Investigation of drug-induced cell cycle responses in high-risk neuroblastoma

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IN

DEGREE PROJECT BIOTECHNOLOGY, SECOND CYCLE, 30 CREDITS

STOCKHOLM SWEDEN 2020,

Investigation of drug-induced cell cycle responses in high-risk

neuroblastoma

MARYAM SAHI

KTH ROYAL INSTITUTE OF TECHNOLOGY

SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

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Investigation of drug-induced cell cycle responses in high-

risk neuroblastoma

Master Thesis

Maryam Sahi

Master thesis project in Medical Biotechnology KTH Royal Institute of Technology May 26^th, 2020, Stockholm, Sweden

Project location: Department of Women’s and Children’s Health | Division of Paediatric Oncology, Karolinska Institutet Supervisor (external, KI): Shahrzad Shirazi Fard | PhD

Supervisor (KTH): Cristina Al-Khalili Szigyarto| PhD Examiner (KTH): Yves Hsieh | PhD

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Abstract

The childhood cancer neuroblastoma mostly affects children under the age of 2 and comprises 6% of all childhood cancers. Neuroblastoma has very diverse phenotypes caused by both inter- and intra- tumour heterogeneities. The phenotypes are classified as being either low- or high-risk. This project focuses on high-risk NB cell lines with various chemotherapy sensitivity. Titration studies with chemotherapy agents cisplatin or doxorubicin showed a proneness of p53 mutated cell lines to arrest in either the S- and/or the G2/M-phase, depending on the drug and the drug dosage, indicating on a dose-dependent cell cycle response. To potentially inhibit the cells from arresting a treatment assay with 3 cell cycle key-components, pATM, Chk1 and Wee1 inhibitors was done. An initial immunocytochemistry staining of the expression levels of pATM and Wee1 showed that pATM was upregulated for 5 out 7 tested cell lines, namely SK-N-SH, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, upon chemotherapy treatment with doxorubicin. Wee1 was however only upregulated for 3 out 7 cell lines;

Kelly, SK-N-DZ and BE(2)-C. The upregulation of pATM and Wee1 showed a potential confirmation of their involvement in CT induced cell cycle arrest. Upon inhibition of pATM, Chk1 and Wee1 diverse effects were observed for each cell line (SK-N-SH, SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C). Wee1 showed the most promising results were the cell viability decreased for all 5 p53 mutated cell lines and the confluency over time decreased for 4 out 5 p53 mutated cell lines. The p53 wild type cell line SK- N-SH was less sensitive towards Chk1 and Wee1 inhibition indicating that cell lines with functional p53 might not be as dependent on the Chk1 and Wee1 pathways compared to cell lines with non-functional p53. Thus, targeting the cell cycle arrest might be a promising therapeutic target for high-risk neuroblastoma.

Key words

Neuroblastoma, chemotherapy resistance, cell cycle arrest, cisplatin, doxorubicin, ATM, Chk1, Wee1

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Sammanfattning

Barndomscancern neuroblastom utgör 6% av all barncancer. Majoriteten av de drabbade är under 2 år. Neuroblastom har en stor mångfald av fenotypiska utryck som orsakas av dess inter- och intra- tumör heterogenitet. Fenotyperna klassificeras antigen som låg- eller högrisk. Här har 7 högrisks- neutoblastom cellinjer med varierande grad av känslighet mot kemoterapi analyserats.

Titreringsstudier med kemoterapierna cisplatin och doxorubicin påvisade en benägenhet för de p53 muterade cellinjerna att arrestera i S- och/eller i G2/M-fasen, beroende på behandlingen samt behandlingsdosen, vilket indikerar på en dos-beroende cellcykel respons. En behandlingsanalys med de 3 nyckelkomponenterna fosforylerat ATM, Chk1 samt Wee1 gjordes för att potentiellt inhibera cellerna från att arrestera. Efter en initial immunocytokemi infärgning av pATM samt Wee1 visade 5 av 7 cellinjer (SK-N-SH, SK-N-FI, Kelly, SK-N-DZ samt BE(2)-C) en uppreglering av pATM-uttryck till följd av doxorubicin behandling. Däremot var Wee1 endast uppreglerat för 3 av 7 cell linjer (Kelly, SK-N-DZ samt BE(2)-C). Uppregleringen av pATM och Wee1 påvisar ett potentiellt samband mellan kemoterapi- inducerad cellcykelarrest och ökat utryck av pATM och Wee1. Vid inhibering av pATM, Chk1 samt Wee1 gav Wee1 de mest lovande resultaten där cellviabiliteten minskade för samtliga 5 p53-muterade cellinjer och där konfluensen över tid minskade för 4 av 5 p53-muterade cellinjer. SK-N-SH med funktionerande p53 var mindre känslig gentemot Chk1 och Wee1 inhibering, vilket indikerar att cellinjer med funktionerande p53 inte är lika beroende av reaktionsvägarna för Chk1 och Wee1 jämfört med cellinjer som har icke-funktionerande p53. Därmed kan riktad behandling mot cellcykelarrest vara en lovande behandling för högrisks-neuroblastom.

Nyckelord

Neuroblastom, kemoterapiresistans, cellcykelarrest, cisplatin, doxorubicin, ATM, Chk1, Wee1

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Abbreviations

ATM – Ataxia telangiectasia mutated kinase CDK– Cyclin dependent kinase

Chk1 – Checkpoint kinase 1

Chk1i – Checkpoint kinase 1 inhibitor Chk2 – Checkpoint kinase 2

DAPI – 4',6-diamidino-2-phenylindole Cisplt – Cisplatin

CT – Chemotherapy Doxo – Doxorubicin

Edu – 5-Ethynyl-2'-deoxyuridine

FACS – Fluorescence-activated cell sorting ICC – Immunocytochemistry

MTS – 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium MYCN – N-myc proto-oncogene protein

NB – Neuroblastoma NS – Not significant

pATM – Phosphorylated ataxia telangiectasia mutated kinase

pATMi – Phosphorylated ataxia telangiectasia mutated kinase inhibitor PFA – Paraformaldehyde

PH3 – Phosphohistone H3 SD – Standard deviation Wee1i – Wee1 inhibitor

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1. Introduction

Neuroblastoma (NB) is a cancer where majority of the cases are children under the age of 2 (1). In Sweden alone NB constitute 6% of all childhood cancer cases, each year approximately 20 new cases are reported (2). The tumours are formed from the peripheral nervous system, and both genetic and non-genetic mechanisms have been suggested as the cause of resistant phenotypes (3). It is common for NB to appear in the adrenal gland and in the nerve tissue around the spinal cord (2). Neuroblastoma has both inter- and intra-tumour heterogeneities that results in several different phenotypes with low- and high-risks (3). The low- and high-risk phenotypes express different kinds of disease symptoms.

Low-risk phenotypes include tumours that do not have N-myc proto-oncogene protein (NMYCN) amplification, are confined to one area and can be completely removed surgically (4). Whereas, metastasised or MYCN amplified phenotypes commonly are classified as high-risk (4). High-risk NB is treated both surgically and together with different chemotherapy (CT) combinations, such as the first line treatment cisplatin (cisplt) (5) and the second line treatment doxorubicin (doxo) (5), in combination with stem-cell transplantation (2). Cisplatin is a cytotoxic, alkylating agent (6). It adds alkyl groups to the bases in the DNA, resulting in cross-linkage of guanine and thereby prohibiting the separation of DNA for replication. The DNA is instead enzymatically fragmented in an attempt of the cell to replace the alkylated bases. Furthermore, both DNA synthesis and RNA transcription are blocked. The alkylation could also contribute to DNA base mis-pair, which could cause mutations (7)(8)(9). The cytotoxic doxo is an antineoplastic anthracycline. It has several different suggested mechanisms of action. The most common is that it forms complexes with the DNA causing intercalation, strand breakage and topoisomerase II inhibition through stabilisation of the DNA- topoisomerase II complex (10)(11). The survival rate for all NB patients, both low- and high-risk is currently at 75% (2).

The leading cause of death for cancer patients is relapse after treatment. This is believed to be linked to intra-tumour heterogeneity (12)(13). Chemotherapy (CT) resistance is commonly built up through random mutations required by some cells during treatment. According to novel research almost all relapses are caused by CT resistance. Usually when a tumour is treated with CT the majority of the tumour cells dies, but a small fraction of cells that have required resistance towards the therapy remain. Following treatment, when the competing cells are gone, the resistant cells can proliferate more easily, resulting in a relapse (12)(13). There are two leading theories for the CT acquired resistance of NB: the stem cell theory and the cell cycle arrest theory. The stem cell theory claims that the cells responsible for the relapse stems from the non-proliferating G0-pool, were they acquire different mutations during CT, making them resistant towards it and thereby able to proliferate, causing a relapse (14). However, the cell cycle arrest theory, which will be the focus of this study argues that the cells arrest at different phases in the cell cycle upon CT treatment (3). The arrest protects the cells from the CT which then can enter the cell cycle again when the CT has dissipated (3).

A common mode of action for CTs is to induce DNA damage in order to stop the cell cycle (proliferation) and induce cell death. The cell cycle consists of 4 phases. The G1-phase, where the cell size increases, cell specific biomolecules are manufactured, and the cell prepares itself for DNA-synthesis. The S- phase, where the DNA synthesis and replication occur. The G2-phase, consisting of several different control mechanisms which makes sure that all the required components are present and assures the DNA quality. And lastly the M-phase, where the cell division occurs through mitosis. When the cells have stopped dividing and are focused on their normal housekeeping functions, they are in the G0- phase (quiescence) (figure 1) (15)(16).

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6 Each transition between phases has a checkpoint with different control mechanisms in order to make sure that the required processes for the current phase has been correctly completed, otherwise the cell cycle will be arrested. For cancer research and therapy the two most relevant checkpoints are the G1/S-phase and the G2/M-phase transition points (15)(16). An important checkpoint for DNA damage inducing CTs, such as cisplt and doxo, is the G2/M-checkpoint, where the cell arrests if there is any DNA damage (15). Previous papers have identified a doxo induced G2/M-phase arrest in a subpopulation of high-risk NB cell lines. This arrest preceded regrowth and was suggested to protect the tumour cells from further CT treatment (3)(17). In this study the cell cycle phase will be determined by fluorescence-activated cell sorting (FACS) in combination with Hoechst, which is a blue fluorophore that binds to dsDNA (18). Hoechst is very useful for cell cycle phase analysis since the amount of DNA varies between the phases and the intensity of Hoechst varies depending on the DNA concentration.

One potential way to eliminate the resistant subpopulations and thereby inhibit their regrowth could be to prohibit them from arresting. The Ataxia Telangiectasia Mutated (ATM) kinase, is activated through phosphorylation (pATM) upon double strand breakage (19). The ATM signal transduction activates several different enzymes such as the Checkpoint kinase 1 (Chk1), Checkpoint kinase 2 (Chk2) and p53 (figure 2) (20). Chk2 for example, has shown to function as a negative regulator of mitotic catastrophe, where inhibition of it in combination with CT has been shown to induce apoptosis in cancer cells (21). The serine kinase Chk1 is active during both the S and G2/M-phase (19)(22). When Chk1 is activated the phosphatases Cdc25A and Cdc25C in the cell cycle cyclin cascade are phosphorylated, causing them to either degrade or become unfunctional (22). This leads to cyclin dependent kinases (CDKs) to accumulate and thereby cause a cell cycle arrest (22). Chk1 has also shown to be over expressed in several different cancer types (22).

The tyrosine kinase Wee1 works in a parallel pathway to the ATM-pathway (20) and is involved at several phases of the cell cycle (figure 2) (19). One of its functions is to regulate the G2/M-phase transition to the mitotic stage at the G2-checkpoint were the cell cycle is arrested if there is any DNA damage (19). This is done through inactivation of the CDK2/cyclin B complex via phosphorylation of CDK2 (19). The G2/M-phase arrest caused by Wee1 gives the cells an opportunity to repair DNA

Figure 1. The cell cycle (figure from Chin, C.F and Yeong F.M. (45)). Cell cycle phases with phase transitions checkpoints (red) and other internal checkpoints (green and grey).

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7 damage, which is unwanted for cancer cells treated with DNA damaging chemotherapies. Wee1 has, as Chk1, shown to be over expressed in NB cell lines (22). If Wee1 is over expressed it could be used as an inhibition target in order to inhibit the cells to arrest in the G2/M-phase. They would instead be able to continue into the M-phase where they could undergo mitotic catastrophe (23). The expression of the enzymes can be measured through, for example immunocytochemistry (ICC), where a targeted antibody either directly or indirectly linked to a fluorophore, binds to the desired antigen. ICC consists of 4 main steps, namely cell seeding, fixation and immunostaining, imaging, and lastly annotation and image analysis (24). The cells are seeded through cultivation on for example glass slides, then they are fixated (24). The fixation method varies depending on the desired application. Some common ways of fixation are acetone, ethanol, methanol, and paraformaldehyde (PFA) (25). PFA is useful when no permeabilization is required since it keeps the cells intact though cross-linkage of the membrane surface proteins (25). However, a pitfall of PFA is over fixation where the cross-linkage masks the epitopes of the antigens if fixated for too long (25). After the fixation, the cells are immunostained with targeted antibodies. This can be done with either direct immunostaining where the fluorophore is bound to the primary antibody or the more specific method with indirect immunostaining where a primary antibody first binds to the antigen and then a secondary antibody with the fluorophore binds to the primary antibody (24). The cells are usually mounted with 4',6-diamidino-2-phenylindole (DAPI), which is a nuclear stain (26) and then analysed in a fluorescent microscope (24). In this study the expression of pATM and Wee1 will be measured through indirect immunostaining.

Figure 2. Cell cycle regulators (figure from Lin, A.B. et al.(24). Pathways for ATM, Chk1 and Wee1 upon DNA damage. ATM is in most cases activated upon double stranded break, whilst Chk1 is more focused on single strand breaks.

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8 The parallel Wee1 and ATM pathways are both able to cause cell cycle arrest at the G2/M-phase, which makes them good potential targets for combination treatment (22). This is particularly interesting for p53 mutated (p53mut) cell lines, where the mutation in the tumour suppressor p53 causes the G1- checkpoint to not function properly, thereby making the G2/M-phase checkpoint even more crucial (22)(27). This could be one of the reasons for the overexpression of the G2/M-phase regulating Wee1 and Chk1 indicated by previous papers (22), meaning that cells with an over expression of G2/M-phase regulation could repair CT induced DNA damage more efficiently (22), leading them to become therapy resistant. Thereby, pATM inhibition makes an interesting combination alternative together with Wee1 inhibition, Chk1 inhibition and doxo, targeting both main pathways of the cell cycle regulators.

This projects aims at investigating the cell cycle behaviour for a panel of high-risk NB cell lines upon CT treatment with a titration series of cisplt or doxo, together with potential expression and

inhibition of the cell cycle regulators pATM, Wee1 and Chk1. Previous papers have identified a CT induced G2/M-phase arrest in a subpopulation of high-risk NB cell lines. This arrest preceded regrowth and was suggested to protect the tumour cells from further CT treatment (3)(17). This was shown for the NB cell line BE(2)-C where the Ki-67 positive and both phosphohistone H3 (PH3) and 5- Ethynyl-2'-deoxyuridine (EdU) positive cells substantially increased compared to the mock upon a single treatment of 1 µM Doxorubicin (3). The molecular marker Ki-67 is present in all phases except G0 and is therefore used to see if the cell has entered the cell cycle (28). The thymidine analogue Edu is incorporated into the DNA in the S-phase and can therefore be used to detect if the cell has

progressed through the S-phase (29). Histone phosphorylation occurs in late G2/M-phase, which makes PH3 a good evaluation marker for those phases (30). On top of the overexpression of Wee1 and Chk1 in NB (22), it has also been shown that inhibition of the Chk1 pathway in combination of cisplt or doxo enhances the antitumor effects of cisplt and doxo (31), as well as ATM deficient cells being more sensitive towards certain CTs (32). Inhibiting resistant cells from arresting in G2/M-phase through inhibition of cell cycle regulators such as pATM, Wee1 and Chk1, could thereby be a way of eliminating this subpopulation and thereby inhibiting regrowth, wherefore the inhibition of these 3 key components will be investigated in this project.

2. Material and Methods

2.1 Cell lines and cell culture

Seven NB cell lines; SK-N-SH, IMR-32, SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, were cultured in complete medium consisting of RPMI 1640, 10% fetal bovine serum, 1% L-glutamine and 1%

pencillin+streptomycin, at 37^oC, with 5% CO2. All of the medium components were obtained from Thermo Fisher Scientific. The cell lines were kindly gifted by Per Kogner at the department of Women’s and Children’s health, Karolinska Institutet. Authentication was done through STR analysis of the extracted DNA from the respective cell lines. The STR was done with the QiAmp Micro kit from Qiagen after the FACS analysis and ICC, but prior to the confluency assays. The extracted DNA was then sent for authentication to an external company.

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9 Table 1: The 7 NB cell lines used, with a selection of different mutations

The table, which can also be found in the currently unpublished article “Chemotherapy-provoked cell cycle arrest precedes regrowth in high-risk neuroblastoma cell lines.” from Jönsson, L.Ö. et al. (17), lists relevant common mutations found in NB cell lines.

* Due to the mutation the ALK phosphorylation increases which causes cell growth and downstream signalling (33).

Cell line p53 status NMYC status 11q status 17q status Alk-

mutations Origin Ref

SK-N-SH Functional (wild type)

Wild type Wild type Gain F1174L* Metastasis location: Bone marrow

Patient age: 4 years Gender: Girl

(34) (35)

IMR-32 Functional (wild type)

Amplified Deletion Gain Wild type Metastasis location:

Abdominal mass Patient age: 13 months Gender: Boy

(34) (36)

SK-N-AS Non- functional (mutated)

Wild type Deletion Gain Wild type Metastasis location: Bone marrow

(34) (37)

SK-N-FI Non- functional (mutated)

Wild type Wild type Gain Wild type Metastasis location: Bone marrow

Patient age: 11 years Gender: Boy

(34) (38)

Kelly Non-

functional (mutated)

Amplified Deletion Gain F1174L* Metastasis location: Brain Patient age: 1 year Gender: Girl

(34) (39) (40) SK-N-DZ Non-

functional (mutated)

Amplified Deletion Gain Wild type Metastasis location: Bone marrow

(34) (41)

BE(2)-C Non- functional (mutated)

Amplified Wild type Gain Wild type Metastasis location: Bone marrow

Patient age: 22 months Gender: Boy

Additional info: Cloned subline of SK-N-BE(2).

Retrieved after repeated chemo and radiotherapy.

(34) (42)

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2.2 Cisplatin and Doxorubicin titration with FACS analysis

To mimic the drug distribution in patient tumours a titration series of the first line CT cisplt (Apoteket AB, Sweden) and the secondary CT doxo (Apoteket AB, Sweden) was prepared with the following concentrations; 10 µM cisplt, 1 µM cisplt, 0.1 µM cisplt, 1 µM doxo, 0.1 µM doxo and 0.01 µM doxo.

All the drug dilutions were made in complete medium. In the negative controls 1xPBS (DPBS GIBCO ™ Life technology) was used as a mock treatment. Five-hundred thousand cells were plated in total 10 ml complete media per plate for all cell lines except BE(2)-C, which had 200 000 cells per plate since it had a faster proliferation rate in comparison to the other cell lines. The treatment was given 24h post plating (48h for SK-N-FI), 48h after treatment the cells were ethanol fixed (70%). The fixed cells were then (at least 24h post-fixation) stained with Hoechst 33342 (ThermoFisher) in 0.1% Trition-X with 1 000 000 cells/ml stain for ~15 min. A live single cell analysis with FACS (NovoCyte), with the software NovoExpress was then done to observe in what cell cycle phase the cells were in upon fixation. Each cell line was made in 3 replicates (except Kelly which had 6).

2.3 Immunocytochemistry

ICC was done on pre-PFA (4%) fixed glass-coverslips (VWR) of all 7 cell lines. Each cell line was made in triplicates. Before fixation, the triplicates were treated with either single treatment of mock (1xPBS) and 1 µM doxo or double treatment of 1xPBS or 1 µM doxo. The treatments were added 24h post plating and then again 48h after first treatment, they were then fixated 48h post last treatment. Five- hundred thousand cells were plated in total 10 ml complete media per plate for all cell lines except BE(2)-C, which had 200 000 cells per plate. The fixated glass-coverslips were incubated for 30 min (room temperature) in TNB buffer consisting of 0.5g blocking reagent (PerkinElmer) in 100 ml TBS buffer (Tris and NaCl with a pH of 7.4). The coverslips were then incubated overnight at +4^oC in the primary antibody solution: 1’ab, 0.3% TX-100, 0.1% NaN3 and PBS. After the incubation, the cells were washed in 1xPBS and then incubated for 2h at room temperature with the secondary antibody solution: 2’ab in TNB buffer. Then, the coverslips were washed with 1xPBS and mounted with Prolong Gold anti-fade (DAPI).The samples were analysed with a Metafer Slide Scanning Platform.

A baseline was done on cells fixated 24h post plating in order to compare the pATM and Wee1 expression between the different cell lines (figure S1, table S3).

Table 2: Primary and secondary antibodies used for ICC

The primary and secondary antibodies applied to measure pATM and Wee1 expression levels in the cell lines.

Antigen Product

specifications

Dilution 1’ab Secondary antibody Dilution 2’ab

pATM ab36810 Anti-ATM

(phospho S1981) mouse antibody [10H11.E12] from Abcam.

1:500 Goat Anti-Mouse IgG

(H+L)

(ImmunoResearch laboratories)

1:200

Wee1 13084S Rabbit ab

from Cell Signaling

1:500 Goat Anti-Rabbit IgG (H+L)

(ImmunoResearch laboratories)

1:800

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2.4 Confluency assay

SK-N-SH (10 000 cells/well), SK-N-AS (10 000 cells/well), SK-N-FI (6 500 cells/well), Kelly (10 000 cells/well), SK-N-DZ (10 000 cells/well) and BE(2)-C (5 000 cells/well) were plated in separate 96-well plates in 150 µL complete media. SK-N-FI was plated 3 days prior to the rest of the cell lines due to its slower proliferation rate. After all of the cell lines were plated a confluency scan was done with the IncuCyte bright-field, and then every 24h for 72h in total for the titration assay and every 48h, 15 days in total for the combination treatment. The drugs were added 24h post plating (72h for SK-N-FI). IMR- 32 was not compatible with the cell culture plates, it did not attach to the plate, and was therefore excluded from further experiments.

2.4.1 Titration and MTS assay

The cells were treated with a titration series of 10 µM, 1µM, 0.1 µM and 0.01 µM pATM inhibitor (pATMi) (KU 60019, Tocris), Chk1 inhibitor (Chk1i) (PF-477736, Sigma Aldrich) and Wee1 (Wee1i) (Adavosertib, MedChemTronica) inhibitors. After 72h an 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was done were 20 µL CellTiter 96® AQueous One Solution Assay (Promega) was added per 100 µL media. The plates were then incubated for 3h and analysed in the microplate reader FLUOstar Omega (BMG LABTECH). Both the 490 nm and the 690 nm absorbance were measured to assess the cell viability post treatment. Each treatment was done in triplicates. The inhibitors stock solutions were made in DMSO and the dilutions in complete media. DMSO was used as a negative control. Each concentration treatment was done in 3 replicates.

2.4.2 Treatment with cell cycle inhibitors

Each cell line was treated with either a single dose of 10 µM pATMi (KU 60019, Tocris), 1 µM Chk1i (PF- 477736, Sigma Aldrich), 1 µM Wee1i (Adavosertib, MedChemTronica), 10 µM DMSO (mock) or 1 µM DMSO (mock). Each treatment was done in 6 replicates. The inhibitors stock solutions were made in DMSO and the dilutions in complete media.

2.5 Statistical analysis

All the statistics were done with GraphPad Prism 8.4.0. Either a Student’s t-test, or a two-way-ANOVA with Dunnett post-hoc was applied.

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3. Results

Seven high-risk NB cell lines were cultivated and then treated with either cisplt or doxo in order to investigate their cell cycle response. This was done to confirm if the cells had a proneness to arrest at different phases upon CT treatment. The FACS analysis confirmed that the cells tended to arrest at either the S- or G2/M-phase depending on the CT and the CT dosage. In order to inhibit the cells from arresting, inhibition of the cell cycle regulators pATM, Wee1 and Chk1 was evaluated.

3.1 Cisplatin and Doxorubicin titration with FACS analysis

All of the following figures (3A and 3B) and tables (S1A and S1B) related to the cisplt and doxo titration with FACS can also be found in the currently unpublished article “Chemotherapy-provoked cell cycle arrest precedes regrowth in high-risk neuroblastoma cell lines.” from Jönsson, L.Ö. et al. (17).

The mean and standard deviation of the fraction of cells in the different cell cycle phases, for the 3 series of the mock, 1-10 µM cisplatin and 0.01-1 µM doxo treated NB cell lines were calculated (table S1A-B). The two p53 wild type (p53wt) NB cell lines SK-N-SH and IMR-32 showed the highest fraction of sub-G1 cells following the higher concentrations of cisplt or doxo treatment. For SK-N-SH (p53wt) the cells underwent cell death following treatment with 1 µM doxo (78% ± 22), 0.1 µM doxo (95% ± 7.5), 10 µM cisplt (94% ± 4.7) and 1 µM cisplt (37% ± 4.6) (table S1A, figure 3A). For IMR-32 (p53wt) the cells underwent cell death following treatment with, 1 µM doxo (96% ± 2.9), 0.1 µM doxo (87%

3.9) and 10 µM cisplt (72% ± 0.3) (table S1A, figure 3A).

SK-N-AS (p53mut) had a significant increase of cells in the sub-G1 phase after treatment with 10 µM cisplt (21% ± 1.5) and 1 µM doxo (7.4% ± 2.3) compared to 0.5% in the mock. Similarly, there was a decrease in the 2N after 10 µM cisplt (29% ± 4.4), 1 µM cisplt (27% ± 4.8), 1 µM doxo (8.8% ± 2.4) and 0.1 µM doxo (7.2% ± 3.6) compared to 51% in the mock. Thirty-two % of the cells were in the S-phase after mock treatment. The number of cells in the S-phase increased after treatment with 10 µM cisplt (40% ± 5.4) and 1 µM doxo (77% ± 4.4) but decreased after 0.1 µM doxo (18% ± 3.0). The majority of cells resided in the G2/M-phase after treatment with 1 µM cisplt (37% ± 0.5), 1 µM doxo (5.4% ± 3.5), 0,1 µM doxo (66% ± 5.7) and 0.01 µM doxo (24% ± 0.6), compared to the mock (15%). This shows that the following doses of cisplt (1 µM) and doxo (0,1 µM and 0.01 µM) increased the fraction of cells in the G2/M-phase (table S1B and figure 3B).

The p53mut SK-N-FI showed an increase fraction of cells that underwent cell death after 10 µM cisplt (30% ± 11). The number of cells in 2N decreased after 10 µM cisplt (33% ± 5.9), 1 µM cisplt (52% ± 5.0), 1 µM doxo (26% ± 6.3) and 0.1 µM doxo (37% ± 3.3). However, they increased in both the S-phase and 4N after 10 µM cisplt (25% ± 3.8, S-phase), 1 µM cisplt (25% ± 4.4, S-phase), 0.1 µM doxo (27% ± 5.1, 4N) and 1 µM doxo (30% ± 4.4 S-phase and 31% ± 7.2 4N). Compared to the mock treated SK-N-FI were 6.6% of the cells were in the G2/M-phase (table S1B and figure 3B).

In Kelly (p53mut) an increase in the sub-G1 phase was shown after 10 µM cisplt (12% ± 6.4), 1 µM doxo (11% ± 6.1) and 0.1 µM doxo (8.4% ± 2.6). However, there was a decrease in the 2N with 30% ± 3.5, 32% ± 11 and 13% ± 3.6, respectively. Similarly to SK-N-AS, there was both an increase and a decrease in the S-phase after 10 µM cisplt (47% ± 6.2), 1 µM doxo (67% ± 3.9) and 0.1 µM doxo (21% ± 4.2), in comparison to the mock (31% ± 6.7). In the 4N Kelly only showed a substantial increase after 0.1 µM doxo (37% ± 5.4), compared to 6.6% for the mock (table S1B and figure 3B).

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13 For the p53mut, SK-N-DZ there was an increase of cells in sub-G1 after 10 µM cisplt (18% ± 1.5), 1 µM doxo (18% ± 11) and 0.1 µM doxo (26% ± 2.5). There was a decrease in 2N after 10 µM cisplt (7.6% ± 2.0), 1 µM doxo (8.0% ± 3.3) and 0.1 µM doxo (16% ± 5.1), however, there was an increase after 1 µM cisplt (43% ± 1.0). Fraction of cells in S-phase or 4N significantly increased after treatment with 10 µM cisplt (39% ± 3.8, 4N), 0.1 µM doxo (32% ± 12, 4N) and 1 µM doxo (39% ± 3.4 S-phase, 35% ± 11 4N), compared to the mock (28% S-phase, 11% 4N) (table 1B and figure 3B) .

For BE(2)-C, which has p53mut, there was a decrease of cells in 2N after treatment with 10 µM cisplt (16% ± 3.6), 1 µM doxo (8.8% ± 3.5) and 0.1 µM doxo (13% ± 11) compared to the mock (62% ± 4.8).

The majority of the cells were in the G2/M-phase for the 2 highest concentrations of doxo treatment of 1 µM (49% ± 6.2) and 0.1 µM (52% ± 27), compared to the mock treated cells were only 10% of the cells were in the G2/M-phase.

Four out of the 5 p53mut cell lines, SK-N-AS, SK-N-FI, Kelly and SK-N-DZ which showed increased fraction of cells in the G2/M-phase (table S1B and figure 3B) also had an increase of cell death post treatment for at least one of the concentrations of either cisplt or doxo (table S1B and figure 3B), indicating that there were at least 2 different phenotypic subpopulations within the cell lines. One that were treatment sensitive and one that was treatment resistant.

Figure 3A. Mock (PBS), cisplt or doxo treated p53wt cell lines FACS analysis

Cell cycle graphs generated through HOECHST flow cytometry of p53wt cell lines SK-N-SH and IMR32, together with corresponding bars graphs showing the cell cycle fraction for each titration assay treatment of 0.1, 1 and 10 µM cisplt and 0.01, 0.1 and 1 µM doxo, from left to right, with mock (PBS) furthest to the left.

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Figure 3B. Mock (PBS), cisplt or doxo treated p53mut cell lines FACS analysis Cell cycle graphs generated through HOECHST flow cytometry of p53mut cell lines SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, together with corresponding bars graphs showing the cell cycle fraction for each titration assay treatment of 0.1, 1 and 10 µM cisplt and 0.01, 0.1 and 1 µM doxo, from left to right, with mock (PBS) furthest to the left.

BE(2)-C

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3. 2 Expression of pATM and Wee1

The FACS analysis showed a confirmation of cell cycle arrest upon cisplt or doxo treatment. Therefore, the expression of the cell cycle regulators pATM and Wee1 was investigated in order to evaluate if they were fitting targets for inhibition.

In the p53wt (table S2A and figure 4A) pATM is significantly upregulated for SK-N-SH after both single and double treatment of doxo, 20% ± 7.0 (p<0.05) and 16 % ± 1.7(p<0.05) respectively, compared to the mocks, 5.4% and 8.5% (table S2A). However, no significant change is observed for Wee1. In IMR- 32 no changes were observed for either pATM or Wee1. In the p53mut cell lines pATM is significantly upregulated for SK-N-FI (single and double treatment) with 82% ± 8.4 (p<0.001) and 76% ± 1.0 (p<0.0001) compared to 12% and 4.1% respectively for the mock, Kelly (single treatment) with 74% ± 2.2(p<0.0001) compared to 3.1% for the mock, SK-N-DZ (single and double treatment) with 89% ± 1.5 (p<0.0001) and 49% ± 25(p<0.05) respectively compared to the 1.2% and 0.9% for the mocks and for BE(2)-C (single and double treatment) with 86% ± 9.7(p<0.0001) and 90% ± 13(p<0.0001) compared to 7.2% and 1.3% for the mocks (table S2B and figure 4B). Upregulation of Wee1 was observed for Kelly (single treatment, 1.2% ± 0.4 p<0.05), SK-N-DZ (single treatment, 9.5% ± 4.3p<0.05) and BE(2)-C (single, 12% ± 5.3p<0.05 and double treatment, 17% ± 1.7p<0.01) (table S2B and figure 4B).

Figure 4A. pATM and Wee1 expression of mock (PBS), cisplatin or doxorubicin treated p53wt cell lines. Fraction of pATM and Wee1 positive cells were analysed with a Student’s t-test. The number of replicates was 3 for all. * = p<0.05

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3.3 Titration and MTS assay

An upregulation of both pATM and Wee1 expression was shown for several of the NB cell lines after doxo treatment, indicating that they potentially could be involved in the cell cycle arrest shown by the FACS analysis. In order to evaluate the most effective dosage for the long-time assay a titration series of pATMi, Wee1i and Chk1i together with an MTS-assay was done.

After inhibition of the cell cycle regulators, an inhibition in confluency was observed for all 6 cell lines, as well as a reduction of cell viability higher than 50% (figure 5). Inhibition of pATM did not give a significant decrease (≥ 50%) in cell viability for any of the 4 tested concentrations (10, 1, 0,1 and 0,01 µM) in all 6 cell lines. However, the cell viability was reduced for all cell lines after treatment with Chk1i. SK-N-SH and BE(2)-C showed ≥ 50% reduction after 10 µM Chk1i, SK-N-FI and SK-N-DZ after 1 µM Chk1i and SK-N-AS and Kelly after 0,1 µM Chk1i. Treatment with Wee1i reduced cell confluency and decreased the viability ≥ 50% for all 6 cell lines. For BE(2)-C the cell viability was reduced ≥ 50%

after 10 µM Wee1i. For SK-N-SH, SK-N-AS, SK-N-FI and SK-N-DZ the reduction was seen after 1 µM Wee1i and lastly for Kelly it was observed after 0.1 µM Wee1i. Based on these results the following concentrations were selected for a long-term assay: 10 µM pATMi, 1 µM Chk1i and 1 µM Wee1i.

Figure 4B. pATM and Wee1 expression of mock (PBS), cisplatin or doxorubicin treated p53mut cell lines. Fraction of pATM and Wee1 positive cells were analysed with a Student’s t-test. The number of replicates was 3 for all.

***= p<0.001, ****= p<0.0001.

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17 Figure 5. Confluency and cell viability control on NB cell lines following inhibitor treatments.

NB cell lines were treated with inhibitors of pATM, CHk1 and Wee1 at 10 µM, 1 µm, 0.1 µM and 0.01 µM. Confluency was measured over time and cell viability was measured through an MTS assay 72h post treatment. N = 3-6.

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3.4 Long-time inhibition assay

pATM inhibition (figure 6) did not show any long-term decrease in cell confluency for any of the 6 cell lines. Chk1 reduced confluency in Kelly. However, SK-N-SH, SK-N-AS, SK-N-FI, SK-N-DZ and BE(2)-C were unaffected by Chk1 inhibition. The Wee1 inhibitor reduced confluency in 4 out of 6 cell lines, SK-N-AS, SK-N-FI, Kelly and SK-N-DZ.

Figure 6. Long-time inhibition assay of NB cell lines. All NB cell lines were scanned 24h after plating, when the cells had attached to the wells, this corresponds to day 1 in the inhibition graphs. Inhibition treatment of 10 µM pATMi, 1 µM Chk1i and 1 µM Wee1i was given directly after the first scan. N=3-6.

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4. Discussion

The results confirmed a proneness of the p53mut NB cell lines to arrest at the S- and/or G2/M-phase upon CT treatment with either cisplt or doxo. It was also confirmed that the NB cell lines tended to upregulate pATM upon doxo treatment, where 5 out 7 cell lines showed a significant upregulation.

However, Wee1 was only upregulated for 3 out 7 cell lines. Furthermore, pATMi, Chk1i and Wee1i showed diverse effects. Wee1 showed most promising results were the cell viability decreased for all 5 p53mut cell lines and the confluency over time decreased for 4 out 5 p53 mutated cell lines. The p53wt cell line SK-N-SH was less sensitive towards Chk1 and Wee1 inhibition indicating that cell lines with function p53 might not be as dependent on the Chk1 and Wee1 pathways compared to cell lines with non-functional p53. pATMi on the other hand did not show any long-term decrease in cell confluency for any of the 6 cell lines.

Relapse is commonly caused by CT resistant subpopulations for high-risk neuroblastoma (NB) (14). For the p53wt SK-N-SH and IMR-32 the majority of the cells were CT sensitive and underwent cell death upon cisplt or doxo treatment. However, for the p53mut cell lines SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C the cells arrested in either S-phase and/or in G2/M-phase. The cell response was dose dependent, where different doses and CT gave arrest at different phases for the different cell lines.

This corroborates the effect of different drug concentrations on cell cycle regulation. Cisplatin has a triphasic elimination from the plasma with half-times of 14 minutes, 274 minutes and 53 days (43).

Similarly, doxo also has a triphasic plasma elimination, with the half-times 12 minutes, 3.3 hours and 30 hours (44). This indicates that different parts of the tumour will be hit with different concentrations of the drugs when it disperses in the tumour, causing the cells to arrest at different phases, creating several different CT resistant subpopulations which needs to be targeted differently. This cellular phenotypic heterogeneity together with genotypic heterogeneity makes it hard to find general CT treatments that are effective.

One way to circumvent the cell cycle arrest, and the subsequent DNA damage repair is to inhibit the pathways that regulates the arrest (20). For this purpose, the 3 cell cycle key-components pATM, Chk1 and Wee1 were chosen. pATM is mostly responsible for apoptosis, senescence or DNA-damage repair responses, whereas activation of the Chk1 and Wee1 often results in cell cycle arrest (20). Chk1 causes G1/S, S and G2/M-phase arrest, whilst Wee1 is more focused on the G2/M-phase arrest (22). The pATM and Wee1 expression levels significantly increased upon single or double treatment of doxo for 5 out 7 cell lines. This indicates that the pATM and Wee1 pathway cascades are activated upon doxo treatment in the different NB cell lines. However, the activation levels seem to be at different extent depending on the cell line. This could be explained by the specific mutations harboured by them, such as NMYCN amplification (table 1). Four out of 5 p53mut cell lines showed an upregulation of pATM, compared to the p53wt. pATM activates Chk1 upon double strand breakage. Through the titration experiments with cisplt and doxo it was shown that the p53mut cells arrested at either S- or G2/M- phase, further confirming the involvement of pATM and Wee1.

Since both pATM and Wee1 have increased expression levels upon doxo treatment they could be potential targets for treatment of NB. Chk1 is downstream of pATM (20) which makes it a good potential target as well. Six NB cell lines were treated with a titration series of pATMi, Chk1i and Wee1i.

Inhibition of pATM did not show any effects on the cell viability. However, treatment with either Chk1i or Wee1i showed a reduction in cell viability. This is in line with previous studies on Chk1i and Wee1i for NB cell lines, were they have shown to be synergistically efficient with each other as well as other DNA damaging CTs such as gemcitabine (22).

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20 To investigate if the inhibitors are able to inhibit regrowth a long-time assay over 15 days with selected concentrations of pATMi (10µM), Chk1i (1 µM) and Wee1i (1 µM) was done for all cell lines except IMR-32. Kelly was the only cell line affected by Chk1i, which gave the highest decrease in confluency.

The proliferation rate of the multi-resistant BE(2)-C was unaffected by all 3 inhibitors, a similar behaviour was observed for SK-N-SH. SK-N-SH has a functioning p53 and might thereby not as dependent on the Chk1 and Wee1 pathways compared to the p53mut cell lines, explaining why SK-N- SH was less sensitive for Chk1i and Wee1i treatments. For the p53mut cell lines SK-N-AS, Kelly and SK- N-DZ, were Wee1i showed the best potential to cell proliferation decrease. This further confirms that the cells are protected by cell cycle arrest activated by Wee1. To investigate this phenomenon in more detail a combination study with different CTs such as cisplt and doxo together with pATMi, Chk1i and Wee1i would be required. The differentiating behaviour of p53mut BE(2)-C could be explained by it harbouring different mutations compared to the other 4 p53mut cell lines (34).

To summarise the p53mut NB cell lines showed an increased proneness to arrest at the S- and/or G2/M-phase upon CT treatment. However, the dose-dependent cell cycle response indicates on a complex resistance expression for NB which makes it hard to find general treatment strategies.

Contradictory to the upregulation of Wee1, where only 3 out 7 cell lines showed a significant upregulation, inhibition of Wee1 showed the highest decrease of cell confluency for the long-time assay, which makes it an interesting target for future CT and inhibitor combination assays.

5. Future perspectives

Moving forward, a good starting point would be to do combination assays of the pATMi, Chki and Wee1i together with each other and the CTs cisplt or doxo, as well as comparing the efficacy for the single treatments of all drugs. Looking at other cell cycle regulators, in particular for BE(2)-C, which was seemingly unaffected by pATMi, Chk1 and Wee1i would also further enhance the study.

Thereafter, when a promising combination treatment has been identified in the cell lines, in vivo experiments (i.e. 3D cultures or animal models) could be applied.

Here we argue that the cells tend to arrest at different cell cycle phases upon CT treatment and are thereby later responsible for causing relapse. However, as mentioned above, another widely accepted theory is the cancer stem cell theory which claims that the cells responsible for the relapse stems from the non-proliferating G0-pool (14). This could be a potential explanation to the observed dose- dependent cell cycle arrest behaviour of the studied NB cell lines and needs to be further investigated.

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6. Acknowledgments

I would like to thank my supervisors Shahrzad Shirazi Fard (Department of Women’s and Children’s Health | Division of Paediatric Oncology, Karolinska institutet) and Cristina Al-Khalili Szigyarto (Systems biology, CBH, KTH), as well as Lars Ährlund-Ritcher (professor emeritus at Karolinska institutet) for their guidance and support throughout the project. A special thanks to Nikolas Herold and Nikolaos Tsesmetzis at the Department of Women’s and Children’s Health | Division of Paediatric Oncology, Karolinska institutet and Jan Mulder and his group at the department of neuroscience at Karolinsksa insitutet, for lending me both their time and equipment. Also, thanks for the collaboration to Tina Dalianis group at the department of oncology-pathology, who we shared the main labs and equipment with. Thank you, Ximena Lopez Lorenzo (M.Sc. student, KTH), for the company and the discussions at the lab, thank you Adelina Rabenius (M.Sc. student, KTH) for reading through all of my drafts and lastly, I would like to thank my friends and family, who have supported me throughout this project.

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7. References

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2. Neuroblastom barn - symptom & behandling | Barncancerfonden | Barncancerfonden [Internet]. [cited 2020 Apr 26]. Available from: https://www.barncancerfonden.se/for- drabbade/om-barncancer/neuroblastom/

3. Hultman I, Haeggblom L, Rognmo I, Jansson Edqvist J, Blomberg E, Ali R, et al. Doxorubicin- provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma. Castresana JS, editor. PLoS One [Internet]. 2018 Jan 17 [cited 2020 Apr 26];13(1):e0190970. Available from: https://dx.plos.org/10.1371/journal.pone.0190970 4. Newly Diagnosed with Neuroblastoma [Internet]. [cited 2020 Apr 26]. Available from:

https://childrensoncologygroup.org/index.php/newly-diagnosed-with-neuroblastoma 5. Habib EE, El-Kashef AT, Fahmy ES. Management of neuroblastoma: A study of first- and

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Investigation of drug-induced cell cycle responses in high-risk neuroblastoma

Investigation of drug-induced cell cycle responses in high-risk

neuroblastoma

MARYAM SAHI

Investigation of drug-induced cell cycle responses in high-

risk neuroblastoma

Master Thesis

Table of contents

Abstract

Key words

Sammanfattning

Nyckelord

Abbreviations

1. Introduction

2. Material and Methods

2.1 Cell lines and cell culture

2.2 Cisplatin and Doxorubicin titration with FACS analysis

2.3 Immunocytochemistry

2.4 Confluency assay

2.5 Statistical analysis

3. Results

3.1 Cisplatin and Doxorubicin titration with FACS analysis

3. 2 Expression of pATM and Wee1

3.3 Titration and MTS assay

3.4 Long-time inhibition assay

4. Discussion

5. Future perspectives

6. Acknowledgments

7. References