Tankyrase inhibition regulates MITF expression in melanoma

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Master’s Thesis 2021 60 ECTS

Faculty of Chemistry, Biotechnology, and Food Sciences (KBM)

Tankyrase inhibition regulates MITF expression in melanoma

Nann Louise Kristoffersen

Biotechnology – Molecular Biology

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Tankyrase inhibition regulates MITF expression in melanoma

Nann Louise Kristoffersen

Supervisors:

Dr. Jo Waaler

Ph.D. student Shoshy Alam Brinch Prof. Harald Carlsen

Master thesis

Faculty of Chemistry, Biotechnology and Food science

Norwegian University of Life Sciences

November 2021

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©Nann Louise Kristoffersen 2021

Tankyrase inhibition regulates MITF expression in melanoma https://nmbu.brage.unit.no

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Acknowledgment

The work in this thesis was carried out in collaboration with the unit for Cell Signaling and Drug Discovery, Department of Immunology and Transfusion Medicine - Oslo University Hospital and Hybrid Technology Hub, Center of Excellence – Oslo University Hospital, as part of a master’s degree in Biotechnology at the Norwegian University of Life Sciences (NMBU), the primary affiliation being the Faculty of Chemistry, Biotechnology and Food Science (KBM), from May 2020 to October 2021.

I wish to express my sincere appreciation to my primary supervisor Jo Waaler, who has given me the opportunity to participate in his research group. I want to acknowledge his encouragement, enthusiasm, and understanding when the road got tough. He has also guided me through the writing process.

I wish to express my gratitude to my co-supervisor, Shoshy Alam Brinch. I am thankful for her teaching and assistance in the laboratory, leaving me with highly improved laboratory skills. She has also provided feedback for my thesis. Additionally, I would like to thank my supervisor at NMBU, Harald Carlsen, for his support and advice.

I also want to thank all my colleagues and the collaborators for support, lab training, and communication. I especially want to acknowledge Enya Isaksen and Lone Holmen for their motivation, support, and helpful feedback. Thanks to Marlén Aurora Nerli and for proofreading my thesis.

Lastly, I would like to express my greatest appreciation to my family, especially my sister Lill Katrin Kristoffersen, and friends for always being there.

Oslo, November 2021

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Nann Louise Kristoffersen

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Abstract

Malignant melanoma is an aggressive form of skin cancer with a poor prognosis for patients with advanced disease. Standard care today has high rates of severe side effects and low success rates.

Currently, immunotherapy is revolutionizing how cancer is treated, yet only a subset of melanoma patients responds to immunotherapy. Melanoma, along with several other cancer types, shows WNT/β-catenin signaling-mediated immune evasion. The regulatory protein tankyrase impacts both WNT/β-catenin and YAP signaling, and by inhibiting tankyrase, we can suppress these oncogenic pathways. Recent studies have shown promising results in increasing the susceptibility for immunotherapy with combined therapy tankyrase and immune checkpoint inhibitor therapy. This study aims to investigate the mechanism behind the combined treatment effect in melanoma cells. During the investigation, we identified tankyrase inhibitor-mediated stabilization of the protein MITF. MITF is a master regulator in melanoma that affects immune cell migration, promotes phenotype switching, and controls antigen presentation. Altered antigen presentation can turn a checkpoint inhibitor-resistant “cold” tumor into a “hot” and responsive tumor. Through immunoblot, real-time RT-qPCR, bioinformatic, and immunofluorescence analyses, we have started to put MITF in context with WNT and YAP signaling to get a

comprehensive understanding of how MITF expression changes upon tankyrase inhibition. The data showed that in vitro treatment for 72 hours with G007-LK accumulated MITF protein in the nucleus. We show that the stabilization of MITF is independent of WNT/β-catenin signaling and that MITF expression is not regulated at the transcriptional level. The results form a basis for further research on a protein that may dictate sensitivity to checkpoint inhibitor treatment in melanoma.

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Sammendrag

Malignt melanom er en aggressiv form for hudkreft med dårlig prognose for pasienter med avansert sykdom. Standardbehandling i dag har høye forekomster av alvorlige bivirkninger og lave suksessrater. For tiden revolusjonerer immunterapi måten kreft behandles på, men bare en undergruppe av melanompasienter responderer på immunterapi. Melanom, sammen med flere andre krefttyper, viser WNT/β-catenin signalmediert immununnvikelse. Det regulatoriske proteinet tankyrase påvirker både WNT/β-catenin og YAP-signalering, og ved å hemme

tankyrase kan vi også hemme disse onkogene veiene. Nyere studier har vist lovende resultater for å øke mottakelighet for immunterapi med kombinert terapi med tankyrase og

immunsjekkpunkthemmer-terapi. Denne studien har som mål å undersøke mekanismen bak den kombinerte behandlingseffekten i melanomceller. Underveis i undersøkelsen identifiserte vi tankyrasehemmermediert stabilisering av proteinet MITF. MITF er en masterregulator innen melanom kjent for å påvirke immuncellemigrasjon, fremme fenotypebytte samt kontroll av antigenpresentasjon. Endret antigenpresentasjon kan gjøre en "kald" svulst som er resistent mot sjekkpunkthemmere til en "varm" og responderende svulst. Gjennom immunoblot, sanntids RT- qPCR, bioinformatisk og immunfluorescens analyser har vi begynt å sette MITF i sammenheng med WNT- og YAP-signalering for å få en helhetlig forståelse av hvordan MITF-uttrykk endres ved tankyrase-hemming. Dataene viste at in vitro behandling i 72 timer med G007-LK

akkumulerte MITF-protein i kjernen. Vi viser at stabiliseringen av MITF er uavhengig av WNT/β-catenin-signalering og at MITF-ekspresjon ikke er regulert på transkripsjonsnivå.

Resultatene danner grunnlag for videre forskning på et protein som kan diktere følsomhet for sjekkpunkthemmer behandling ved melanom.

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Abbreviations

α-MSH α-Melanocyte-stimulating hormone β-TrCP β-transducin repeat-containing protein

ABS Absorbance

ADP Adenosine diphosphate

AKT AKT serine/threonine kinase 1 ALX3 Aristaless-like homebox 3

AMOT Angiomotin

AMOTL1 Angiomotin-like 1 AMOTL2 Angiomotin-like 2 ANOVA Analysis of variance ARC Ankyrin repeat cluster APC Adenomatous polyposis coli APCs Antigen presenting cells

ATF4 Activating transcription factor 4 AXIN Axis inhibition protein

bHLH-Zip Basic helix-loop-helix leucine zipper BRAF B-Raf Proto-Oncogene

cAMP cyclic AMP response

CCN1 Communication network factor 1 CCN2 Communication network factor 2 CDH1/2 Cadherin 1 and 2

CDK2 Cyclin-dependent kinase 2

CDKN1A Cyclin Dependent Kinase Inhibitor 1A CDKN2A Cyclin Dependent Kinase Inhibitor 2

cDNA complementary DNA

CK1α Casein kinase 1 alpha

CREB cAMP-response element binding protein

CRISPR Clustered regularly interspaced short palindromic repeats CTGF Connective tissue growth factor

CTLA-4 Cytotoxic T-Lymphocyte Associated Protein 4 CYR61 Cysteine-rich angiogenic inducer 61

DC Dendritic cell

DCT Dopachrome Tautomerase

DEC1 Deleted in esophageal cancer 1 DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DVL Dishevelled segment polarity protein EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

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viii EMT Epithelial to mesenchymal transition

ERK1/2 Extracellular signal-regulated kinase 1/2 esiRNA endoribonuclease-prepared siRNA FBS Fetal bovine serum

FDA Food and drug administration

FZD Frizzled

GAPDH Glyceraldehyde-3-phosphate dehydrogenase GLI2 GLI Family Zinc Finger 2

GPR143 G Protein-Coupled Receptor 143 GSK3ß Glycogen synthase kinase 3ß GPCR G-protein-coupled receptor HOXA1 Homebox transcription factor1 HPS Histidine, Proline, Serine HRP Horseradish peroxidase ICT Immune checkpoint therapy IL-2 Interleukin-2

IL-R1 Interleukin-1 receptor

KARS Lysyl-tRNA synthetase (KARS), LATS1 Large tumor suppressor kinase 1 LATS2 Large tumor suppressor kinase 2 LATS1/2 Large tumor suppressor 1 and 2 LEF1 Lymphoid enhancer-binding factor 1

LRP5/6 Lipoprotein receptor-related protein 5 and 6 MC1R Melanocortin 1 Receptor

MEK Mitogen-activated protein kinase kinase MET Mesenchymal to epithelial transition MHC Major histocompatibility complex

MITF Microphthalmia-associated transcription factor

MLANA Melan-A

MOB1A/B MOB kinase activator 1A and 1B

mRNA messenger RNA

MST1/2 Mammalian sterile 20-like protein kinase 1 and 2 NAD+ Nicotinamide adenine dinucleotide

NF- κB nuclear factor kappa-light-chain-enhancer of activated B cells NK cell Natural killer cell

NOTCH Neurogenic locus notch homolog protein PAR Poly (ADP-ribose)

PARP Poly (ADP-ribose) polymerase

PARsylation Poly adenosine-di-phosphate (ADP)-ribosylation PBS Phosphatate-buffered saline

PD-1 Programmed cell death

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ix PD-L1 Programmed cell death ligase

PFA Paraformaldehyde

PI3K Phosphoinositide 3-kinase

RIPA Radio Immune precipitation assay

RT-qPCR Real-time quantitive reverse transcription-polymerase chain reaction RNF146 Ring finger protein 146

PBS-T Phosphate-buffered Saline Tablets PTEN Phosphatase and TENsin homolog PVDF Polyvinylidene difluoride

SAM Sterile alpha motif

SAV1 Salvador family WW domain-containing protein 1

SDS Sodium Docecyl Sulfate

SOX10 SRY-related high-mobility group box 10 TBM Tankyrase binding motif

TAZ Transcriptional co-activator with PDZ-binding motif TAD Transactivation domain

TBS-T Tris buffered Saline Tablets

TCF T cell factor

TCR T cell receptor

TEAD1 TEA domain transcription factor 1 TEAD2 TEA domain transcription factor 2

TEAD 1/2 Tef-1 and abaA (TEA) domain family members 1 and 2 TFE3 Transcription Factor Binding To IGHM Enhancer 3 TFEB Transcription factor EB

TGFβ Transformating growth factor-beta TLE Transducing-like enhancer protein

TMB Tumor mutational burden

TNKS1 Tankyrase 1

TNKS2 Tankyrase 2

TNKS1/2 Tankyrase 1 and 2

TYR Tyrosinase

TYRP1 Tyrosinase Related Protein 1

UV Ultraviolet

WNT Wingless-type mammary tumor virus integration site WNT signaling WNT/β-catenin signaling

YAP Yes-associated protein

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List of figures

Figure 1. Overview of the hallmarks of cancer………. 2

Figure 2. Different types of skin cancer and melanoma identification chart……….... 3

Figure 3. Melanoma tumor progression using the Clark model……….... 4

Figure 4. The WNT/β-catenin signaling pathway………. 6

Figure 5. AXIN coordinates the assembly of the destruction complex in WNT signaling... 7

Figure 6. The Hippo-YAP/TAZ signaling pathway……….. 9

Figure 7. Structure of TNKS1/2 peptide………... 10

Figure 8. PARsylation activity of tankyrase………. 11

Figure 9. Tankyrase inhibition suppress WNT signaling………. 12

Figure 10. Tankyrase inhibition suppress YAP signaling…….………. 13

Figure 11. Phenotype switching between epithelial phenotype and mesenchymal phenotype……….. 15

Figure 12. MITF gene isoform structures………... 17

Figure 13. Multiple factors regulate MITF activity and cellular programs……… 18

Figure 14. Transcriptional regulation of MITF-M expression……….... 19

Figure 15. Key regulatory mechanism on MITF-M protein………... 20

Figure 16. Innate immunity VS adaptive immunity………... 22

Figure 17. Mechanism of action of immune checkpoint inhibitors……… 24

Figure 18. Immune response in melanoma through interactions of MITF at post-translational level……….. 26

Figure 19. Detection of target protein through chemiluminescent immunoblotting……….. 35

Figure 20. Combinational therapy with G007-LK and PD-1 shows synergic anti-tumor effect……….. 37

Figure 21. High activity of YAP signaling correlates with low baseline MITF expression and potential for decreased MITF transcription upon tankyrase inhibition……. 38

Figure 22. Effect of G007-LK on YAP target proteins and genes in B16-F10 cells ……... 40

Figure 23. MITF expression increases upon treatment with G007-LK in vivo………. 41

Figure 24. Tankyrase inhibitor G007-LK can increase MITF expression in murine B16-F10 cells………42

Figure 25. Regulation of MITF expression by G007-LK is β-catenin independent………. 43

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xi Figure 26. Effect of G007-LK on the localization of MITF, YAP, and β-catenin in

B16-F10 cells……… 44/45 Figure 27. WNT3a can induce morphology change in B16-F10 cells………... 46 Figure 28. Reversible morphology changes in B16-F10 cells……… 47 Figure 29. G007-LK stabilizes both Cdh1 and Cdh2 genes in B16-F10 cells in vitro …….. 48 Figure 30. Effect of G007-LK on EMT target genes in B16-F10 and Clone M-3^Z1

cells in vivo……… 49 Figure 31. Effect of G007-LK on nuclear and cytoplasmic fractions on a panel of human

melanoma cell lines………... 51 Figure 32. Effect of G007-LK treatment on WNT and YAP signaling in a panel of

human melanoma cell lines………... 52 Supplementary figure 1. Regulation of Cdh1 and Cdh2 with G007-LK is not β-catenin

dependent………... 77 Supplementary figure 2. Overview of all immunoblots from nuclear fractions of human

melanoma cell lines………... 78 Supplementary figure 3. Overview of all immunoblots from cytoplasmic fractions of

human melanoma cell lines……….. 79 Supplementary figure 4. Effect of treatment with G007-LK on the localization of MITF

and β-catenin in B16-F10 cells………. 80 Supplementary figure 5. Effect of treatment with G007-LK and WNT3a on the

localization of MITF and YAP in B16-F10 cells……….. 81

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List of tables

Table 1. A panel of human melanoma cell lines………. 29

Table 2. Plate seeding ratio of cells………. 30

Table 3. Real-time RT-qPCR mix………... 32

Table 4. Mouse probes for real-time RT-qPCR……….. 32

Table 5. Human probes for real-time RT-qPCR………. 32

Table 6. Primary antibody used for immunoblotting……….. 34

Supplementary Table 1. SDS loading buffer... 71

Supplementary Table 2. 10x Protein transfer buffer... 71

Supplementary Table 3. 1x Protein transfer buffer... 71

Supplementary Table 4. 4% PFA (1L)... 71

Supplementary Table 5. Primary antibodies diluted in 4% BSA/PBS………. 72

Supplementary Table 6. Aqueous Mounting Medium……….. 72

Supplementary Table 7. List of equipment……… 72

Supplementary Table 8. List of instruments... 75

Supplementary Table 9. Characterization of human melanoma cell lines……… 75

Supplementary Table 10. List of software used... 76

Supplementary Table 11. MITF target genes………. 76

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1

Introduction

1.1 Cancer

Cancer is defined as cells that proliferate in an uncontrolled matter caused by genomic instability, such as damages or mutations in the cell’s genetic material [1]. Cancer cells do not collaborate with normal cells to construct normal tissues, and these uncontrolled cell divisions may result in the formation of tumors [2]. Cancer cells arise from our body’s existing cells and give rise to hundreds of different forms of the disease [2].

The first step of cancer development is tumor initiation, where a single cell has acquired

abnormal shape and function [3]. This cell may outgrow other normal cells, which leads to tumor progression where further mutations arise in cancer-critical genes [3]. Cancer cells acquire advantages such as resistance to apoptosis and rapid growth. These traits become dominant to the descendants; a process called clonal selection [3]. Tumors are categorized into two groups:

benign and malignant tumors. Benign tumors grow without spreading, whereas malignant tumors are cancerous and can invade other adjacent tissues of the body, a process called metastasis [4].

Benign tumors can still cause damage by growing and compressing healthy tissue [4]. Two genes play critical roles in tumor progression: tumor suppressor genes and proto-oncogenes [5]. Tumor suppressor genes usually inhibit cell proliferation, protecting cells from becoming cancerous.

Loss of function in such genes can promote cancer development. Proto-oncogenes convert into oncogenes by the gain of function mutations which induce cell growth [5]. In addition, mutations in genes controlling deoxyribonucleic acid (DNA) repair increase tumor development by causing further instability to the genome [6].

In 2000, Douglas Hanahan and Robert A Weinberg organized the complex nature of cancer into hallmarks, illustrated in Figure 1 [1]. The first and most fundamental hallmark is sustained proliferate signaling, where a cancer cell mutation results in cell division and growth without receiving this signal. Second, cancer cells can evade growth suppressors, which typically act as a growth stop signal for themselves or neighboring cells. When these cancer cells are

uncontrollably growing and dividing, the next step is resistance to cell death, defined as the third hallmark. Normal cells have internal sensors to detect dangerous mutations and initiate apoptosis.

Mutation in these signaling proteins may help the cancer cells to evade apoptosis. The fourth hallmark is enabling replicative immortality, also termed immortalization, as the cells can divide indefinitely. In the interest of sustaining uncontrollable division and growth, the cancer cells need nutrients and resources. By accomplishing this, cancer cells secrete molecules to stimulate blood vessel growth, a process called angiogenesis – the fifth hallmark of cancer. The sixth hallmark is activating invasion and metastasis, which causes cancer cells to migrate and invade neighboring tissues. The two enabling factors are genome instability and tumor-promoting inflammation. In 2011, two new emerging hallmarks were proposed [7]. The first emerging hallmark is

deregulating cellular energetics, in which cancer cells are replacing the metabolic program in

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2 normal cells. The second hallmark is avoidance of immune destruction, where cancer cells evade immune attack [1, 7]. [7]

1.2 Malignant melanoma

Melanoma is a rare and aggressive type of skin cancer where the 10-year survival rate for patients with stage IV metastatic melanoma is 10-15% [8, 9]. The six most common skin cancers are listed in Figure 2. Basal cell carcinoma is the most common skin cancer, followed by squamous cell carcinoma [10, 11]. Merkel cell carcinoma is associated with developing other skin cancers such as basal or squamous cell carcinomas [12]. Nodular melanoma is the most aggressive type

Figure 1. Overview of the hallmarks of cancer. The schematic overview shows arguably acquired properties to develop cancer. Traits as sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, replicative immortality, inducement of angiogenesis, and activating invasion and metastasis be necessary to manifest cancer, depending on the type of cancer. Later, reprogramming of energy metabolism and evasion of immune destruction were proposed as emerging hallmarks of cancer. Enabling factors are genome instability and mutation, and tumor-promoting inflammation. Adapted from “Hallmarks of Cancer template” by BioRender.com (2021), inspired by [7].

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3 of melanoma, growing faster than the different skin cancer types [13]. Nodular melanoma

accounts for 15 percent of all skin cancer cases [13]. Superficial spreading melanoma grows slowly and horizontally and is the most common melanomas, accounting for 70 percent of melanoma [14]. Lastly, lentigo maligna melanoma often develops in older people and is usually harmless [15]. Melanoma is more dangerous when compared to carcinoma since the skin cancers differ from each other in the types of cells in which cancer originated and the rate of

aggressiveness. Melanoma is the deadliest type of skin cancer, developing from the skin’s

melanocytes [16]. Melanocytes derive from neural crest cells and exist in the epidermis, iris, hair follicles, the nervous system, heart, and inner ear [16]. Melanoma has a high likelihood of

becoming malignant if not treated at an early stage. Figure 2 also illustrates a melanoma identification chart to differentiate melanoma from normal skin moles.

Causes of malignant melanoma are high UVA, UVB, and UVC radiation exposure over time, family history, genetics such as fair skin and red hair, lower socioeconomic status resulting in more advanced disease, and atypical mole syndrome [17]. Sun exposure is relatively easy to prevent with sunscreen, yet cutaneous melanoma exhibits high amounts of the C > T UV signature mutation. Hence, prolonged exposure to UV radiation is the primary source of malignant melanoma [18, 19]. UV radiation can cause cancer without sunburn because of the formation of reactive oxygen species, impaired continuing immune function, and increasing production of growth factors [18]. However, UV radiation also benefits health by increasing the natural synthesis of vitamin D [20].

Figure 2. Different types of skin cancer and melanoma identification chart. Skin cancer is categorized into basal cell carcinoma (most common carcinoma), Merkel cell carcinoma (rare disease), squamous cell carcinoma (second most common carcinoma), nodular melanoma (most aggressive of all), superficial spreading melanoma (most common melanoma), and lentigo maligna melanoma (least dangerous melanoma). Sun exposure is the primary source of all melanoma and carcinomas. For identifying melanoma, one can use the “ABCDE” chart to differentiate melanoma from normal skin moles. Figure adapted from “Skin cancer/Identifying melanoma template” by BioRender.com (2021).

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4 Melanoma affects people with lighter skin at a higher rate compared to people of color [21]. The pigment that protects against UV radiation is the skin-darkening melanin, of which we find a higher amount in the darker skin tone [21]. Regardless of genetic background, a series of

alterations and lesions must occur for melanocytes to become invasive melanoma. Although the process is not entirely mapped out, there are some common alterations illustrated in Figure 3.

The first step of metastatic melanoma development is the formation of a benign nevus in a melanocyte [22]. The second step is the proliferation of the benign nevus to a dysplastic nevus about 5 mm in size and irregular borders. The melanocytes then proliferate into the epidermis, and vertical proliferation and invasion into the basement membrane in the radial growth phase, becoming metastatic. The final step is the spreading of the malignant melanocytes to distant body parts [23].

Before 2011, no treatment of metastatic melanoma patients had shown a significant response [24]. In 2011 the first paradigm shift in melanoma treatment came from knowledge of the activating B-Raf Proto-Oncogene V600E (BRAF^V600E) mutation in BRAF, leading to the

discovery and Food and Drug Administration (FDA) approval of BRAF^V600Einhibitors [24]. The BRAF mutation arises in the benign nevus. Because of secondary resistance, a second paradigm shift came from using immune checkpoint inhibition, which improved outcomes for advanced- stage melanoma patients [24]. (A detailed description of malignant melanoma treatment is described in 1.8.2 Metastatic melanoma ICT). However, many melanoma patients fail to respond to immune checkpoint inhibition monotherapy [25]. Previous studies have shown that targeting wingless-type mammary tumor virus integration site (WNT)/β-catenin signaling

Figure 3. Melanoma tumor progression using the Clark model. The schematic illustration of a benign nevus in the epidermis ultimately becoming metastatic melanoma. The benign nevus becomes a dysplastic nevus with irregular borders before starting a radial growth phase, followed by a vertical growth phase. The result is distant metastases of melanoma cells. Figure created with BioRender.com, inspired by [23].

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5 pathway (in this thesis referred to as WNT signaling pathway) may sensitize melanoma to

immune checkpoint blockade [25].

1.3 The canonical WNT/β-catenin signaling pathway

The WNT signaling pathway is an evolutionarily conserved pathway that is central to vital cellular functions such as differentiation, proliferation, cell development, and tissue homeostasis [26]. Elevated WNT signaling increases cell proliferation and has been associated with the development of human diseases such as cancer [26, 27]. Aberrant WNT signaling is found in several cancer types such as melanoma, colorectal cancer, breast cancer, and prostate cancer [27].

The WNT signal cascade consists of 19 identified human WNT genes secreted from cell to cell for communication and activation of WNT signaling [28, 29]. The first discovery of WNT proteins happened in 1982, where the proto-oncogene Int1 (now known as Wnt1) was identified in mice [30]. The segment polarity gene wingless, a homolog of Int1, was later independently discovered in flies [30]. The wingless gene was found to be required for wing development, while the Int1 gene was shown to promote tumor development in mammals [30]. In 1991, the first direct link between WNT signaling and human cancer was established when mutations of the adenomatous polyposis coli (APC) gene were the underlying cause of the hereditary colon cancer syndrome termed familial adenomatous polyposis [30]. The interaction between the APC gene and β-catenin led to the loss of function of APC and increased T cell factor (TCF)/β-catenin signaling [30].

The WNT signaling pathway is grouped into two categories: β-catenin dependent (canonical) and independent (non-canonical) signaling [31]. The canonical WNT signaling is the most studied pathway and regulates the stabilization and accumulation of the transcriptional co-activator β- catenin. Melanoma cells utilize the non-canonical WNT signaling for metastasis and the canonical WNT signaling for growth and transformation [32].

WNT proteins are secreted through exocytosis and work as intracellular signal molecules to bind to neighboring cells’ receptor complex (Figure 4) [33]. WNT signaling pathway switches

between an “on” and “off” state, depending on the presence of WNT ligands bound to receptors.

In the absence of a WNT ligand in the canonical WNT signaling (off-state), β-catenin is attached to a destruction complex in the cytoplasm [33]. The most significant constituents of the

destruction complex are glycogen synthase kinase 3β (GSK3β), casein kinase 1 alpha (CK1α), APC, and the concentration-limiting compound axis inhibition protein 1 and 2 (AXIN1/2) [33].

APC and AXIN1/2 act as scaffolding proteins in the destruction complex. GSK3β phosphorylates bound β-catenin resulting in recognition by E3-ubiquitin ligase β-transducing repeat-containing protein (β-TrCP200), which ubiquitinates and tags β-catenin for degradation by the proteasome [30]. Consequently, there is no transcription of WNT target genes in the nucleus [30]. [34]

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6 WNT signaling is activated upon binding between WNT ligands and Frizzled receptors (FZD) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors [27, 30, 35].

Bound WNT ligands generate a dimerization and conformational change of FZD and LRP5/6, causing phosphorylation of LRP5/6 by CK1α and GSK3β. The Binding of WNT ligands also leads to the recruitment of AXIN1/2 and, therefore, the destruction complex. Together with the recruitment of the scaffolding protein dishevelled segment polarity protein (DVL), this complex

Figure 4. The WNT signaling pathway. Left Panel: In the presence of the ligand, β-catenin degradation is inhibited by recruitment of the destruction complex to FZD and LRP5/6 receptors through AXIN. Hence, β-catenin is free to translocate into the nucleus and transcribe WNT target genes. Here, β-catenin displaces the repressive complex TLE/Groucho and forms the active transcription complex with TCF/LEF. The destruction complex consists of AXIN, APC, DVL, CK1α, and GSK-3β.Right panel:In the absence of the ligand, β-catenin is phosphorylated, ubiquitinated, and degraded by the proteasome through the destruction complex in the cytoplasm. TLE/groucho represses the transcription of WNT target genes. Figure created with BioRender.com, inspired by [34].

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7 formation prevents the phosphorylation of β-catenin [36]. Accumulation of β-catenin in the cytoplasm eventually leads to the translocation of β-catenin into the nucleus where β-catenin displaces co-repressor transducing-like enhancer protein (TLE/groucho) and bind to transcription factors T cell factor/ Lymphoid enhancer-binding factor 1 (TCF1/LEF1), and the transcription of WNT target genes [35, 37].

AXIN2 is one of many target genes of the WNT signaling pathway, and one of few target genes that is non-specific regarding cell type [36]. AXIN2 acts as a negative feedback loop in most cells, where activation of WNT signaling leads to transcription of AXIN2, which further degrades β-catenin [37]. Loss of function for negative regulators such as AXIN2 and gain of function to positive regulators such as β-catenin and TCF can result in aberrant WNT signaling [38]. Both AXIN1 and AXIN2 are suppressors of the WNT signaling pathway [39]. AXIN2 is required for degradosome formation, while AXIN1 is neither needed for degradosome formation nor

degradation of β-catenin [40].Figure 5 shows a schematic diagram of AXIN and its interacting partners in the WNT signaling pathway. [41]

Figure 5. AXIN coordinates the assembly of the destruction complex in WNT signaling. The functional domains of AXIN act as scaffolding domains, including binding sites for several WNT signaling proteins; Tankyrase (TBM), APC (RGS), GSK-3β (GID), and β-catenin (CID). AXIN also contains the MID domain (MEKK1 bindings site) and DIX domain. Tankyrase inhibition stabilizes AXIN by inhibiting PARsylation activity of tankyrase, allowing the destruction complex to form, ultimately degrading β-catenin and suppressing WNT signaling. Abbreviations: tankyrase-binding domain 1 and 2 (TBM1/2 Regulators of G protein signaling (RGS), Mitogen-activated protein kinase kinase kinase 1 (MEKK1). Figure created with BioRender.com, inspired by [41].

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1.4 The Hippo-YAP/TAZ signaling network

The Hippo signaling pathway regulates tissue regeneration, self-renewal, tissue growth, and organ size – and eventually tumorigenesis [42]. Like the WNT signaling pathway, the Hippo pathway is evolutionarily conserved and works in an “on” and “off” state. The discovery of the Hippo signaling network in Drosophila showed its regulatory role of organ size [42, 43]. Later, mutations in the Hippo signaling in mice demonstrated the regulation of organ overgrowth and tumorigenesis [44, 45]. The network is of great importance during cell proliferation,

differentiation, and tissue homeostasis. Co-activator paralogs Yes-associated protein

(YAP)/transcriptional coactivator with a PDZ-binding domain (TAZ) are downstream effectors in the Hippo signaling pathway and function as prime mediators as well as oncoproteins, and like WNT, elevated YAP signaling is characteristic in numerous human cancers [46, 47]. When the Hippo signaling pathway is “off,” YAP signaling is “on” [46, 47]. The Hippo signaling pathway is complicated and interacts with many other pathways. Recent studies have identified new upstream regulatory components such as G-protein-coupled receptor (GPCR), WNT,

transforming growth factor-beta (TGFβ), epidermal growth factor (EGF), and neurogenic locus notch homolog protein (Notch) [47].

The Hippo signaling pathway is a serine/threonine kinase signaling cascade with a highly conserved core, consisting of mammalian sterile 20-related 1 and 2 kinases (MST1 and MST2) and large tumor suppressor 1 and 2 kinases (LATS1 and LATS2) (Figure 6) [43, 47, 48]. MST kinases have a SARAH domain that mediates dimerization of MST1/2. Several upstream signals activate the Hippo signaling pathway, such as cell-cell contact, stress signals, stiffness of the extracellular matrix, and cell polarity. These signals regulate and activate the phosphorylation and dimerization of MST1/2 and LATS1/2 either by other kinases or autophosphorylation [43, 48]. Upon activation of the Hippo signaling pathway, the MST1/2 dimer, together with SARAH domain-containing protein Salvador 1 (SAV1), initiates phosphorylation of MOB kinase activator 1A, and 1B (MOB1A and MOB1B) [43, 47, 48]. Both MOB1A/B and LATS1/2 are

phosphorylated by MST1/2 which in turn leads to interaction between MOB1A/B and LATS1/2.

At the same time, phosphorylation of MOB1A/B initiates autophosphorylation of LATS1/2 [43].

Substrates for LATS kinases are YAP and TAZ, and phosphorylation of YAP/TAZ by LATS kinases keeps YAP/TAZ in the cytoplasm and prevents gene transcription in the nucleus [47].

LATS kinases are AGC kinases that recognize and phosphorylate the substrate consensus sequence HXRXXS/T [43]. YAP and TAZ have five and four HXRXXS/T motifs, respectfully, which are phosphorylated by LATS1/2. Phosphorylation of YAP/TAZ can either result in binding to 14-3-3 proteins leading to cytoplasmic retention or ubiquitin-mediated protein degradation [43, 47]. Consequently, activation of the Hippo signaling pathway turns YAP signaling off. To further complicate the Hippo signaling pathway, the activation occurs upon crosstalk between other pathways such as Notch and WNT signaling pathways. For instance, in the WNT signaling- inactive cells, YAP and TAZ can accumulate in the β-catenin degradosome [49]. YAP and TAZ

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9 can dislocate from the β-catenin degradosome in the WNT signaling-active cells, resulting in a YAP/TAZ accumulation in the nucleus and thereby activating YAP signaling [49]. [50] [51]

When the Hippo is inactivated, YAP/TAZ are in a non-phosphorylated state and are free to translocate to the nucleus and bind with TEA domain family member (TEAD) transcription factors [43, 52]. Connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and angiomotin proteins (AMOTL1/2) are examples of target genes of YAP signaling.

[43].

Figure 6. The Hippo-YAP/TAZ signaling pathway. Left panel: Several different upstream signals activate the Hippo signaling pathway, leading a serine/threonine kinase cascade consisting of two groups of kinases: MAST1/2 and LATS1/2, together with their activating adaptor proteins SAV1 and MOB1. The cascade ultimately causes cytoplasmic retention of YAP/TAZ or the proteasomal degradation of YAP/TAZ. Right panel: In the hippo “off” state, the serine/threonine kinases are not phosphorylated by the upstream signals, leaving YAP/TAZ free to translocate to the nucleus and form the active transcription complex with TEAD, leading to increased YAP signaling. Abbreviations: ubiquitin (Ub), a phosphate group (P). Figure created with BioRender.com, inspired by [50,51].

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10

1.5 Tankyrase 1.5.1 Structure

Tankyrase 1 and 2 protein (TNKS1/2) both belong to the poly(adenosine diphosphate (ADP)- ribose) polymerase (PARP) family, which are structurally conserved enzymes that participate in both the WNT signaling pathway and Hippo signaling pathway [53]. The TNKS1 isoform differs from TNKS2 by an additional histidine, proline, serine-rich (HPS) domain in the TNKS1 (Figure 7) [53]. The function of the HPS domain is unknown. Both enzymes contain five ankyrin repeat clusters (ARC), a sterile alpha motif (SAM), and a catalytic PARP domain [53, 54]. TNKS1/2 interacts with target proteins through ARC domains. The SAM domain is responsible for homo- oligomerization, while the catalytic domain contains the ADP-ribosyltransferase activity [53, 55].

[53]

1.5.2 Cellular function and enzymatic activity

Tankyrases are enzymes with multiple, overlapping cellular functions, including regulating different signaling pathways, mitosis, telomere maintenance, glucose metabolism, and tumor suppressors[55]. Tankyrases can also function as scaffolding for other proteins through their SAM domains, providing structural functions [56].

The catalytic activity of TNKS1/2 controls different regulations in the cells by poly-Adenosine Diphosphate-ribosylation (PARsylation) [55]. PARsylation results in post-translational

modification, which regulates other cellular processes by stabilizing the target genes [55].

PARsylation involves the hydrolysis of nicotinamide adenine dinucleotide (NAD+) to ADP-

Figure 7. Structure of TNKS1/2 peptide. The HPS domain is one of the structural differences between TNKS1 and 2, while the function of this domain remains unknown at this point. The five ARC domains contribute to the interactions between TNKS1/2 and other target proteins. SAM domain controls self-oligomerization. PARP is the catalytic domain that performs PARsylation. TNKS1/2 domain structures are highly conserved. Figure created with BioRender.com, inspired by [53].

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11 ribose and nicotinamide, attaching the ADP-ribose to a target protein which regulates cellular processes including gene transcription, DNA damage repair, and cellular stress (Figure 8) [53].

The growing poly(ADP-ribose) chain is recognized by the E3 ubiquitin ligase RING finger protein 146 (RNF146), which tags the protein for degradation by the proteasome [57].

Figure 8. PARsylation activity of tankyrase. Tankyrase orchestrates the hydrolysis of NAD+ to ADP ribose and nicotinamide. Tankyrase PARsylates the target protein by attaching an ADP-ribose to the target. Figure created with BioRender.com

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12

1.5.3 Tankyrase inhibition suppress WNT signaling

TNKS1/2 interacts with the TBM domain of AXIN1/2 and PARsylate AXIN1/2, leading to the recognition of PARsylated AXIN1/2 through E3 ligase RNF146 (Figure 9) [34]. RNF146 mediates ubiquitination and degradation of AXIN1/2 through the proteasome. This ultimately destabilizes the destruction complex, resulting in the accumulation of β-catenin, which

translocates to the nucleus [53]. Dysregulated WNT signaling and overexpression of WNT target genes are often found in multiple cancer types [30]. By inhibiting TNKS1/2, AXIN proteins stabilize [34]. Small-molecule tankyrase inhibitors, such as G007-LK, therefore, destabilize β- catenin and decreases WNT signaling. [34]

Figure 9. Tankyrase inhibition suppress WNT signaling. Left panel: Without tankyrase inhibitors, active tankurase binds the TBS domain of AXIN and PARsylates AXIN, leading to phosphorylation, ubiquitination, and degradation by RNF146 and 26S proteasome. Hence, AXIN is not allowed to form the destruction complex, lading to accumulation of β-catenin and gene transcription in the nucleus. Right panel: Tankyrase inhibition stabilizes AXIN, allowing the destruction complex to form and perform ubiquitin-mediated proteosomal degradation of β-catenin, thereby suppressing WNT signaling. Abbreviations: ubiquitin (Ub), Figure created with BioRender.com, inspired by [34].

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13

1.5.4 Tankyrase inhibition suppress YAP/TAZ activity

AMOTs are interacting partners with YAP and TAZ in the Hippo signaling pathway. AMOT1/2 proteins can regulate YAP signaling directly (Figure 10) [47]. Similar to the WNT signaling pathway, TNKS1/2 PARsylates AMOT proteins lead to their degradation by E3 ligase RNF146, leaving YAP/TAZ free to translocate to the nucleus [48]. Here, YAP/TAZ forms the active transcription complex with TEAD [48]. Multiple efforts have been made to find effective YAP/TAZ activity inhibiting drugs, whereas tankyrase inhibition has shown YAP/TAZ

suppressing functions. By inhibiting TNKS1/2, AMOT proteins can associate with transcription co-factors YAP/TAZ and leave the nucleus. Thus, YAP/TAZ signaling gets suppressed [48]. [48]

Figure 10. Tankyrase inhibition suppresses the Hippo signaling network. Left panel: Without tankyrase inhibitor, active tankyrase PARsylates AMOT proteins, leading to their degradation by RNF146 and 26S proteasome, leaving YAP/TAZ free to translocate into the nucleus. Here, YAP/TAZ forms the active transcription complex with TEAD. Right panel: Tankyrase inhibition leads to an increased level of AMOT proteins, which binds YAP/TAZ and either causes cytoplasmic retention or activation of LATS ½ and thereby ubiquitin-mediated proteasomal degradation. Abbreviations:

phosphate group (P), ubiquitin (Ub), Figure created with BioRender.com, inspired by [48].

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14

1.5.5 Tankyrase inhibition and tankyrase inhibitors

The druggable effect on the catalytic domains in TNKS1/2 makes the inhibition of the enzymes a topic of interest in cancer treatment, leading to less active pro-oncogenic signaling pathways.

Inhibitors bind to the catalytic domain and can be classified into drugs that attach to the

nicotinamide binding pocket or those that bind to the catalytic domains adenosine binding pocket [58]. The nicotinamide binding pocket is similar for all types of PARPs, and an example of a developed inhibitor that binds there is XAV939. TNKS1/2 are the only proteins in the PARP family that contain adenosine binding pockets, whereas other developed inhibitors such as G007- LK and OM-153 can bind [59]. Hence, G007-LK and OM-153 are more specific TNKS1/2 inhibitors compared to XAV939.

Multiple tankyrase inhibitors have been developed because they inhibit key cancer-promoting signaling pathways [60]. In addition to WNT and Hippo signaling pathway, TNKS1/2 can act on various other signaling pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase

(PI3K)/AKT serine/threonine kinase 1 (AKT) [48, 60, 61]. Although TNKS1/2 seems like promising target candidates in many of those pathways, there are currently no tankyrase inhibitors available for clinical practice.

Tankyrase inhibitor monotherapy has shown limited responsiveness in malignant melanoma. A synergic effect may be achieved by combining tankyrase inhibition with other therapies, such as chemotherapy, programmed cell death (PD-1), PI3K inhibitor BKM120, and epidermal growth factor receptor (EGFR) inhibitor erlotinib against the different types of cancer [25, 53, 62]. For example, combined inhibition of TNKS1/2 with G007-LK and the breakthrough of immune checkpoint inhibitors in anti-cancer therapy have been reported to sensitize melanoma tumors to anti-PD-1 immune checkpoint therapy [25].

1.6 Phenotype switching

Several distinct phenotypic states of melanoma have been identified, depending on the microenvironment state. Nutrient limitation, hypoxia, inflammation, and immunotherapy are some of the causes that can induce a phenotype switch in melanoma, leading to de-differentiation and resistance to therapy [63].

Cancer cells must undergo a switch in phenotype during metastasis to acquire new properties such as motility and invasiveness. One of these reversible transitions is termed epithelial to mesenchymal transition (EMT), where the cells shed many of their epithelial characteristics to become mesenchymal cells (Figure 11) [64, 65]. A typical marker for the epithelial phenotype is E-cadherin, an adhering molecule between epithelial sheets creating structural integrity [64]. The CDH1 gene that specifies E-cadherin is often promoter methylated in invasive carcinomas, leading to either inhibited expression or mutation of E-cadherin [66]. Loss of E-cadherin occurs

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15 in the vertical growth phase of melanoma tumorigenesis (Figure 2) [67]. A typical marker on the mesenchymal transition side is the CDH2 gene that specifies N-cadherin, another adhering molecule when melanocytes transform to melanoma [64]. Normally, E-cadherin binds melanocytes to keratinocytes (epithelial cells) through homodimers [64, 68]. Since both E- cadherin and N-cadherin bind by homophilic interactions, replacing E-cadherin with N-cadherin would favor homotopic interactions between melanoma cells and various types of mesenchymal, such as fibroblasts and endothelial cells [64, 68]. Bonds between N-cadherin molecules are weaker compared to E-cadherin molecules, giving the cells higher motility. It has been proposed that this shift promotes invasion of stroma since N-cadherin are continuously connecting

mesenchymal cells. Hence, an epithelial to mesenchymal transition leads to more invasive melanoma cells [64].

Other markers for mesenchymal phenotype are vimentin and fibronectin. Vimentin is an intermediate filament expressed in mesenchymal cells, while fibronectin is an extracellular matrix protein often expressed by epithelial cells that have undergone phenotype switching [66, 69].

During embryogenesis, cells migrate to the interior of the embryo, forming germ layers [68]. This process is called gastrulation, where the cells detach, acquire motility and round shapes [68]. A shift between E-cadherin and N-cadherin also occurs here [68]. This comparability between embryogenesis and tumor invasiveness suggests that carcinomas activate an already encoded program in their genome to undergo this transition. In addition, the stroma around a tumor and

Figure 11. Phenotype switching between epithelial phenotype and mesenchymal phenotype. The transition from epithelial phenotype to mesenchymal phenotype is a reversible biological process where the cells go from

proliferative/differentiated cells to invasive and de-differentiated cells, and vice versa. E-cadherin is a characteristic epithelial phenotype marker, whereas the cells shed this characteristic as they switch towards mesenchymal phenotype.

Here, N-cadherin, Vimentin, and Fibronectin are typical markers. MITF expression tends to be high in epithelial cells and decreasing towards mesenchymal phenotype. Figure created with BioRender.com, inspired by [64].

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16 inflammatory cells can release phenotype switch promoting signals [68]. If cells undergo EMT to detach and invade an environment lacking these signals and inflammatory cells, the transition can be reversed. This transition is called mesenchymal to epithelial transition (MET). Several

pathways play a role in promoting either epithelial or mesenchymal states in cells, including the WNT signaling pathway [68].

1.6.1 WNT signaling mediated regulation of phenotype switching in melanoma

Melanoma cells can exhibit different states, being either invasive or proliferative as those two are uncoupled (Figure 11) [70]. Proliferative melanoma cells are less likely to be invasive, highly depending on the major melanoma coordinator microphthalmia-associated transcription factor (MITF). MITF^high expressing cells have a proliferative behavior, whereas MITF^low expressing cells are invasive and tumor-initiating [63]. Cells must undergo a phenotype switch to acquire these different states [63].

Changes in WNT signaling have been associated with phenotype switching in melanoma [70].

Studies show contradictory results whether nuclear β-catenin correlates with aggressive disease or better prognosis. Loss of β-catenin has been linked to a mesenchymal phenotype, therefore more invasive cells, yet both activation and inhibition of WNT signaling have been suggested for melanoma therapy [71]. In our studies, the loss of β-catenin has been necessary to achieve the synergistic effect of tankyrase and checkpoint inhibitor treatment [25].

1.7 MITF

MITF functions as a master regulator of melanocyte development, survival, migration, invasion, and proliferation, and is expressed in 80% of melanoma [72, 73]. Furthermore, MITF may play a key role in solving the obstacles in immunotherapy resistance. It is also crucial for melanoma cell plasticity, tumor heterogeneity, and modulation of coinhibitory receptors such as programmed cell-death ligand 1 (PD-L1) [50].

1.7.1 MITF-structure and expression

MITF is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor and belongs to the MYC family [74]. MITF gene consists of 526 amino acids, and MITF locus is mapped to chromosome 3 in humans [75]. Twelve isoforms of MITF have been reported: MITF-A, MITF-B, MITF-C, MITF-D, MITF-E, MITF-H, MITF-J, MITF-MC, MITF-CX, MITF-CM, MITF-Mdel, and MITF-M, distinguishing from each other at the N-terminal due to alternative splicing in the first exons and alternative starting point for promoters. (Figure 12) [76]. The MITF-M isoform is limited to melanocytes and melanoma cells, whereas the others are widely expressed in other cell types [75]. MITF-M is the most studied isoform and plays an important role in tumor progression and survival [75]. [77]

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17 MITF activity is affected by immune cells in the tumor microenvironment, genetic alterations, modifications of signaling pathways, and epigenetics (Figure 13) [78]. Combinations of all these factors contribute to variable MITF expression across melanoma specimens and different areas within the same tumor [78]. Increasing evidence suggests that the cancer genome can switch phenotype in response to these regulations, whereas the critical determinant for melanoma phenotype is the activity of MITF [72, 73, 78]. High MITF activity in melanoma cells has been linked to proliferation and differentiation, while low MITF activity may lead to invasion [78].

[78]

Figure 12. MITF gene isoform structures. Due to alternative splicing and different starting sites for transcription, MITF is produced in multiple isoforms differing from each other at the N-terminal. Each isoform differs in the first exon and is driven by different promoters. Exon 2-9 are identical in all MITF isoforms and includes the functional domains. MITF-M and MITF-Mdel are specific for melanoma cells, whereas the others are expressed in several cell types. Figure created with BioRender.com Inspired by [77].

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1.7.2 Transcriptional regulation of MITF

Together with related family members such as transcription Factor Binding To IGHM Enhancer 3 (TFE3) and the transcription factor EB (TFEB), MITF constitutes the “MiT” transcription factor family [79]. MITF binds as homo- or heterodimer to M-boxes (5’TCATGTG-3’) and E-boxes (5’CACGTG-3’) in the target genes. An important target gene is tyrosinase (TYR) and

tyrosinase-related protein 1 (TYRP1), which plays an essential role in melanocyte development by controlling differentiation in several cell types [80]. MITF-M is the most studied isoform, and little is known about the upstream promoters regulating the other isoforms. Therefore, we focus on transcriptional regulation controlling messenger ribonucleic acid (mRNA) expression of MITF-M. The MITF promoter is located upstream of several transcription factors that either repress or activate MITF expression [51]. Figure 14 illustrates a schematic diagram of the most relevant factors and signaling pathways in melanoma biology [51]. [78] [51]

Figure 13. Multiple factors regulate MITF activity and cellular programs. Transcriptional activity of MITF is regulated by a wide range of factors, which either activate or repress MITF activity. Epigenetics, microenvironment, modication of signaling pathways and genetic alterations are along these regulatory factors. Melanoma cells expressing high levels of MITF seem to differentiate or proliferate, whereas low MITF activity promotes stem cell-like properties or invasive potential. Figure created with BioRender.com, inspired by [78].

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19 SRY-related high-mobility group box 10 (SOX10), cAMP-response element-binding protein (CREB), and paired box homeodomain transcription factor (PAX3) are among the activating regulators of MITF-M. Loss of function of MITF can result in Waardenburg syndrome type IIA due to mutations in transcription factors PAX3 and SOX10, which include melanocyte loss and depigmentation due to disrupted dimer formation [79]. A low number of melanocytes affects the hearing and is one of the characteristics in both Tietz and Waardenburg syndrome, along with pale skin and light hair [81]. Cyclic AMP response (cAMP)- CREB signaling pathway activates MITF-M transcription by a series of events and is critical for UV response [51]. α-Melanocyte- stimulating hormone (α-MSH) activates the cAMP-CREB signaling pathway, which triggers MITF-M transcription. This, in turn, activates tyrosinase expression, resulting in increased pigmentation. MITF-M transcription can also be activated by WNT signaling due to the LEF- 1/TCF binding site in the MITF-M promoter, which is central for generating neural crest-derived melanoblasts and establishing melanocyte lineage through regulation of β-catenin [51].

Furthermore, MITF-M transcription can be activated by the Hippo signaling effector protein TAZ and YAP65 which work as PAX3 co-activators [79].

Notably, activating transcription factor 4 (ATF4) may act as a negative regulator by competing with cyclic AMP regulatory element-binding protein (cAMP) [51]. Other repressors of MITF-M expression are aristaless-like homeobox 3 (ALX3), which modulates pigmentation pattern in rodents [51]. GLI Family Zinc Finger 2 (GLI2) suppress MITF which promotes invasion and BRAF inhibitor resistance [51]. Deleted in esophageal cancer 1 (DEC1) is a bHLH transcription factor that represses MITF during hypoxia [51]. BRN2 is widely expressed in melanoma but not in melanocyte development, and can both up-and downregulate MITF expression [51]. BRN2 can bind elements within the MITF promoter but may also regulate transcription by cooperative

Figure 14. Transcriptional regulation of MITF-M expression. MITF has several transcriptional regulators which either activate or repress MITF expression through diverse signaling pathways. Positive regulators promoting MITF expression are indicated in green, while a red represents negative regulators suppressing MITF.

Notably, activating transcription factor 4 (ATF4) may negatively regulate by competing with CREB. Figure created with BioRender.com, inspired by [51,78].

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20 binding with other transcription factors [51]. Post-translational modifications of MITF such as phosphorylation enhance the transcriptional activity of MITF in general [82].

1.7.3 Post-translational regulation of MITF

The transcriptional activity described above depends on post-translational modifications and co- operating partners [82]. Several signaling pathways phosphorylates and thereby regulate MITF expression on protein level, including extracellular signal-regulated kinase 1/2 (ERK1/2) at Ser73, GSK3β at Ser298, p38 at Ser307, and p90^RSK at Ser409 (Figure 15) [82]. Phosphorylation will in general enhance the transcriptional activity of MITF. However, double phosphorylation of Ser73 and Ser409 promotes proteasome-mediated MITF degradation [82]. This mechanism is dependent on BRAF^V600E mutation, which causes enhanced activation of ERK1/2 pathway [82].

Unphosphorylated mutants at Ser73 and Ser409 are more stable yet transcriptionally

incompetent, suggesting a coupled mechanism of transcriptional activity degradation of MITF in melanoma cells [82]. MITF contains two positively charged fragments involved in DNA binding called transactivation domain (TAD), where co-activators such as p300/CBM interact [82].

p300/CBP affects these Serine phosphorylations and can lead to enhanced and attenuated MITF transcription activity [82]. Complex interactions between these phosphorylations can further inhibit these effects. p300/CBP works by linking transcription factors bound to DNA with basal transcriptional machinery [82]. SUMOlyation at Lys182 and Lys316 also regulates MITF expression through E1 SUMO-activating enzyme SAEI/SAEII and E2 SUMO-conjugating enzyme UBC9, which additionally regulate Lys201 [82]. Non-SUMOlyatible MITF mutants displayed increased transcriptional activity on multiple target genes, although it has been reported contradictory results [82]. [78]

Figure 15. Key regulatory mechanisms on MITF-M protein. Post-translational activities such as phosphorylation and SUMOlyation regulate the transcriptional activity of MITF. These regulations may affect each other (not shown) by inhibiting or enhancing effect. Figure created by BioRender.com, inspired by [78].

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21 Recently, several activators of MITF activity such as SOX10 and β-catenin have been identified.

MITF can redirect β-catenin from WNT signaling to engage activation of MITF dependent genes [82]. Homeobox transcription factor 1 (HOXA1), interleukin-1 receptor (IL-1R), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) have been connected to a suppressive role in MITF activity [82]. Several interacting partners regulate MITF on protein level, yet mechanisms of MITF regulations are still being explored.

1.8 Immunotherapy

Historically, the three most common treatments for cancer are surgery, radiation therapy, and chemotherapy. Future cancer treatment includes more personalized treatment based on genes, tumor heterogeneity, and resistance against the traditional treatments [83]. Immunotherapy uses medicines to activate our immune system to recognize and kill cancer cells, and is currently changing cancer treatment in melanoma [84]. For a better understanding of immunotherapy, a short overview of the immune system can be useful.

1.8.1 The immune system

The immune system consists of two parts: the innate immune system and the adaptive immune system. The innate immune system is the body’s first line of defense against non-self-pathogens, being nonspecific [85]. The response time is fast, and innate immunity causes inflammation at the site of the infection [85]. The innate immunity consists of neutral killer cells (NK-cells),

macrophages, neutrophils, basophils, eosinophils, mast cells, and dendritic cells (DC) (Figure 16) [85]. Some infections outrun the innate immune system; that’s when the adaptive immune system takes over. The adaptive immune system identifies and “remembers” germs, making the response more accurate and specific. The response time is longer, and the adaptive immunity develops against one pathogen, which is of little use for different pathogens [85]. Once the body has been in contact with the pathogen for the first time, the adaptive immune cells can react immediately next time [85]. The adaptive immune system consists of T and B lymphocytes (T and B cells). T cells can activate the adaptive immune system through chemical messengers (T helper cells/CD4+ helper T cells), detect and destroy viruses or tumorous cells (cytotoxic T cells/CD8+ killer T cells), or become memory T cells. B cells are activated by T helper cells, leading to the multiplication of the B cells and transforming them into plasma cells, producing high amounts of antibodies and releasing them into the bloodstream [85]. B cells can also become memory cells] [85]. Both cytotoxic T cells and NK-cells can recognize cancer cells and kill them directly. However, cancer cells can evade either being recognized or being attacked by immune cells [86]. In the latter, cancer cells can express membrane proteins that interact with proteins expressed on CD8+ killer T cells and dampen the immune response [86]. This mechanism is called the immune checkpoint, which the immune system uses to avoid immune activation against its cells (a more detailed description in 1.8.2 Metastatic melanoma ICT). A

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22 breakthrough within cancer treatment was thus to inhibit this immune checkpoint apparatus and stimulate the immune system to mount an immune response against cancer cells.

1.8.2 Metastatic melanoma ICT

Immune checkpoint therapy (ICT) has revolutionized cancer research and led to significant progress in melanoma and other advanced cancers; however, primary and acquired resistance has resulted in many patients not responding to treatment.

In 2002, most metastatic melanomas were known to harbor the BRAF^V600Emutation, but it was not until 2011 specific BRAF^V600Einhibitors vemurafenib and dabrafenib were FDA approved [24]. The results of these first targeted therapies were striking, with a 60% response rate.

However, secondary resistance developed only a few months later [24]. The BRAF^V600Emutation decreased MITF expression, whereas BRAF inhibitors increased MITF expression, driving melanoma towards differentiation and a less invasive state [50]. By combining BRAF^V600E inhibitors with mitogen-activated protein kinase kinase (MEK) inhibitors, we could now delay

Figure 16. Innate immunity VS adaptive immunity. The immune system consists of two parts: the innate and the adaptive immune system. The innate immune system is present at birth and causes rapid, non-specific immunity against foreign pathogens. The innate immunity consists of macrophages, NK-cells, DC, neutrophils, eosinophils, and basophils. If the innate immune system fails to destroy the pathogens, the adaptive immune system takes over.The adaptive immunity is created in response to exposure to a foreign substance, being specific, and fights specific infections. The response time is slow, yet the adaptive immune system can remember the particular pathogen and defeat it faster the next time. The adaptive immune system consists of B and T cells. Figure created with BioRender.com

Tankyrase inhibition regulates MITF expression in melanoma

Tankyrase inhibition regulates MITF expression in melanoma

Nann Louise Kristoffersen

Tankyrase inhibition regulates MITF expression in melanoma

Supervisors:

Dr. Jo Waaler

Ph.D. student Shoshy Alam Brinch Prof. Harald Carlsen

Master thesis

Faculty of Chemistry, Biotechnology and Food science

Norwegian University of Life Sciences

November 2021

Acknowledgment

Table of contents

Abstract

Sammendrag

Abbreviations

List of figures

List of tables

Introduction

1.1 Cancer

1.2 Malignant melanoma

1.3 The canonical WNT/β-catenin signaling pathway

1.4 The Hippo-YAP/TAZ signaling network

1.5 Tankyrase 1.5.1 Structure

1.5.2 Cellular function and enzymatic activity

1.5.3 Tankyrase inhibition suppress WNT signaling

1.5.4 Tankyrase inhibition suppress YAP/TAZ activity

1.5.5 Tankyrase inhibition and tankyrase inhibitors

1.6 Phenotype switching

1.6.1 WNT signaling mediated regulation of phenotype switching in melanoma

1.7 MITF

1.7.1 MITF-structure and expression

1.7.2 Transcriptional regulation of MITF

1.7.3 Post-translational regulation of MITF

1.8 Immunotherapy

1.8.1 The immune system

1.8.2 Metastatic melanoma ICT