Metastatic melanoma ICT - Tankyrase inhibition regulates MITF expression in melanoma

1. Introduction

1.8 Immunotherapy

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

23 the secondary resistance [24, 50]. Triple combinational therapy with BRAF, MEK, and PD-1 inhibitors has improved antitumor activity [87, 88].

Today´s standard care of metastatic melanoma patients are anti-PD-1 drugs, anti-Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) antibody, combinational therapy of both PD-1 and CTLA-4 inhibitors and interleukin-2 (IL-2) [89]. These ICT´s are not optimal as they have a low success rate and a high rate of severe side effects. CTLA-4 inhibitors can cause liver problems, nerve and hormone gland inflammation, eye problems, and skin reaction [89]. IL-2 can cause flu-like symptoms, gastrointestinal side effects, and capillary leak syndrome [89]. PD-1 and PD-L1 inhibitors are a better target for melanoma cells, shrinking melanoma for 25-45% of patients with fewer side effects and reduced risk of cancer coming back after surgery [89]. Immune checkpoint monotherapy shows less impressive outcomes, while combined, the response rate may reach 50%

[50]. In our studies, our goal is to further increase the response rate to PD-1 inhibitors by combining this therapy with tankyrase inhibitors and stabilizing MITF.

Antigen-presenting cells (APCs) present tumor antigens to T cells via major histocompatibility complex (MHC) proteins on the APCs and the T cell receptor (TCR) (Figure 17). PD-1 is

expressed on activated T cells, and when it binds to the PD-L1 ligand on the tumor cell, the T cell is inactivated [90]. Immune therapies use immune checkpoint inhibitors to prevent the specific binding between PD-L1 on the tumor cell and PD-1 on the T cell, which otherwise results in survival of the cancer cells [90]. The blockade allows the T cell to recognize and kill the tumor cells [90]. In the endolysosomal pathway, antigenic peptides are produced from degraded

proteins [50]. In melanomas, lysosome-related organelles called melanosomes carry this function [50]. Interestingly, MITF drives melanosomal and endolysosomal biogenesis and function in melanoma [50]. In addition, melanosomes and endolysosomes can modify the presentation of MHC-restricted epitopes [50].

Although immunotherapy may seem promising and increases the survival rate compared to chemotherapy/targeted therapy, 40-70% of cancer patients do not respond to immune checkpoint inhibition [25]. Combinational therapies can sensitize cells to immune checkpoint blockade and increase the survival rate even more [25].

1.8.3 WNT/Hippo signaling regulations of immune cells

Development of immune cells and WNT signaling activity are connected as the immune cells originate from hematopoietic stem cells, which can differentiate to any blood cell, which in turn can mature into immune cells [91]. WNT signaling tightly regulates stem cell development, differentiation, tissue repair, and immune cell function [91]. The primary role of WNT signaling in B cells, T cells, and NK cells is development [91]. WNT signaling affects T cells in several ways. T cells are produced in the bone marrow and mature in the thymus, where WNT signaling substantially affects thymopoiesis [91]. In addition, WNT transcription factors TCF1 and LEF1 are essential for establishing CD8+ T cell identity [91]. Furthermore, β-catenin induces self-renewal and differentiation for CD8+ T cells [91]. WNT signaling regulates B cell proliferation through LEF1, and WNT3a stimulation decrease the proliferation potential of B cells, although WNT signaling in B cells isless understood. Macrophages utilize WNT signaling for tissue repair and regeneration and are regulated during infection or inflammation [91].

Recent studies show a connection between the Hippo-YAP/TAZ network and the regulation of innate and adaptive immune responses [84]. The complexity of the Hippo signaling pathway involves it in many biological functions such as cell fate determination [92]. MST1/2 kinases have been shown to independently of YAP/TAZ, and LATS1/2 regulate lymphocyte biology, affecting T cell development and differentiation and B cell homeostasis [92]. Patients with MST deficiency show susceptibility to virus and bacterial infections and signs of lower amount T and B cells in the body [92]. For DCs, MST1/2 regulates adhesion and cell motility and, therefore, the migration of DC. YAP and TAZ affect various T cells’ differentiation, and T cell transcription

Figure 17. Mechanism of action of immune checkpoint inhibitors. Left panel: MHC on the APC binds to TCR on the tumor cell. Without an immune checkpoint inhibitor, the PD-1 expressed on an active T cell will bind to ligand PD-L1 on the tumor cell, leading to T cell exhaustion and therefore survival of the tumor cell. Right panel: Blockade of the PD-1 or PD-L1 by immune checkpoint inhibitors result in antitumor responses by the T cell. Figure adapted from “Immune Checkpoint Inhibitor against Tumor Cell”, BioRender.com (2021).

25 factors [92]. YAP/TAZ are rarely expressed in multiple immune cells, and further investigation of their regulatory roles is needed.

1.8.4 MITF, the immune system, and immunotherapy

The use of monoclonal antibodies to prevent CTLA-4 or PD-1 engagement led to a paradigm shift in melanoma treatment, yet immune checkpoint inhibition is limited in melanoma because of acquired resistance. Resistance mechanisms of ICT include poor immunogenicity of the tumor, altered T cell infiltration, and reduced T cell killing activity [50]. In addition, the tumor

microenvironment (inflammation, hypoxia, nutrients) also affects resistance towards ICT in melanoma. These signals in the tumor microenvironment may decrease MITF levels and promote phenotype switching towards more invasive melanoma cells [50]. Regarding ICT, melanomas with a higher tumor mutational burden (TMB) due to UV radiation respond better to anti-PD-1 therapy than those with intact DNA repair systems [50]. MITF promotes genomic stability by regulating various DNA repair genes. Hence, MITF^low expressing cells should be favoring genomic instability [50]. MITF^lowexpressing cells is associated with invasiveness and mesenchymal state of the melanoma cells, and MITF^highexpressing cells push cells towards differentiation [73]. Studies also indicate that MITF^low cells are more resistant to

immunotherapies, being more dedifferentiated and harboring lower expression of immunogenic target antigens, in addition to producing inflammatory secretomes, leading to decreased immune cell recruitment [50]. Thus, MITF^lowcells may be harder to detect for T cells. However, not all patients with high TMB respond to ICT, and the clinical benefit of a high TMB alone is low [50].

UV radiation may also favor aggressive melanoma [50]. Figure 18 illustrates the interactions of MITF and oncogenic pathways such as WNT, BRAF, and PI3K/AKT. Active β-catenin, the gain of function mutation in β-catenin, loss of function in negative regulators in WNT signaling, and increased expression of β-catenin effectors can induce a “cold tumor” phenotype, meaning non-immunogenic tumors that have not been infiltrated by T cells [50]. The PI3K/AKT pathway is frequently activated in melanoma due to loss of function in the tumor suppressor Phosphatase and TENsin homolog (PTEN) [2]. Mutation in PTEN often correlates with a mutation in BRAF, rending melanoma cells resistant to T cell-mediated immunotherapy [50]. Previous studies have also shown that MITF regulation influences chemokine expression on both transcriptional and protein levels. Reduction of cytokine secretion and PD-L1 expression both favor

immunosurveillance [50]. However, there is a need for more detailed research on different levels of MITF and its impact on chemokine expression and immune cell infiltration in melanoma cells

26 [50, 73]. [50]

Figure 18. Immune response in melanoma through interactions of MITF at post-translational level. Oncogenic signaling pathways such as WNT, PI3K/AKT, and BRAF in melanoma regulate MITF activity, which regulates immune response. Most melanomas harbor the BRAF^V600E mutation, whereas BRAF inhibitors increase MITF levels, favoring immunosurveillance. The PI3K/AKT pathway is frequently activated in melanoma, leading to resistance towards T cell-mediated immunotherapy. PI3K inhibition has been shown to increase MITF levels and differentiation antigens.

Downstream of PI3K is WNT signaling pathway, where overly active β-catenin can result in T cell exclusion, showing resistance to ICT. No inhibitors targeting WNT signaling pathway exist in clinical practice. KARS1 operates in

melanocytes and regulates antigen expression through the regulation of MITF. Reduction of cytokine secretion and PD-L1 expression favors immunosurveillance. Figure created by BioRender.com, inspired by [50].

2. Aims of the study

WNT/β-catenin-induced immune evasion is found in multiple cancer types. Still, no approved drugs targeting this pathway are in clinical practice today. This project is preliminary research where the main objective is to evaluate if tankyrase inhibitor-induced regulation of WNT and YAP signaling, as well MITF, renders melanoma more sensitive to checkpoint inhibition in vivo.

To achieve the main objective, we formulated eight sub-aims to start the process:

I. Examine how G007-LK treatment would affect components of the WNT/β-catenin and YAP signaling pathways in B16-F10 melanoma cells

II. Investigate whether G007-LK efficacy is dependent on β-catenin

III. Evaluate if YAP and WNT/β-catenin signaling pathways can regulate MITF expression

IV. Examine whether MITF is accumulated in the nucleus or the cytoplasm

V. Investigate if G007-LK could affect localization of MITF, YAP and β-catenin proteins VI. Evaluate if G007-LK can affect EMT-like and phenotype switching in B16-F10

melanoma cells

VII. In a pilot study, we wanted to investigate how G007-LK affects components of the WNT/β-catenin and YAP signaling pathways in human melanoma cell line panel VIII. In a pilot study, we wanted to investigate if tankyrase inhibition can regulate MITF

expression in human melanoma cell line panel

3. Materials and Method

3.1 Cell culture and maintenance

Equipment and instruments with product name, catalogue number and producer are listed in Appendix B, Supplementary Table 7.

3.1.1 Cell lines

The murine melanoma cell line B16-F10 derived from the skin of the albino mice strain

C57BL/6N was used in experiments to evaluate MITF and phenotype switching upon tankyrase inhibitor treatment. To investigate β-catenin-mediated immune evasion and whether WNT signaling affects MITF expression in B16-F10, two β-catenin knockout cell lines were produced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based knockout (KO). B16-F10^Ctnnb1KO was designed using CHOPCHOP v2, a web tool of CRISPR genome engineering [25, 93]. The knockout sequence from wild-type B16-F10 was 5′-

GATTAACTATCAGGATGACG-3′, and by amplifying this gene fragment in a polymer chain reaction (PCR), we could verify a successfully performed knockout. Immunofluorescence with β-catenin was also performed for additional verification [25].

The effect of tankyrase inhibitors on WNT and YAP signaling depends on cell lineage and context [25]. Previous studies have shown synergistic anti-PD-1/tankyrase inhibitor treatment effects in B16-F10 tumors [25]. However, genetic background and cell signaling pathways may cause variable results between different melanomas [25]. Furthermore, the B16-F10 murine mouse model only partially recapitulates human melanoma. Therefore, a panel of human melanoma cell lines shared by Dr. Eivind Hovig (Oslo University Hospital-Radiumhospitalet) was utilized further to investigate the effects of tankyrase inhibitors in vitro. This master thesis used the following human cell lines listed in Table 1 to analyze transcriptional and protein level regulation.

29 Table 1. The panel of human melanoma cells

Human melanoma cell lines

3.1.2 Cultivation of cells

All cell lines were cultured in cell culture flasks at 37 °C in 5% CO2. Cells were cultured in Roswell Park Memorial Institute – 1640 medium (RPMI) with 5% fetal bovine serum (FBS), providing amino acid and growth factors. To prevent bacterial contamination, 1%

penicillin/streptomycin solution (Pen-strep) was added to all cell culture mediums.

3.1.3 Cell splitting and passaging

All cell cultures were inspected daily using a microscope to keep cells in an exponential growth phase and a maximum confluence below 80-90%, before passaging the cells in a 1:10 split ratio about twice a week. After removing the used cell culture medium, the cells were washed once using phosphate-buffered saline (PBS) and split with trypsin/EDTA solution detaching the cells from the plastic surface of cell culture flasks. The flasks were next incubated for ~10 minutes at 37 °C until the cells were visibly detached. The addition of new medium containing FBS leads to the inactivation of trypsin. An increasing passaging number was noted on the flask for each cell culture passage, and all cell cultures were kept below 20 passages. All work with cell culture was performed in sterile environments.

3.1.4 Cell seeding

Cells were seeded one day before treatment in an appropriate amount to reach ~80-90%

confluence after 72 hours of treatment.10 µl of detached cell suspension sample were inserted into each chamber on Bio-Rad counting slides for determining cell concentration before seeding.

The slides were inserted into a TC20™ automated cell counter. After determining cell

30 concentration, an appropriate amount of cells were calculated and seeded depending on the concentration and size of the seeding plate ratio (Table 2).

Table 2. Plate seeding ratio of cells Plate Cell number

Aliquots cell cultures, with low passage numbers, were stored in liquid nitrogen at -196ºC for long-term storage. After detaching the cells with trypsin, the cells were resuspended in 1 ml medium containing 10% Dimethyl Sulfoxide (DMSO), a cryoprotective agent, and transferred to a CryoTube. The aliquots were transferred to a Mr.Frosty™ Freezing container, with 100%

isopropyl alcohol and kept at -80°C. Storage of Mr.Frosty overnight at -80°C leads to a slow cooling rate at about 1°C/min. CryoTubes were transferred to liquid nitrogen the next day for optimal preservation.

3.1.6 Cell thawing

Frozen cells were taken from the nitrogen tank and thawed in a 37°C water bath for about 1 minute. 1 ml of cells were transferred to 15 ml Falcon tubes and centrifuged at 2000 rounds per minute (rpm) for 3 minutes. After removing the supernatant, the remaining pellet was

resuspended in 1 ml pre-warmed cell culture medium and transferred to a cell culture flask.

3.1.7 Cell line authentication and mycoplasma detection

Mycoplasma testing of all cell cultures was performed monthly, to ensure the absence of mycoplasma contaminations, using a MycoAlert™Mycoplasma Detection Kit. The kit detects common contaminants through luciferase enzymes in the Mycoalert TM substrate. Cell cultures infected with mycoplasma can acquire abnormal cell physiology, metabolism, and cell growth.

Further effects of mycoplasma contamination are chromosomal aberrations, change of gene expression patterns, cell death, and changes in cell membrane antigenicity. Significant sources of mycoplasma contamination in cell cultures are medium, improper sterilization, laboratory

personnel, and incubation [94]. Two chambers were used to minimize mycoplasma spreading in the 5% CO2 chamber used for cell incubation. One was mycoplasma-free cultures (tested), and the other was cultures yet to be tested (quarantine). All cultures used in this study were free of mycoplasma.

3.1.8 Drugs and substances

The first tankyrase inhibitors discovered for repressing WNT signaling were XAV939, and IWR-1 [95]. Specific tankyrase inhibitors such as G007-LK were later discovered, a hydrophobic small-molecule that dose-dependently inhibits cell growth. G007-LK suppresses WINT signaling by blocking the PARsylation activity of tankyrase (see 1.5 Tankyrase) and suppressing YAP signaling by upregulating AMOT proteins (see 1.5.4 Tankyrase inhibition suppress YAP/TAZ activity). In our lab, G007-LK (molecular weight of 529,96) was dissolved in DMSO, resulting in a 10 mM stock solution, and stored at -4°C. For in vitro experiments, the stock was further diluted to 10 nM in a cell culture medium.

WNT3a is one of 19 members of WNT ligands, which activates the WNT signaling pathway [96]. In our studies, recombinant WNT3a was used. WNT3a was diluted with 0.1% BSA.

Combinational treatment with WNT3a and G007-LK was used to investigate if G007-LK could counteract the WNT signaling activating effect of WNT3a.

3.2 Real-Time RT-qPCR

Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to quantify the gene expression level of purified RNA molecules, using reverse transcription to synthesize RNA to DNA via a series of heating and cooling cycles. Each cycle amplifies the DNA, where the DNA expression is measured with fluorescence that correlates with the amplification of PCR products.

3.2.1 RNA isolation and cDNA synthesis

Isolation of RNA from cell cultures was performed using column-based Qiagen RNeasy Mini Kit. RNA purity was determined using NanoDrop 2000 spectrophotometer. To achieve equal RNA purity before synthesizing complementary DNA (cDNA), each isolated RNA sample was diluted with the appropriate amount of nuclease-free water. The cDNA was synthesized from 0.5-2 µg isolated RNA using a High-Capacity cDNA Reverse Transcription Kit

3.2.2 Preparation of real-time RT-qPCR

Real-time RT-qPCR was performed using TaqMan Gene expression Master Mix (Table 3) on ViiA^TM 7 Real-Time PCR systems with an amplification protocol sat to 40 cycles with three replicates of samples and probes. Tables 4 and 5 show the probes used in this study.

32 Table 3. Real-time RT-qPCR mix

Reagent Volume

Master Mix

(buffer, dNTP, primers, reverse transcriptase, nuclease-free H2O) 5 ul

DEPC water 3 ul

Probe (1:1) 1 ul

cDNA 1 ul

Total volume 10 ul

Table 4. Mouse probes for RT-qPCR Probe Catalogue number

Table 5. Human probes for RT-qPCR Probe Catalogue number GADPH Hs02758991_g1 AXIN2 Hs00610344_m1 CTGF Hs00170014_ml AMOTL2 Hs01048101_ml

Analysis of the real-time RT-qPCR data was performed using the comparative quantification method ( Ct). All samples were normalized to the housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The difference between Ct values of the gene of interest and Gapdh was calculated ( Ct), after that the difference between the Ct value for each sample value and the Ct of the control sample was calculated:

Next, the Ct value was used to calculate the relative quantification (RQ) value, showing relative gene expression changes:

3.3 Immunoblot analysis

Immunoblotting is a laboratory technique used to separate and visualize specific proteins from a mixture of proteins, extracted from cells. After separating proteins by size through

electrophoresis, the proteins are transferred to solid support where specific antibodies can bind.

After incubation of both primary and secondary antibodies, detection and quantification of the proteins follow [97].

3.3.1 Sample preparation

After 72 hours of treatment, cells were washed with cold PBS and fractionated into cytoplasmic and nuclear extracts. First, the cells were lysed in NP-40 buffer containing cOmplete protease inhibitors and PhosSTOP phosphatase inhibitors (1 tablet per 10 mL NP40) for 30 minutes on ice. After incubating, the cells were scraped and collected into 1.5 μl Eppendorf tubes. NP-40 disrupts the plasma membrane, allowing pelleting of the nuclei and cell debris to the bottom of the Eppendorf tube, while the cytoplasmic extracts remain in the supernatant. Cell lysates were centrifuged at 15,000 rpm for 15 minutes to collect the pellet, separating the nuclei and

cytoplasmic fractions. After transferring the supernatant into a second tube, radio immune precipitation assay (RIPA) buffer was added to the pellet to disrupt the nuclear envelope. The pellet containing the atomic extract was sonicated for 3x10 minutes with Biorupter Plus

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