has been shown to be downregulated by various mechanisms, such as loss of Chromosome 13q, loss of function mutation in the RB gene, mutation in cyclin-dependent kinase inhibitor 2B (CDKN2B) gene (inhibitor of CDKs) and amplification of cyclin-dependent kinases (CDKs)44–46.

Figure 5: Schematic representation of therapeutic targets and its drugs against cancer. Reprinted with permission47 The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-Akt pathway is regulated by a tumor suppressor protein called phosphatase and tensin ana-log (PTEN). PTEN is involved in cell proliferation, differentiation, and mi-gration3249. In general, stimulation of PI3K pathway is triggered by activated RTKs. Stimulated PI3K (class IA PI3K) converts phosphatidylinositol (4,5)-bisphosphate to trisphosphate (PIP2 to PIP3), which is negatively regulated by PTEN. Further, PIP3 activates Akt–mTOR pathway, which leads to cell proliferation and survival. In the tumor cells, the PI3K-Akt pathway is dysregulated facilitating uncontrolled cell proliferation and migration. In GBM, this pathway is malfunctioned due to loss of PTEN, mutation in phos-phatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA, catalytic subunit of class IA PI3K), mutation in Phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1, regulatory subunit of class IA PI3K), wild-type EGFR am-plification (one of the RTKs), and mutation in EGFR (EGFRvIII mutant). These genetic changes induce aberrant activation of PI3K-Akt pathway lead-

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ing to GBM tumorigenicity. Hence, identifying and targeting potential up-stream and downstream regulators of these pathways could be one of the possible ways to suppress GBM49.

RTKs such as EGFR and PDGFR is reported to be functionally disrupted in 45% and 13% respectively of 251 GBM cases studied, which makes them as a potential therapeutic target against GBM32. Some of the existing drugs targeting RTKs are gefitinib, lapatinib, bevacizumab, sorafenib, imatinib and vatalanib44,47,50 (Figure 5).

GBM disease models To decipher the pathophysiology of GBM and to identify new therapeutic targets, we need an appropriate disease model, which should resemble GBM in several important regards. Available modes to study GBM are broadly grouped into in-vitro, in-vivo, and ex-vivo models.

Cell cultures are an important tool in all forms of tumor biology51,52. In GBM research, the use of GBM cell lines, grown in serum-supplemented conditions, has been a major in-vitro research model until the late 2000’s. In recent decades, however, patient-derived GBM cell cultures (GC) grown in defined medium are preferred over traditional serum-supplemented cell cul-tures52–54. One of the major concerns is how well the serum-supplemented cultures represent its parental tumor?55–59.There are reports shown that the serum induce differentiation of GBM cultures,60,61 which ultimately losses its credibility of cancer stem-like cells. In the new era to overcome these problems, GCs are maintained in defined growth factors media (EGF and FGF growth factors as supplements) instead of serum52,54,62. It consists of heterogeneous mixtures of cell population such as a pool of GBM cancer stem-like cells (reason for treatment resistance and tumor relapse) and a bulk tumor cells53. The GC can be cultured as both neuro-sphere suspension cul-tures and laminin coating dependent adherent cultures in a serum free de-fined media. These adherent defined-medium cultures better approximate the in vivo transcriptional profile of human GBM tumor samples52 until certain passages and also shows a high capacity to initiate GBM tumors in xenograft mouse models as similar to human GBM63. Hence, GC are used as a major in-vitro disease model from basic functional studies to high-throughput compound and genetic screens64,65. Recently, we have established an open-source GC biobank called human glioblastoma cell cultures (HGCC) which consists of 48 genomically well-characterized GC lines63.

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The main caution that we should be aware of while using cell lines is, its authenticity (match with parental source). For instance, our lab showed that the U87MG cell line (widely used serum-supplemented GBM cell culture), established at Uppsala University66 in 1966, does not match with the original tissue source, and it is derived from a different individual than the U87MG cell culture source found in standard repositories such as ATCC 67,68. GBM rodent models have been explored from late 1940s onwards 69,70. The first type of model was based on chemically induced (via N-nitroso com-pounds) brain tumors in rats71,72. Today, the main focus is on genetically engineered mouse models (GEMM), in which GBM like brain tumors are induced by manipulations of signaling pathways that are commonly mutated in GBM such as TP53, RTK, PI3K-AKT and pRB73,74. Different types of GEMM are available to model GBM such PDGFB-driven glioma tumors75,76 (using virus), PDGF- driven glioma tumors using RCAS/tv-a technology to induce tumors at different cell of origin77,78 (Nestin/tv-a, GFAP/tv-a, CNP/tv-a background), NF1 models79,80, and EGFR models81. It has been shown that mouse models engineered to cause somatic mutations in Nf1, p53, and Pten genes produced human like GBM tumors74. Patient-derived tumor xenografts (PDXs) and patient-derived cell lines or-thotopically injected to immunodeficient mice are the other in-vivo options to study GBM. It has been shown that tumors generated by these mouse models are histologically and genetically similar to patient GBM63,82–84. The use of GBM mouse models is complemented with imaging techniques. One example is CLARITY is a recently developed method that clarifies lipid bilayers of a biological tissue and replaced by hydrogel-based compounds. By using this method we can clarify entire adult mouse brain to study visual-ly any interactions in the brain by using fluorescent microscope85–87. This technique may be used to investigate for instance tumor vasculature before and after treatment with angiogenesis inhibitors etc. Organoid culture is a three-dimensional ex-vivo culturing technique in which cells grow inside matrigel support88. Recently, such models were established for GBM using patient-derived cells. These organoids capitulate GBM stem cell properties, in-vivo tumor initiation and it mirror genetic composition of parental tumor. One of the benefits of this method is, the possibility to create organoids from tumor cells isolated from brain metastasis biopsies originat-ed from different tumor otherwise difficult to grow as cell culture. By using this model, we can study in-vivo mimic condition of tumor microenviron-ment89.

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Organotypic brain slice model is another ex-vivo model used to conduct invasion related studies90. In a recent report, this method is successfully em-ployed by seeding GCs (1x105 cells) on top of brain slice (corpus callosum) to study invasion capacity of GCs after the drug treatment. After a week, the brain slice was fixed and studied for relevant markers91. To summarize, we have range of advanced in-vitro, in-vivo, and ex-vivo disease models to perform high resolution GBM research. In this thesis, we have used GCs (in-vitro) and GCs orthotopically injected into immunodefi-cient mice (in-vivo) as a disease models to study GBM.

Clinical trials Translational studies aimed at new therapies typically progress from preclin-ical investigations to clinical trials in a structured manner. In preclinical drug development, a targeting mechanism or drug of interest is studied in appro-priate in-vitro and (non-human) in-vivo models to define its functional mechanism, efficacy and toxicity. The drugs that perform well are consid-ered as candidates to enter into clinical trial in human subjects 92.

Clinical trials are divided into different phases (phase 0, I, II, III, and IV). In phase 0, a very low suboptimal dose of drug will be administered to a very small group of patients to study drug tolerance by the human body, drug reachability to the tumor spot, and initial tumor cell response. In some cases, phase 0 can be skipped and a drug will directly enter into phase I. In phase I, a group of patients will be recruited to define a range of doses that can be safely administered in patients. This is typically achieved by escalating dos-es in an ascending order to every next group of subjects. Side affects, biolog-ical half-life of the drug and food effects will also be monitored. A phase II trial aims to investigate the efficacy, pharmacokinetics and pharmacodynam-ics of the drug at an optimal therapeutic dose in a larger cohort of patients (more than phase I). Data from this study will give more insights about im-portant biological aspects of the drug. Notably, a large percentage of thera-pies failed to pass phase II. In phase III, the effectiveness of the drug will be interrogated along with efficacy of the drug. The treatment outcome from the trial will be compared to the most efficient available medical treatment. Phase III trials are mostly performed in a randomized manner in a much larger cohort of patients (than phase II) and typically run over many years. The successful drug from phase III trial will get license approval for com-mercial production and treating patients. In last or phase IV trial, the li-censed therapy will be monitored for long-term beneficial and adverse ef-fects in the patients undertaking the treatment. If any life threatening side

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effects are being found in this phase, the drug in question will receive a ban or restricted application92,93.

The scientific community is conducting many clinical trials to improve ther-apeutic treatments against GBM by utilizing different biological strategies such as immunotherapy, combination therapy, antibody-drug conjugated therapy (ADCs)94 and targeted therapy etc. Below, a selected set of recent examples are discussed: Adoptive immunotherapy is a technology based on modified chimeric anti-gen receptor T cells (CAR-T). Under the commercial name KYMRIAH, CAR-T cells targeting CD19 for B-cell precursors have been used as treat-ment for acute lymphoblastic leukemia (ALL) patients with less than 25 years of age. It has been shown that 83% of B-cell ALL patients treated with Kymriah was remission from the disease95,96. Adapting this concept as a possible strategy against GBM, efforts are underway to exploit CAR-T tech-nology against EGFRvIII, a mutant form of EGFR (RTK) diagnosed in 20-30% of GBM cases97. EGFRvIII lacks exon 2 to 7 and is continuously ac-tive, thus promoting cancer cell proliferation and survival98. A phase I clini-cal trial has been conducted to test the safety assessment of CAR-T cell di-rected against EGFRvIII mutant in a total of 10 patients positive for EG-FRvIII. The results indicate tolerance to the therapy and that CAR-T cells reached the antigen specific tumor cells 99. Another immunologically based approach; Rindopepimut (a cancer vaccine) was tested in phase II clinical trial to target EGFRvIII-positive GBM. But the study was terminated since there was no difference in OS compared to the control group100. Antibody-Drug Conjugates (ADCs) is another interesting methodology to target GBM cells. An ADC involves a cytotoxic drug conjugated to a mono-clonal antibody (mAb) with a particular specificity 94. Clinical trials has been reported in GBM involve AMG 595, built from anti-EGFRvIII conjugated to maytansinoid DM1 (a cytotoxic drug). The phase I/II clinical trial results shown that the patients tolerate ADC and noticed treatment response in two out of 24 patients101. A different conjugate, ABT-414, is a conjugation of mAb against EGFR amplification (ABT-806) with monomethyl auristatin F. A Single-photon emission computed tomography images from Phase I trial confirmed that the ABT-414 was well targeted the EGFR amplified cells only102.

Recently a phase III clinical trial for treating GBM with TTFs in adjuvant with standard of care treatment was approved. The treatment increased OS and progression free survival of 4.9 months and 3.2 months respectively compared to patients only received current treatment regimens. The TTFs

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are produced by a device called optune (head wearable device), which will slow down the mitosis of GBM cells, as a final outcome programmed cell death will happen26. Marizomib is a second-generation irreversible proteasome inhibitor that has shown promising GBM preclinical results in both in-vitro and in-vivo. This drug is able to penetrate blood brain barrier compare to its predecessor bortezomib and carfilzomib103,104. In an ongoing phase 1 trial, therapeutic relevant dose of marizomib is being evaluated as a single agent or in combi-nation with bevacizumab (angiogenesis inhibitor)105. In addition to this, there is a new phase 1b clinical trial going to be launched to study marizo-mib in combination with TMZ and optune in 12 GBM patients. The trial is planning to purse in third quarter of 2017106. It is worth mentioning that in one of our studies, we noticed that GC cells are both sensitive (nM concen-tration) and resistant (uM concentration) to proteasome inhibitors.

GBM therapeutic challenges Despite the several advances discussed above, the development of GBM therapies still faces many hurdles. This section discusses some of the key obstacles ahead, including recurrence, intra-tumor cell heterogeneity, lack of good targets, and the blood brain barrier. Patients who undergo treatment for GBM typically relapse in their disease. The relapse is grown from a residual population of cancer cells that are re-sistant to the original treatment. Generally, patients with complete tumor resection live longer (median survival=15.2 months) than patients with a less than complete resection (median survival=9.8 months)107. In most of the cases, the reason behind less than complete resection is due to location of tumor in sensitive part of the brain108. This indicates that it is generally not possible to completely remove a GBM tumor. This is problematic, since a recurrent GBM does not respond well to TMZ and there is thus a lack of options 109. To a degree, drug resistance is mediated by drug resistance genes such as multi drug resistance related protein (MRP1), and multi drug re-sistance protein (MDR1) in cancer stem cells armor them to develop re-sistance against the treatment110. It is also quite possible that subclones pre-sent from the primary tumor dominate the recurrent tumor. Both GBMs and GC cultures have been shown to exhibit intra and inter genomic heterogenei-ty63,111, with observable consequences at the level of drug responses, in-vivo tumor initiation capacity and in-vitro cell culture properties112,113. It has been reported that when analyzing tumor biopsies isolated from different sites of the tumor from a same patient displayed different molecular subtypes111.

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This intra-tumor heterogeneity is one of the major problems for poor treat-ment outcomes. This motivates attempts to map the functional and therapeu-tically relevant heterogeneity of GBM, which is one of the topics pursued in this thesis. Such stratification must go hand in hand with systematic analysis of data from GBM and other cancers. The analysis of cancer networks and cancer databases enabled by several tools such as cancerlandscapes.org114, L1000 database, hgcc.se63, oncoscape, NCI-ALMANAC115 is one compo-nent of this discovery effort.

Figure 6: Complications in delivering drugs to brain parenchyma. A) Representa-tion of intact blood brain barrier in a normal scenario. B) Drugs passing disrupted BBB in brain tumor patients C) High uptake of drugs by brain tumor cells when employing Convection-enhanced delivery method. Reprinted with permission94

The blood-brain barrier (BBB) is a term that refers to the specialized organi-zation of endothelial cells, pericytes and astrocytes, in the brain vasculature. The BBB is, generally, difficult to penetrate by molecules larger than 400 Da94 and the brain vasculature also has active transport processes to void the brain of toxic chemicals. Accordingly, passing the BBB is the major hurdle that many drugs may fail to cross even it exhibits excellent preclinical effi-cacy. In Figure 6, two possibilities of drug penetrance through BBB to brain parenchyma is showed (6b): in case of tumor, the BBB is compromised to some extent, which then be utilized by the drug to reach tumor cells. Even after reaching brain parenchyma, overexpression of MDR genes and efflux pumps on tumor cells expel the drugs out. (6c) Convection-enhanced deliv-ery (CED) is an invasive catheter based infusion of drug to the tumor area. This is one of the promising methods to deliver drugs in brain parenchyma comes with its own limitation such as need of highly skilled surgical exper-tise, and catheter-related surgical complications94,116.

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ZBTB16/PLZF/ZNF145 Zinc finger and BTB domain-containing protein 16 (ZBTB16), also known as promyelocytic leukemia zinc finger protein (PLZF), is a transcription factor located on chromosome 11q23. The ZBTB16 protein contains three functional domains: a BTB-POZ domain, an RD2 domain and a zinc finger domain (9 zinc finger motifs). The zinc finger domain of the PLZF mediates DNA binding, protein interaction and nuclear-cytoplasmic shuttling117 (Fig-ure 7).

Figure 7: The pictorial representation of ZBTB16 protein and functional role of each of its domain. Reprinted with permission118

ZBTB16 has been reported to be both an oncogene and a tumor suppressor gene, depending on the biological context118. It was first identified as a fu-sion protein along with retinoic acid receptor alpha protein (RARα) in acute promyelocytic leukemia (APML) patients119. The reciprocal chromosomal translocation of ZBTB16 t(11;17)(q23;21) results in two variants of fusion protein i)PLZF-RARα, ii) RARα-PLZF in APML118,119 PLZF-RARα behaves as an oncogene and acts as a transcriptional repressor of tumor suppressor genes in APML and promotes leukemia by blocking myeloid cell differentiation. The tumor express PLZF-RARα shows poor response to all-trans retinoic acid treatment. Interestingly, however, the sen-sitivity of retinoic acid treatment is restored in a mouse model expressing PLZF-RARα protein by treating it with histone deacetylase (HDAC) inhibi-tors 120,121.

In clear renal cell carcinoma, ZBTB16 expression has shown to be abundant-ly overexpressed and reported as an oncogene. In a flank xenograft mouse model of renal cell carcinoma, ZBTB16 overexpression increased tumor burden. It has been shown to regulate tumor growth by transcriptionally repressing the expression of CDKN1A (translate into P21 protein) and TP53122. By contrast, in prostate cancer and NSCLC (non-small cell lung cancer), ZBTB16 is reported as a tumor suppressor gene123–125 . Downregula-tion of ZBTB16 in prostate cancer has shown to be caused by direct interac-

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tion of ZBTB16 with Kallikrein-related peptidase 4 (KLK4 an oncoprotein). Knocking down KLK4 is shown to increase the expression of ZBTB16 and inhibits tumor growth125.

Figure 8: Transduction cascade of ZBTB16. Focusing on GBM, ZBTB16 (PLZF) is highly expressed in GBM compared to neural stem cells (NSC) (Oncomine database) and human astrocytes. To exhibit the transcription factor activity, ZBTB16 has to be transported to the nucleus. This transport is reported to depend on activation of the ren-in/prorenin receptor126. Translocated ZBTB16, in turn, binds to PIK3R1 promoter region126 and promotes the gene expression, which is necessary for PI3K/AKT pathway activation and thereby to maintain cell proliferation and cell survival (Figure 8). This translocation process has shown to be inhibited by genistein drug and renin inhibitors127,128. It has also been shown that the treatment of renin inhibitors induce apoptosis and inhibits the growth of GBM cells129. Based on all this information, it is interesting to perform per-turbation studies of ZBTB16 in valid experimental models to explore its role (e.g. transcription factor, PI3K/AKT and TP53 pathway) in GBM.

P85 P110P

PIP3PTEN

PIP2

Akt

P53

P21

MDM2ZBTB16

ZBTB16

P21 PIK3R1ATP6AP2

x x

Renin

Pror

enin

rece

ptor R

TK

Ligand

Cell cycle/proliferation

Genistein drug

Aliskiren drug

(P85 alpha)

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Aims

This thesis aims to identify new molecular therapies against GBM by using most clinically relevant GBM models. In order to achieve our aim, we per-formed four individual studies: Article I: Studying the synergistic treatment effects of pterostilbene with gefitinib or sertraline in patient-derived GBM cell lines. Article II: High throughput screening of FDA approved pharmacological compounds in a larger cohort of genomically well-characterized patient-derived GBM cell lines to establish a novel computational drug prediction model for new GBM cases. Article III: To identify novel gene targets against GBM by performing a large scale screening of gene targets and elucidating its functional mecha-nism in a panel of genomically well-characterized patient-derived GBM cell lines. Article IV: To map the genomic stability changes induced by cell culture passaging of clinically relevant patient-derived GBM cell lines.

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Present Investigations

Article I: Case-specific potentiation of glioblastoma drugs by pterostilbene

Aim To investigate the synergism treatment effect of pterostilbene (P) in combi-nation with gefitinib (G) or sertraline (S) on patient-derived GBM cell lines (GC). Methods In previous work, our lab had found that the three compounds pterostilbene, gefitinib and sertraline functionally interact to suppress GBM cells. In this study, we aimed to understand this interaction and its variability in patient-derived GBM cells. GC cultures were treated with P, S and G individually and also combinations of PG and PS for 72 hrs. After 72 hrs, we measured cell viability estimated functional interaction scores. Following a dose-finding experiment, the identified most efficient concentration of drug com-binations (PG and PS) were validated with respect to their effect in GC phe-notypes including proliferation (EdU assay), sphere formation, migration (Trans-well assay) and cell cycle in GCs. In order to test the efficiency of drug combinations in a larger panel of GCs, we performed cell viability as-say after treating 41 GC lines with PG and PS (11 dose series concentration). The predictive biomarker for PS combination was identified by correlating 41 GCs drug response data with their gene expression data (untreated GCs). RNA sequencing and protein expression assay (NanoPro 1000) was per-formed to identify differential gene and protein expression induced by treatment. Major findings Pterostilbene at 20uM in combination with gefitinib (10uM) or sertraline (7uM) suppressed cell viability, migration, proliferation and sphere forming ability of tested GC lines. RNF11 transcript and its DNA copy number level were identified as a predictor biomarker of PS response in GC. Pterostilbene potentiates gefitinib and sertraline in GCs by cell cycle arrest and suppres-sion of the MAPK pathway. Thioredoxin interacting protein (TXNIP) is a tumor suppressor gene and mediator of ROS responses was consistently

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upregulated after pterostilbene treatment. We found that downregulation of TXNIP by gene silencing and NAC treatment abrogated the effect of pterostilbene in GC. Taken together, the study deepens our understanding of pterostilbene as a possible potentiating compound in GBM, and also sup-ports the idea of case-specific prediction of drug synergism.

Article II: Targeting tumor heterogeneity: multi-omic modeling of glioblastoma drug response using an open-access library of patient-derived cells Aim: To develop a novel computational drug prediction model for GBM by utiliz-ing high throughput drug vulnerabilities and genomics data from 106 IDH1 wildtype glioblastoma cases. Methods How predictable are drug effects in GBM cells, and do differences in drug response overlap with known classification systems? To address these ques-tions, we performed a sequence of drug screening experiments using a panel of up to 1544 FDA approved pharmaceutical compounds across several doz-ens of patient-derived GBM cell lines (n=106) to generate unique drug vul-nerability data. We performed DNA copy number array, methylation array, gene expression array and DNA sequencing to create genomics data of GCs. A selected set of GC cultures (n=10) were further studied to understand the different molecular mechanism induced by the proteasome inhibitors using PEA (protein extension assay), WB, ICC, apoptosis assay, ROS assay, pro-teasome assay, aggresome assay and flow cytometry. Immunodeficient or-thotopic xenograft mouse models were used to test in-vivo efficacy of the proteasome inhibitors. By utilizing generated drug and genomics data, we predict the sensitivity of a given compound using two machine learning techniques elnet and random forest. Major findings An integrated analysis showed that the GC cultures stratified in a manner that is distinct from current classification systems. In particular, from the large-scale drug screening data, we identified that almost equal number of patient-derived GBM lines were sensitive (nM concentration) and resistant (uM concentration) to proteasome inhibitors (PI). In both sensitive and re-sistant GCs, proteasomes were inhibited followed by PI treatment, which avoided questioning the functional potential of the PI. We found that basal proteasomal level was directly correlated with GC cell death after PI treat-

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ment. We also identified high levels of ubiquitin tagged proteins in sensitive GC cells after bortezomib treatment. This distinct phenotypic drug response clustering was molecularly explained by p53 and p21 functional loss in re-sistant GCs, and retained p21 function in sensitive GCs. The bortezomib treatment induced apoptosis in sensitive GC cells in response to high level of oxidative stress in the cells. Under in-vivo condition, tumor generated by PI sensitive GC lines responded well to the bortezomib treatment and vice ver-sa. We observed an individual combination of tylosin, spectinomycin, and cilnidipine with bortezomib produced high synergy toxicity response in GCs. Finally, we created a novel computational drug prediction model to predict sensitivity of the drug for given GC by combining our drug response and genomics data. The study shows that the pharmacological classification of GBM is distinct from current subtyping systems, introduces PI sensitivity as a key characteristic of GBM cells.

Article III: Loss of transcription factor ZBTB16 induces cell death in patient-derived GBM cell lines Aim To identify a novel gene target against glioblastoma. Methods We performed a systematic screening of gene targets by loss of function method (siRNA library consists of siRNA against 1112 genes) in patient-derived GBM cells (GCs) to identify essential genes needed for the survival of GCs. In the follow up work, selected candidate genes were studied to assess effects on GC cell viability (Alamar blue assay), proliferation (x-celligence), invasion (collagen invasion assay), and migration (IncuCyte). The knockdown treatment induced molecular changes were investigated by techniques such as WB and qPCR. Lentivirus based stable over-expression of identified gene targets was transduced into a GC. By use of this model, we investigated the physical and molecular properties of the transduced GC by proliferation assay and gene expression array respectively. Major findings We found that the gene ZBTB16/PLZF was essential for GC cell survival. Loss of ZBTB16 inhibits GC cell viability, proliferation, migration, and in-vasion. The treatment induced cell death was due to down regulation of c-MYC and Cyclin B1. Genistein, recently described as a ZBTB16 nuclear translocation inhibitor induced GC cell death at therapeutically relevant dos-es. As expected, over expression of ZBTB16 increased GC proliferation and

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viability. The study thus introduces ZBTB16/PLZF as a new and potentially druggable growth regulator in GBM.

Article IV: Primary glioblastoma cells for precision medicine: a quantitative portrait of genomic (in)stability during the first 30 passages

Aim To investigate the genomic stability, new sub clones expansion and ploidy status of patient derived GBM cell lines (GCs) upon cell culture passaging. Methods We selected a panel of three GC lines at very low passage (p6) and continu-ously passaged them until p30. During passaging at regular intervals we isolated DNA and RNA from GCs to observe any changes induced by cell culture passaging using DNA copy number arrays, Affymetrix gene expres-sion arrays, and DNA methylation arrays. Combining state-of-the-art signal processing methods with a mathematical model of growth, we estimated clonal composition, change of growth rate, affected pathways and correla-tions between altered gene dosage and transcription. Ploidy status of the GCs was analyzed by using fluorescent in-situ hybridization (FISH) and flow cytometry. Major findings All three GC cultures displayed clonal evolution, in which populations with previously undetected DNA copy number alterations completely replaced the original cell population between 7th-25th passages. Individual clones significantly differ by their cell cycle rates, ranging from 41 to 121 hours per doubling. Variable amount of aneuploid cell population were present in all three GC cultures at different passages, which may reflect pseudo-sub clones generated through continuous aberrant cell divisions. As passage increased, we noticed a molecular subtype switch in two GCs. The GC cultures also show significant transcriptional drift in several metabolic and signaling pathways, including ribosomal synthesis, telomere packaging and signaling via the MTOR, WNT and interferon pathways, to a high degree explained by changes in gene dosage. Compared to chromosomal aberrations and gene expression, DNA methylations remained comparatively stable during pas-saging, and may be favorable as a biomarker. Taken together, GC cultures undergo large genomic and transcriptional changes that need to be consid-ered in functional experiments and biomarker studies that involve primary glioblastoma cells.

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Discussion & Future Perspectives

Despite decades of research by many teams, GBM continues to pose a major health problem. The incidence rate of GBM is 3.2/100,00017 and with the current treatment strategy average survival period of patients is between 12 to 15 months19. Hence, identification of new therapeutic gene targets and compounds are highly required to improve current treatment protocols. In this thesis, we have used patient-derived glioblastoma cell cultures52,54 (GCs) as a model and in-vitro research tool to explore therapeutic opportu-nities. While all models have limitations in terms of validity and impreci-sion, patient-derived GC cultures are likely a major step forward compared to traditional cell cultures and better approximates the in-vivo transcriptional profile of GBM tumor samples and initiates tumor in xenograft mouse mod-els63. A large material of GC has been used in some of the studies in this thesis also demonstrates that GC cultures are an interesting model of GBM diversity. During last decades, The research field is rapidly advanced in understanding GBM by molecularly classified them into subtypes (proneural, neural, clas-sical and mesenchymal, G-CIMP +ve, G-CIMP –ve) based on RNA33 and methylation data35. But, the clinical significance of this subtype still remains as a big question. Our results do not strengthen the idea that current systems are useful for therapy stratification, but underline the possibility of pharma-cological classifications, that may vary significantly across classes of drugs. Our novel computational drug prediction model indicates that it is possible in principle to predict a drug response for a given GC. The drug response of GC was predicted well by optimally selected markers than molecular sub-type assignment. The long term clinical relevance of the predicted biomarker therapy, if any, remains to be demonstrated in prospective clinical studies. The broad scope of study II clearly offers many venues of continued investi-gation. For instance, the experimental follow up in the paper thus far focuses on proteasome inhibition, which is one of the seven reported pharmacologi-cal clusters. Understanding the biology behind each of the clusters, consider-ing combinations amongst the studied compounds and proving the in-vivo relevance of the biomarkers are all important goals ahead. Compounds with

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significant differences between GC cultures, where I am currently doing work include a natural compound (romidepsin) that is believed to be a HDAC inhibitor, and two spindle-poisons (vinca alkaloids vinorelbine tar-trate and vinblastine). I would like to explore these drugs in a panel of GC cultures. At the moment, we are developing GBM patient xenograft mouse models, which will be used to test the efficacy of new drugs under in-vivo environment.

Figure 9: (A) ZBTB16 immunoprecipitation results of U3065MG. The optimal con-dition for ChIP protocol and ZBTB16 antibody pulling down efficiency was opti-mized after testing different concentration of formaldehyde, SDS and sonication cycles (PFA/ SDS/ Sonication cycles). (B, C) The bioanalyzer verification of sonicated ChIP DNA pulled down by using ZBTB16 antibody.

In study III, an important extension is to understand how the transcription factor ZBTB16 exerts its function in GBM cells. To explore this, and to bet-ter understand the functional roles of ZBTB16, we are currently performing ChIP-sequencing experiment to identify the (likely many) binding sites of ZBTB16 on the DNA. To purse ChIP-sequencing, we have validated the most crucial steps such as sonication and antibody efficiency (Figure 9). Based on the optimization results, we will use sample 3 condition for preparing ZBTB16 ChIP DNA for sequencing (HiSeq2500 Rapid mode, 50bp paired end sequencing). We have performed gene expression arrays to study the changes in expression of other genes altered by ZBTB16 KD (n=5 GCs) and ZBTB16 over expression (n=1 GC). At the moment bioinformatics analysis of data is ongoing (Figure 10) and we are hoping to identify GBM relevant differentially expressed genes induced by the treatment. We also intend to treat the GCs with renin inhibitors and HDAC inhibitors. These drugs interfere with translocation and transcription factor activity of ZBTB16. Based on the literature studies, we are planning to study the ex-

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pression of P21, PIK3R1, Akt proteins by western blot method after KD of ZBTB16 as these proteins plays a major role in GBM tumorigenicity.

Figure 10: Initial bioinformatics analysis of gene expression data from three GC. The color gradient blue to red denotes deferential expression of genes from down-regulation to upregulation respectively.

Currently, we are also validating an in-vivo tumor initiation capacity of ZBTB16 by orthotopically (right hemisphere stratium) injecting ZBTB16 over-expressing cells in female NMRI nude mice. We have used U3053MG for this study since we already know that this particular GC does not produce tumor in-vivo. We have injected U3053MG control cells in 10 mice and U3053MG ZBTB16 O.E cells in 10 mice. Out of 10 mice injected with U3053MG ZBTB16 O.E cells, two mice were sacrifised after eight and 16 weeks of injection due to neurological symptoms. Other mice will be monitered until it shows neurological symptoms or up to 40 weeks from the date of injection.

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38

Acknowledgements

First of all, I would like to wholeheartedly thank all the Patients who have agreed to provide the invaluable tumor biopsy for scientific research. I would like to wholeheartedly thank Government of Sweden for providing us a world-class education and international friendly government laws. I would like to thank all the Funding Agencies who have supported my research projects. I would like to thank my supervisor Dr.Sven Nelander for giving me an opportunity to acquire all the skills needed to become a successful research-er. Thanks a lot for your excellent scientific guidance, giving me lots of freedom to pursue my own ideas and molded me as an independent re-searcher. I have always admired your presentation skills and scientific writ-ing! I would like to thank my co-supervisor Dr.Karin Forsberg Nilsson for your efficient scientific discussions and suggestions about my projects even dur-ing your busy administration schedule. I would like to thank Dr.Grzegorz Wicher for selected me as a project stu-dent during my Masters program. I would say that's the turning point in my research career. You are such a student-friendly supervisor and I learned a lot from you. I would like to thank “Biochemical and Cellular Assay facility” located in Stockholm and “Uppsala array and analysis facility” for your tremendous support to pursue my project aims.

I would like to thank all the IGP administration people especially to Chris-tina Magnusson and Heléne Norlin for your patience and unlimited admin-istration help from the beginning to end of my Ph.D. studies. I would like to thank all the Neuro-Oncology members for providing such a friendly research environment and all the NO N.O kickoffs.

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I would like to thank Markus Mayrhofer, Anders Isaksson, Voichita Marinescu (Thanks a lot for providing me an opportunity to work in hetero-geneity project), Antonia Kalushkova, Alba Atienza Párraga, Argyris Spy-rou (keep up your smile), Mohanraj Ramachandran for wonderful collabo-ration projects and your support. I would like to thank all my group members: Ludmilla Elfineh (always young and thank you very much for helping me a lot in my projects), Cecilia Krona (thanks a lot for your valuable scientific advice and critics. I always cherish your family presence during my marriage), Patrik Johansson (you are my best friend and wonderful companion during my Ph.D. life, Hope it continues!), Linnea Schmidt (thanks a lot for the collaboration), Elin Almstedt (most cheerful person in the lab and good luck to finish your Ph.D. ON TIME), Soumi Kundu (very very sweet person and thanks a lot for help-ing me with my projects whenever I needed you), Santhanam (thanks a lot for your help during the primary drug screen, good luck for your future) Evgenia Gubanova, Karl Holmberg Olausson (thanks for your valuable scientific and pathological insights), Emil Rosén (good luck with your Ph.D.), Caroline Wärn, Neda Hekmati (thanks for your contribution in my projects), Willemijm Brouwer (good luck for your future) for accompanying me during my Ph.D. journey. I would like to thank all my Indian friends with special mention to G.M. Karthik, R. Mohanraj, M.Vivekanand, S.Aravindh (Rendambathu group), and Darvesh for creating rememberable memories with me. I am truly blessed to have a wonderful Life partner, Amma, Appa, Sister and my new family members Mother-in-law, Father-in-law, Sister-in-law, and Brother-in-law. Thanks a lot for all your great support, believing in me, and your true INFINITE LOVE. Finally, I would like to thank soulfully and dedicate my work to my Maternal Grand Parents for your invaluable moral support and being with me throughout my life. Without you, I will not be able to attain these academic heights.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1389

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”.)

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