Effects of PI3K and FGFR Inhibitors on Human Papillomavirus Positive and Negative Tonsillar and Base of Tongue Cancer Cell Lines

Birthe Katrin Alexandra Lange ______Master Degree Project in Medical Research, 30 credits. Spring 2019 Department: Oncology-Pathology, Karolinska Institutet Supervisor: Prof. em. Tina Dalianis; Dr. Stefan Holzhauser

UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 2 (31)

Abstract Background: The standard treatment, chemoradiotherapy, for patients with human papillomavirus positive (HPV+) and negative (HPV-) tonsillar and base of tongue squamous cell carcinomas (TSCC, BOTSCC) is similar although their sensitivities to therapy vary considerably. It is too harsh for many patients with HPV+ but too weak for some patients with HPV- tumours. PIK3CA and FGFR3 are mutated in fractions of TSCC and BOTSCC patients and represent possible therapy targets. Aim: To test the effects of PI3K and FGFR inhibitors as single and combination treatments on TSCC and BOTSCC cell lines. Methods: HPV- TSCC UT-SCC-60A and HPV+ BOTSCC UPCI-SCC-154 cells were treated with the PI3K/mTOR inhibitor BEZ235 (0.25 µM to 5 µM) and FGFR inhibitor AZD4547 (5 µM to 50 µM). The WST-1 assay, CellTox Green Cytotoxicity assay, Caspase-Glo 3/7 assay and xCELLigence RTCA DP instrument were used to evaluate the treatments regarding viability, cytotoxicity, apoptosis and proliferation. Results: UT-SCC-60A cells were sensitive to treatment with the highest tested doses of BEZ235 and AZD4547, while UPCI-SCC-154 cells were more sensitive to both inhibitors. Combinations of the inhibitors resulted in larger treatment effects in both cell lines. Conclusions: Combinations of BEZ235 and AZD4547 for treatment of UT-SCC-60A and UPCI-SCC- 154 cells were superior compared with single treatments, to which UT-SCC-60A cells were less sensitive than UPCI-SCC-154 cells. It should be investigated further if combination treatments enable the use of lower drug doses to reduce side effects while delaying resistance development. This could be one way to improve treatment for TSCC and BOTSCC patients.

Key words Tonsillar squamous cell carcinoma, base of tongue squamous cell carcinoma, oropharyngeal squamous cell carcinoma, HPV, PI3K inhibitor, FGFR inhibitor, targeted therapies

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Introduction General introduction In 2007, the International Agency for Research on Cancer (IARC) acknowledged human papillomavirus (HPV) type 16 as a risk factor for oropharyngeal squamous cell carcinoma (OPSCC) (1). Tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) accounted for 83 % of the OPSCC cases in Sweden between 2008 and 2012 (2). In the past decades, the incidence of TSCC and BOTSCC increased epidemically in many Western countries, including Sweden, due to an increase of HPV+ cases (3–7). In contrast, the number of HPV- cases, which are caused by smoking and alcohol, has decreased in many Western countries (4, 8–10). Remarkably, patients with HPV+ TSCC and BOTSCC were found to have a much better clinical outcome, than those with HPV- cancers and other head and neck squamous cell carcinomas (HNSCC) (80 % vs. 40-50 % 5-year disease specific survival with conventional radiotherapy) (11, 12). Today, HPV+ TSCC and BOTSCC account for around 30 % of all oropharyngeal cancers (13). The treatment was intensified several years ago and now often consists of surgery followed by hyperfractionated and accelerated, or conventional high dose radiotherapy, with concomitant chemotherapy or, occasionally, epidermal growth factor receptor (EGFR) inhibitors for patients with stage III and IV tumours (2, 14–16). This treatment is used for all HNSCC, irrespective of type, HPV status or prognosis (2, 14, 16). Intensified therapy comes with more serious side effects and is not necessary for most patients with HPV+ TSCC and BOTSCC. This is due to their positive response to conventional radiotherapy (17). Furthermore, the intensified therapy does not improve the survival of patients with HPV+ TSCC and BOTSCC (17). Therefore, other treatment options such as targeted therapies should be investigated and considered for these patients. The aim of this thesis was to test targeted therapies in vitro on HPV+ and HPV- TSCC and BOTSCC cell lines. Human Papillomaviruses Over 200 HPVs have been described and they are commonly divided into five genera (alpha-, beta-, gamma-, mu- and nu-papillomaviruses) (18). Most known HPV types infect the skin, but there are around 50 HPV types that cause mucosal infections (19). These can be divided into high-risk, putative high-risk or low-risk types depending on their ability to induce cancer development. The mucosal high- risk types 16 and 18, which are frequently responsible for the development of cervical cancer, belong to the alpha genus (20). Mucosal HPVs are usually transmitted sexually and infection of basal cells at the local epithelium occurs predominantly after wounding. HPVs are non-enveloped icosahedral viruses with a diameter of approximately 55 nm (21). They belong to the DNA viruses and contain a double-stranded circular genome of approximately 8000 base pairs, which is called the episome (22). The episome encodes for the early regulatory proteins (E1, E2 and E4- E7) and two structural proteins (L1 and L2) (23–25). All HPV types share well-conserved E1 and E2 genes, that are involved in viral DNA replication (26), as well as L1 and L2 genes which are essential for viral packaging (27). The remaining genes E4, with a role in virus release (28), E5, which contributes to immune evasion (29, 30), as well as E6 and E7, the major oncoproteins (31, 32), are more variable. After infection, the E6 and E7 proteins may lead to cell cycle entry in the differentiating cells in the mid- layers of the epithelium (33, 34). This serves the purpose of allowing viral genome amplification and packaging before mature virions are released at the epithelial surface. To promote cell cycle progression, high-risk E7 proteins bind to the retinoblastoma protein to cause its degradation (31, 35–37). This results in E2F-stimulated transcription which in turn causes S-phase entry (38). Uncontrolled expression of high-risk E7 promotes host genome instability due to a high probability of cell cycle aberrations (39, 40). In parallel, high-risk E6 promotes telomerase upregulation and ubiquitin-mediated degradation of p53 via the E6AP ubiquitin ligase (41–43). The latter prevents p53 activation in response to upregulation of UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 4 (31)

p14ARF by E2F-dependent transcription (44). Therefore, deregulated E6 expression prevents p53-induced DNA repair or apoptosis in response to DNA damage (45, 46). This can lead to the accumulation of somatic mutation and carcinogenesis. Another protein that is upregulated due to E2F release, p16INK4A, can be utilised as a biomarker for HPV infections (38, 47). Virus maturation is dependent on the expression of the viral capsid proteins L1 and L2, which enable the virus release from dying cells at the epithelial surface (27). HPV, cancer development and HPV vaccines There are around 25 high-risk or putative high-risk HPV types (48). Most commonly known is the association of HPV with the development of cervical cancer, where it is causative for nearly 100 % of the cases (49). However, HPV can also cause other anogenital cancers such as vulvar (causative factor in 15- 48 % of the cases), vaginal (78 %), penile (51 %) and anal cancer (88 %) (49). More recently, it has also been acknowledged as a risk factor for OPSCC, TSCC and BOTSCC (50–52). It is found in around 75 % of the TSCC and 70 % of the BOTSCC cases in Sweden (6). When expression of the E6 and E7 proteins gets deregulated, e.g., through a loss of E2 upon integration of the episome into the host genome, precancerous lesions progress and may result in carcinogenesis (53–57). To prevent cases of tumour development, vaccines against the most common high-risk HPV types 16 and 18 (as well as other high- risk and low-risk types) have been developed (58–60). TSCC, BOTSCC and HPV In 2000 and 2004 associations between HPV infections and OPSCC, TSCC as well as BOTSCC were established (50–52). In 2007, HPV infections were added as risk factors for OPSCC by the IARC (1). It was gradually established that HPV- tumours, which are often caused by smoking and alcohol consumption (8), are very different from HPV+ tumours, which are formed due to the viral infection (61). The incidence of TSCC and BOTSCC in Sweden and many other Western countries has steadily increased since the 1970s (3–7). At the same time the fraction of HPV+ cases increased from 23 % to more than 70 % (3, 6). This was suggested to be due to changes in sexual habits, such as increasing numbers of sexual partners (62). The incidence of HPV- cases decreased concurrently in many, but not all, countries for the number of smokers in the Western world has decreased (4, 9, 10). As mentioned above, patients with HPV+ TSCC and BOTSCC generally have a higher 5-year disease- specific survival (80 %) than patients with HPV- tumours (40–50 %) (11, 12). In fresh frozen tissue, the detection of HPV E6 and E7 mRNA is the golden standard to diagnose active HPV infections in the tumour (63). However, many patient samples are formalin-fixed and paraffin-embedded and in these cases the combined presence of HPV DNA and p16INK4A overexpression was shown to be almost as accurate as the evidence of E6 and E7 mRNA (64). Diagnosis of these tumours usually occurs relatively late, which is primarily due to the lack of specific early symptoms (65). When diagnosed, the tumour’s size may already have increased substantially and, in HPV+ TSCC and BOTSCC, nodal metastasis has often already occurred (65). As the prognosis for HNSCC patients is generally poor, the treatment has been intensified in the past decade in response to a French multicentre randomised controlled trial (66, 67). This trial showed the advantage of additional concomitant chemotherapy compared with radiotherapy only (66, 67). The current standard treatment includes postsurgical high-dose radiotherapy for 6 to 7 weeks or hyperfractionated and accelerated radiotherapy and often, especially in advanced cases, additional chemotherapy or EGFR inhibitors (2, 14–16). This intensified therapy results in more severe side effects, which can persist long after the treatment has concluded (66). These side effects can prevent patients from returning to normal life. Today, this therapy is usually administered irrespectively of the tumour’s HPV status (2, 14, 16). As patients with HPV+ tumours have a very high 5-year survival rate and respond to therapy comparatively UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 5 (31)

well, they often do not benefit from the intensified treatment (11, 12). Additionally, it does nothing to improve the survival, while the patients suffer from more severe side effects (17). This has led to efforts which aim to reduce the radio- or chemotherapy for patients likely to respond well to the treatment (68, 69). To be able to predict which patients belong to this group, many studies have focused on finding biomarkers that indicate good treatment response. Other approaches focus on the development of targeted therapies that may represent a treatment alternative with less severe side effects. Biomarkers that predict treatment response for TSCC and BOTSCC patients Several studies have focused on finding biomarkers that predict prognosis or treatment response in TSCC and BOTSCC patients. Among the first factors associated with the outcome were the presence of HPV DNA or RNA and p16INK4A levels as well as age, sex and tumour stage (11, 50, 51, 70). Additionally, some of the early studies aimed to associate the presence of aneuploidies and genomic imbalances to outcome, but found these to be secondary to HPV status (71, 72). Some studies combined several markers into models that predict the outcome for OPSCC patients (17, 73, 74). An early model used a combination of HPV status, pack-years of smoking, and tumour as well as lymph node stage to predict which patients are at high, intermediate or low risk of dying within 3 years (73). A later model combined age, stage and diagnosis (TSCC or BOTSCC) with HLA class I expression levels and CD8+ tumour- infiltrating lymphocyte (TIL) counts to predict 3-year progression-free survival for patients with HPV+ tumours (74). In a third model, age, stage, HPV E2 expression levels and CD8+ TIL counts were combined to predict 3-year survival for patients with HPV+ tumours (17). The latter identified 56 % of the patients that have a 3-year progression-free survival with a positive predictive value of 98 % (17). Over the years, several additional prognostic factors have been reported. Positive prognostic factors for HPV+ tumours include high numbers of CD8+ lymphocytes (75, 76), lower HLA class I expression levels (77, 78), decreased LMP7 expression (79), lower nuclear LMP10 staining (80), the presence of HPV16 E2 mRNA (81), lower CD44 (82, 83) and CD98 expression (84) as well as a higher expression of LRIG1 (85). Interestingly, in patients with HPV- tumours the opposite association was observed regarding the HLA class I levels (77, 78). It is assumed that in HPV+ tumours the decrease in HLA expression occurs in response to HPV E5 and E7, to avoid immune recognition (30, 86). However, an increase in HLA expression in response to radiation has been shown in an experimental system, which may render the tumours more sensitive to immune recognition (87). To identify additional differences between HPV+ and HPV- tumours and reveal molecular signatures that could potentially be used to enhance prediction accuracies, genomic analyses that used next generation sequencing (NGS) approaches have been performed. Many studies on microRNA expression, the transcriptome, the proteome and the microbiome lacked successful reproduction of the results as they used small sample sizes (88). The numbers of studies in these fields are still limited.

The mutational profile of HPV+ and HPV- HNSCC HPV+ and HPV- tumours have been characterised by NGS to reveal their mutational profiles. In several studies PIK3CA (89–96), PTEN (91, 93, 96), FBXW7 (91, 93, 96), FGFR3 (92–94, 96) and KRAS (91, 92, 95, 96) mutations were described as being characteristic for HPV+ tumours. In HPV- tumours, in contrast, TP53 (89, 91–97), CDKN2A (89, 91–95), NOTCH (89, 92, 93, 96, 97), EGFR (91, 93, 94, 96), CCND1 (91, 93, 94), FGFR1 (93, 94) and MYC mutations (93, 94) were more common. In HPV- tumours PIK3CA mutations were less common than in HPV+ tumours but were nonetheless found in a fraction of samples (89–96).

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To target PIK3CA and FGFR3 mutations, a number of small molecules have been tested preclinically, some of which have advanced to clinical trials or have even been approved by the food and drug administration (FDA) (98, 99). PIK3CA and FGFR3 represent promising targets for targeted therapies since, depending on the study, between 20 and 56 % of the HPV+ patients had PIK3CA mutations (89– 96) and 7 to 14 % were positive for FGFR3 mutations (92–94, 96). Additionally, FGFR3 mutations were associated with a worse prognosis in TSCC and BOTSCC patients (96). To summarise, finding new therapies for TSCC and BOTSCC patients that do not benefit from the current standard treatment is important, especially to improve the survival of these patients. As PIK3CA and FGFR3 mutations occur in a fraction of OPSCC patients, PI3K and FGFR inhibitors represent promising targeted therapies for both TSCC and BOTSCC patients. Therefore, in this thesis, the dual PI3K and mTOR inhibitor BEZ235 and the FGFR inhibitor AZD4547 were tested in vitro on the HPV- TSCC cell line UT-SCC-60A and the HPV+ BOTSCC cell line UPCI-SCC-154. These cell lines had previously been tested negative for PIK3CA and FGFR3 mutations (C. Bersani, data unpublished). This model system was used for a pilot study to estimate the sensitivity of TSCC and BOTSCC cell lines to these inhibitors, in order to pave the way for further studies to test the sensitivity in cell lines with relevant mutations. In addition to single treatments, combination treatments were tested to evaluate if these would result in additional beneficial effects. To evaluate the treatment effect on the cell lines, the WST-1 assay was used to determine cell viability, the CellTox Green Cytotoxicity assay to determine cytotoxicity, the Caspase-Glo 3/7 assay to determine induction of apoptosis and the xCELLigence RTCA DP instrument to determine cell proliferation. These assays and machines are widely used for such purposes. The expectation was that, by combining two drugs, it would be possible to reduce the dose of each single inhibitor and thereby also the specific side effects. Additionally, this approach might prevent or at least delay resistance development.

Aims  To find targeted therapies with higher efficacies and fewer side effects for the treatment of TSCC and BOTSCC patients.  To test PI3K and FGFR inhibitors on TSCC and BOTSCC cell lines.  To test if these inhibitors have a beneficial effect as combination treatments.

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Material and Methods Cell lines and cell culture The HPV- cell line UT-SSC-60A (obtained from Reidar Grénman, University of Turku, Finland) and the HPV+ cell line UPCI-SCC-154 (obtained from Susanne Gollin, University of Pittsburgh, Pennsylvania, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco via Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 10 % fetal bovine serum (FBS; Gibco via Thermo Fisher Scientific) that was supplemented with 2 mM L-glutamine (Gibco via Thermo Fisher Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco via Thermo Fisher Scientific) at 37 °C and 5 % CO2. To harvest the cells, they were first washed twice with phosphate-buffered saline (PBS; Gibco via Thermo Fisher Scientific), then detached with trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco via Thermo Fisher Scientific), after that centrifuged at 350 × g for 5 minutes and finally resuspended in fresh medium. Treatment with the PIK3CA and FGFR3 inhibitors The cells were seeded and, after an incubation of 24 hours, treated with the PIK3CA and FGFR3 inhibitors. To inhibit PIK3CA the dual ATP competitive PI3K and mTOR inhibitor BEZ235 (Selleck Chemicals, Munich, Germany) was used. The selective FGFR inhibitor AZD4547 (Selleck Chemicals) was administered to inhibit FGFR3. These inhibitors were used for single treatments and in combination on the cell lines UT-SCC-60A and UPCI-SCC-154. To generate the stock concentrations, the drugs were dissolved in dimethyl sulfoxide (DMSO) and dilutions of the stock were prepared by the addition of PBS. The final concentrations for BEZ235 were in the range of 0.25 µM to 5 µM, while the ones for AZD4547 were between 5 µM and 50 µM. WST-1 viability assay For the WST-1 viability assay, 5 × 103 cells in 90 µL DMEM without penicillin and streptomycin were seeded per well of a transparent 96-well plate with flat bottom (TPP Techno Plastic Products, Trasadingen, Switzerland) and treated with the inhibitors as described above. The outermost wells were filled with medium only to avoid edge effects. PBS was added as a positive control and for the background measurements two wells with 90 µL DMEM without penicillin and streptomycin were used. After 24, 48 and 72 hours, 10 µL of the WST-1 reagent (Roche Diagnostics, Mannheim, Germany) was added. WST-1 is a tetrazolium salt, which is extracellularly converted to formazan via an intermediate electron acceptor. The process occurs in response to NAD(P)H production by the mitochondrial succinate-tetrazolium-reductase system of metabolically active cells. After an incubation of 1 hour at 37 °C, the amount of viable cells in each well was determined by the measurement of the absorbance at 450 nm with a VersaMax microplate reader (Molecular Devices, San José, California, USA). The average value of the duplicates (single treatments) and triplicates (combination treatments) was calculated and normalised to the PBS control after subtraction of the background value. Three repetitions were performed for each cell line. The single treatments were performed by Anna Ohmayer, a previous Master student in the group. CellTox Green Cytotoxicity assay For the CellTox Green Cytotoxicity assay, 5 × 103 cells in 80 µL DMEM without penicillin and streptomycin were seeded per well of a 96-well clear flat bottom black polystyrene TC-treated microplate (Corning, Corning, New York, USA) and treated with the inhibitors. The outermost wells were filled with medium only to avoid edge effects. PBS was added as a negative control and three wells with 80 µL DMEM without penicillin and streptomycin were used for the background measurements. After 48, 72, 96 and 120 hours, measurements were performed for the single and combination treatments. Before the read-out, 4 µL lysis solution (Promega Corporation, Fitchburg, Wisconsin, USA) was added to three UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 8 (31)

untreated wells, as a positive control, and incubated at 37 °C for 30 minutes. The CellTox Green reagent was prepared by mixing the CellTox Green dye with the assay buffer (Promega Corporation) in a ratio of 1:100. The asymmetric cyanine dye is able to enter dead cells and bind to the DNA, which, in turn, enhances its fluorescent properties and enables the read-out. To each well, 20 µL of the CellTox Green reagent was added and the plate was incubated, protected from light, on a shaker for 15 minutes. A Spark 10M multimode microplate reader (Tecan Trading, Männedorf, Switzerland) was used to detect the fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. For the analysis, the background values were subtracted from the values of each well and the average values of the triplicates (single and combination treatments) were calculated and afterwards normalised to PBS. The assay was performed four times for each cell line. Caspase-Glo 3/7 assay After the CellTox Green Cytotoxicity assay was performed, 100 µL of the Caspase-Glo 3/7 reagent (Promega Corporation) was added to two wells of each triplicate. The Caspase-Glo 3/7 reagent consists of a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, the Ultra-Glo recombinant luciferase and a buffer that causes cell lysis. Upon caspase cleavage of the substrate, aminoluciferin is released and a luminescent signal is created by the Ultra-Glo recombinant luciferase. The plate was protected from light and incubated on a shaker for 1 hour. A Centro LB 960 Microplate Luminometer (Berthold Technologies, Bad Wildbach, Germany) was used to detect the luminescence. To analyse the data, the background values were subtracted and the average of the duplicates (single and combination treatments) was formed and finally normalised to the PBS-treated wells. Each cell line was analysed in three replicates. Proliferation assay The cell proliferation was followed with the xCELLigence RTCA DP instrument (ACEA Biosciences, San Diego, California, USA). The measurements were performed in plates which contain microelectronic sensor arrays that measure the impedance that arises in response to the interaction between cells and the sensors upon application of an electric potential. The resulting values are expressed as a Cell Index, a dimensionless value which changes, e.g., in response to cell number or cell morphology changes. This allows for inference of the amount of cell proliferation. For background measurements, 50 µL DMEM without penicillin and streptomycin was added to the 16 wells of an E-Plate VIEW 16 (ACEA Biosciences). Afterwards, 5 × 103 cells in 50 µL DMEM without penicillin and streptomycin were added to each well and the plates were incubated at room temperature for 30 minutes. PBS was added between the wells. The impedance of the electron flow was measured every 2 hours for a total of 24 hours. After 24 hours, the programme was paused and the inhibitors were added. PBS was added as a positive control. The experiment was continued for an additional 84 hours with measurements every 2 hours. The average values of the duplicates (single and combination treatments) were calculated for each time point and normalised to the endpoint value of PBS. Three repetitions were performed for each cell line. Determination of the half maximal inhibitory concentration (IC50) The IC50 values were calculated from the results of the single treatments with BEZ235 and AZD4547 for 24, 48 and 72 hours in the WST-1 viability assay. For the calculations, the concentrations were log10 transformed and the values for the relative effects were multiplied by 100 to adjust their range. The GraphPad Prism function “log(inhibitor) vs. normalised response – variable slope” was used to fit a nonlinear regression model to the resulting concentration-response curves and calculate the IC50 values from the equation of the model. The analyses were performed in GraphPad Prism 8.1.1.

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Statistical analysis For the statistical analysis of the treatment effect, multiple unpaired t-tests were performed to compare the effect of each treatment with the inhibitors with PBS across all examined time points. No assumption of consistent standard deviations was made and a correction for multiple comparisons was performed according to the Holm-Šídák method. The analyses were performed separately for each assay and inhibitor concentration in GraphPad Prism 8.1.1. To compare the effect of the combinations with the corresponding single treatments, an analysis according to the “Highest Single Agent” approach was conducted on the results of the viability assay (100, 101). In summary, a two-tailed unpaired Student’s t- test was used to compare each combination treatment (EAB) with the maximum value of the corresponding two single treatments (max⁡(EA,EB)) for all time points of the viability data. A combination treatment had a significant positive effect if its effect size was, according to this test, significantly greater. Additionally, a combination index (CI) was calculated as CI= max⁡(EA,EB)⁄EAB. A CI < 1 indicates a positive effect of a combination compared with the corresponding single treatments, while a CI > 1 denotes a negative effect. The analyses were performed in GraphPad Prism 8.1.1. A P < 0.05 was considered statistically significant for all statistical tests.

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Results Effects of BEZ235 and AZD4547 single and combination treatments on the viability of UT-SCC- 60A and UPCI-SCC-154 cells To determine the effect of the inhibitors BEZ235 and AZD4547 on the viability of the cell lines UT- SCC-60A and UPCI-SCC-154, the cells were treated with different concentrations of the inhibitors and the WST-1 assay was performed 24, 48 and 72 hours after treatment to determine the number of viable cells. The inhibitors were tested as single treatments and in combination and the results are shown in Figure 1. In general, the cell line UT-SCC-60A was less sensitive to treatment with BEZ235 and AZD4547 than the UPCI-SCC-154 cell line. Effects of BEZ235 and AZD4547 on the viability of UT-SCC-60A cells Single treatments The resulting relative viability after single treatments of the cell line UT-SCC-60A with BEZ235 and AZD4547 is displayed in Figure 1A. The treatments with 5 µM BEZ235 as well as 25 µM and 50 µM AZD4547, the highest concentrations of the two inhibitors, resulted in significant decreases of the cell viability in at least two of the time points. For these two AZD4547 concentrations, the decreases were larger than 75 % compared with PBS at all time points and therefore especially pronounced. Treatments with 0.25 µM and 0.5 µM BEZ235, however, did not decrease the cell viability. Instead, the relative cell viability seemed to increase over time which may have occurred in response to a release of the mTOR- mediated negative feedback loop (102). The treatments with 1 µM BEZ235 and 10 µM AZD4547 had intermediate effects. Treatment with 5 µM AZD4547 significantly reduced the cell viability compared with PBS after 48 hours, while after 72 hours the cell viability seemed to be highly increased. Since the agreement between the three replicates was very limited for this treatment at 72 hours, it remains unclear if the observation is reliable. Combination treatments The effects of different combinations of BEZ235 and AZD4547 on the relative viability of UT-SCC-60A cells are presented in Figure 1B. At all examined time points all combinations decreased the viability significantly and by more than 50 % compared with PBS. As a general trend, a reduction in relative viability over time was observed for most of the combinations. Additionally, the viability generally decreased with increasing concentrations of the inhibitors. Addition of a second inhibitor to a single treatment resulted in a decrease in relative viability for most of the treatments which highlights the beneficial effects of the combination treatments. Effects of BEZ235 and AZD4547 on the viability of UPCI-SCC-154 cells Single treatments In Figure 1C the effects of BEZ235 and AZD4547 single treatments on the relative cell viability of UPCI-SCC-154 cells are displayed. All the treatments caused significant decreases of the relative cell viability at most of the time points. The decrease in cell viability was generally larger after treatment with higher concentrations of both inhibitors. Additionally, there were trends towards a decrease in relative viability over time for all the concentrations. Combination treatments In Figure 1D the effects of BEZ235 and AZD4547 combination treatments on the relative cell viability of UPCI-SCC-154 cells are presented. At every examined time point all treatments reduced the cell viability significantly and by more than 50 % compared with the PBS control. The relative viability seemed to decrease over time for all combinations. When a combination treatment was administered, the relative UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 11 (31)

viability was usually lower than with each of the two corresponding single treatments. Additionally, the relative viability decreased for most of the combinations if any of the two drug concentrations was increased.

Figure 1. Cell viability after treatment of the cell lines UT-SCC-60A and UPCI-SCC-154 with the PI3K and mTOR inhibitor BEZ235 and the FGFR inhibitor AZD4547. The cell viability was determined with the WST-1 assay 24, 48 and 72 hours after treatment. (A) Relative viability of UT-SCC-60A cells after single treatments with BEZ235 and AZD4547. (B) Relative viability of UT-SCC-60A cells after treatment with combinations of BEZ235 and AZD4547. (C) Relative viability of UPCI-SCC-154 cells after single treatments with BEZ235 and AZD4547. (D) Relative viability of UPCI-SCC-154 cells after treatment with combinations of BEZ235 and AZD4547. The bar heights represent the means after normalisation to PBS of three independent experiments, while the error bars indicate the standard deviations. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with PBS at the same time point. The experiments in panel A and C were performed by Anna Ohmayer, a previous Master student in the group. Summary of the data for UT-SCC-60A and UPCI-SCC-154 cells In summary, the HPV- cell line UT-SCC-60A appeared to be less sensitive than the HPV+ cell line UPCI- SCC-154. In UT-SCC-60A cells, low doses of the BEZ235 single treatments caused viability increases UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 12 (31)

over time, while the high doses of both inhibitors and all combination treatments caused reductions in relative viability. The cell line UPCI-SCC-154 was more sensitive to both inhibitors and a reduction in viability already occurred in the single treatments. The combination treatments yielded some additional viability reduction, although it was not as pronounced as for the UT-SCC-60A cells. IC50 values for UT-SCC-60A and UPCI-SCC-154 cells The IC50 values, the concentrations at which the half maximal inhibitions occurred, were calculated for the different time points from the single treatments of UT-SCC-60A and UPCI-SCC-154 cells with BEZ235 and AZD4547. The results are displayed in Table 1. The IC50 values for the treatments of UT- SCC-60A cells with BEZ235 were in the range of the highest tested concentrations at all time points. Notably, the value at 24 hours was extrapolated. This confirms a low sensitivity of UT-SCC-60A cells to BEZ235 at the concentrations used. The IC50 values that resulted from the treatments of the cell line UT- SCC-60A with AZD4547 were approximately in the middle of the range of the tested concentrations. This indicates an intermediate sensitivity to AZD4547 at the concentrations used. The treatments of UPCI-SCC-154 cells with BEZ235 resulted in IC50 values in the lower to intermediate range of the tested concentrations, while the values for the AZD4547 treatments were in the lower range. The values for BEZ235 at 48 and 72 hours, as well as AZD4547 at 72 hours were extrapolated, which indicates that UPCI-SCC-154 cells were relatively sensitive to both BEZ235 and AZD4547 at the tested concentrations. This confirms the conclusions from the data presented in Figures 1A and 1C. Table 1. IC50 values for UT-SCC-60A and UPCI-SCC-154 cells after treatment with BEZ235 or AZD4547 for 24, 48 and 72 hours. IC50 (µM) Cell line Drug 24 hours 48 hours 72 hours BEZ235 5.99 3.47 4.29 UT-SCC-60A AZD4547 16.33 8.55 10.41 BEZ235 0.74 0.12 0.14 UPCI-SCC-154 AZD4547 7.24 6.29 2.86

Comparison of the single and combination treatments of UT-SCC-60A and UPCI-SCC-154 cells according to the “Highest Single Agent” approach To compare the effects of the single and combination treatments, an effect-based analysis according to the “Highest Single Agent” principle was conducted. The results are displayed in Figure 2. For this analysis, the effect size of each combination treatment was compared with the greater effect of the two corresponding single treatments. Additionally, the CI was calculated to evaluate the direction of the effect. A significant CI < 1 denotes a significant positive effect of the combination, while a significant CI > 1 indicates a significant negative effect of the two drugs. For both cell lines, few combination treatments caused significant positive effects compared with the corresponding single treatments after 24 and 72 hours. The reason for this was most likely the high variation between the different replicates of the single treatments at these time points. After 48 hours, however, all combinations, apart from 1 µM BEZ235 and 25 µM AZD4547, had significant positive effects on UT-SCC-60A cells compared with the single treatments. Similarly, most combination treatments were significantly positive on UPCI-SCC-154 cells at this time point. No significant negative effects were observed at any of the time points. In general, there were trends towards positive effects for most of the combinations at least at one of the time points. This confirms the conclusions that were drawn from Figure 1. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 13 (31)

Figure 2. Comparison of the single and combination treatments according to the “Highest Single Agent” approach. A significant combination index (CI) < 1 denotes a significant positive effect of the combination, while a significant CI > 1 indicates a significant negative effect. ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. nd not determined. Cytotoxic effects of BEZ235 and AZD4547 single and combination treatments on UT-SCC-60A and UPCI-SCC-154 cells As the combinations of BEZ235 and AZD4547 decreased the relative viability of the cell lines UT-SCC- 60A and UPCI-SCC-154 compared with the single treatments, the CellTox Green Cytotoxicity assay was used to examine if the decrease in viability was due to cytotoxicity. The assay was performed 48, 72, 96 and 120 hours after addition of the inhibitors as single treatments and combinations. As the effects of the treatments on the relative viability were limited after 24 hours, this time point was excluded. Two time points at 96 and 120 hours were added to extend the observation period because initial experiments did not show large effects on the cytotoxicity at 48 and 72 hours. The treatments with 5 µM BEZ235 and 50 µM AZD4547 were excluded for the following assays, as they, already as single treatments, resulted in very large effects in the WST-1 assay. For the combination treatments one low (0.25 µM BEZ235 and 5 µM AZD4547), one intermediate (0.5 µM BEZ235 and 10 µM AZD) and one high (1 µM BEZ235 and 25 µM AZD4547) concentration that showed a stepwise increase in effect in the WST-1 assay were used for the following assays. The results are displayed in Figure 3. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 14 (31)

Figure 3. Cytotoxicity after treatment of the cell lines UT-SCC-60A and UPCI-SCC-154 with the PI3K and mTOR inhibitor BEZ235 and the FGFR inhibitor AZD4547. The cytotoxicity was determined with the CellTox Green Cytotoxicity assay 48, 72, 96 and 120 hours after treatment. (A) Relative cytotoxicity of single treatments with BEZ235 and AZD4547 on UT-SCC-60A cells. (B) Relative cytotoxicity of treatments with combinations of BEZ235 and AZD4547 on UT-SCC-60A cells. (C) Relative cytotoxicity of single treatments with BEZ235 and AZD4547 on UPCI-SCC-154 cells. (D) Relative cytotoxicity of treatments with combinations of BEZ235 and AZD4547 on UPCI-SCC-154 cells. The bar heights represent the means after normalisation to PBS of four independent experiments, while the error bars indicate the standard deviations. The inserts represent the rescaled graphs after exclusion of the lysis control. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with PBS at the same time point. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 15 (31)

Cytotoxic effects of BEZ235 and AZD4547 on UT-SCC-60A cells Single treatments The relative cytotoxicity after BEZ235 and AZD4547 single treatments of the cell line UT-SCC-60A is displayed in Figure 3A. As the values of the lysis controls were very high compared with the cytotoxicity caused by the different treatments, an insert without the positive control is shown. The BEZ235 treatments did not result in any clear increases or decreases in cytotoxicity. However, the treatments with 5 µM and 10 µM AZD4547 seemed to cause a decrease in cytotoxicity compared with PBS, while the cytotoxicity tended to be increased after treatment with 25 µM AZD4547. Combination treatments In Figure 3B, the relative cytotoxicity after BEZ235 and AZD4547 combination treatments of UT-SCC- 60A cells are displayed. An insert without the positive control is shown for clarity. The cytotoxicity seemed to be increased after treatment with 1 µM BEZ235 and 25 µM AZD4547. The two lower concentrations did not increase cytotoxicity. After 96 and 120 hours the detected cytotoxicity was even lower than after PBS treatment, which may reflect higher levels of cell death in the PBS control due to depletion of nutrients. This likely caused the values for the treatments with the inhibitors to appear lower after normalisation. In general, an increase in cytotoxicity on UT-SCC-60A cells was only obtained after single or combination treatments including 25 µM AZD4547. The relative cytotoxicity did not increase after the combination treatments compared with the single treatments in most of the cases. Cytotoxic effects of BEZ235 and AZD4547 on UPCI-SCC-154 cells Single treatments The relative cytotoxicity after BEZ235 and AZD4547 single treatments of UPCI-SCC-154 cells is presented in Figure 3C. As the values for the positive controls were much higher than the ones for the treatments with the inhibitors, an insert without the lysis control is shown. The single treatments with BEZ235 significantly reduced the cytotoxicity compared with the PBS control. Additionally, the BEZ235 treatments seemed to cause slightly lower relative cytotoxicity at later time points. The AZD4547 single treatments, however, showed a trend towards higher cytotoxicity at all the concentrations and time points. For both inhibitors, no clear concentration dependent increases or decreases in cytotoxicity were observed. Combination treatments In Figure 3D, the cytotoxicity after treatments of UPCI-SCC-154 cells with combinations of BEZ235 and AZD4547 is displayed. An insert without the lysis control was included to increase clarity. An increase in inhibitor concentrations seemed to result in a slight increase of relative cytotoxicity, while the cytotoxicity after treatment with 0.25 µM BEZ235 and 5 µM AZD4547 was comparable to PBS. Treatment of UPCI-SCC-154 cells with combinations of BEZ235 and AZD4547 caused higher cytotoxicity than the corresponding BEZ235 single treatments but lower or similar cytotoxicity than the corresponding AZD4547 single treatments. Summary of the data for UT-SCC-60A and UPCI-SCC-154 cells To summarise, the CellTox Green Cytotoxicity assay confirmed a lower sensitivity of the HPV- cell line UT-SCC-60A compared with the HPV+ cell line UPCI-SCC-154. Treatment of UT-SCC-60A cells with a high concentration of AZD4547 (25 µM), but not the lower concentrations, caused higher levels of cytotoxicity than the treatment with PBS. However, the treatments with BEZ235 caused no clear increases of the relative cytotoxicity. Treatment of UPCI-SCC-154 cells with all AZD4547 concentrations caused a slight increase in cytotoxicity. The cytotoxicity seemed to decrease over time after BEZ235 treatments. In general, the addition of BEZ235 to single treatments with AZD4547 did not UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 16 (31)

cause any beneficial effects on cytotoxicity. This, in combination with the results from the viability assay, indicates a cytostatic role of BEZ235, while AZD4547 seemed to have cytostatic and cytotoxic effects on the tested cell lines. Effects of BEZ235 and AZD4547 single and combination treatments on the induction of apoptosis in UT-SCC-60A and UPCI-SCC-154 cells To test the effects of BEZ235 and AZD4547 single and combination treatments on the induction of apoptosis in UT-SCC-60A and UPCI-SCC-154 cells, the Caspase-Glo 3/7 assay was performed 48, 72, 96 and 120 hours after treatment with the inhibitors. This way, mechanistic insights into the cause of the viability reductions and increases in cytotoxicity, at some time points of the high treatment concentrations, could be obtained. The concentrations of the single and combination treatments were the same as in the CellTox Green Cytotoxicity assay. The results are depicted in Figure 4.

Effects of BEZ235 and AZD4547 on the induction of apoptosis in UT-SCC-60A cells Single treatments The effects of single treatments with BEZ235 and AZD4547 on the relative induction of apoptosis in the cell line UT-SCC-60A are displayed in Figure 4A. The highest sensitivity was observed with the 25 µM AZD4547 treatment, although the effect was not significant (after 48 hours P = 0.39). All other treatments reduced the induction of apoptosis significantly compared with PBS at almost all time points, although higher inhibitor concentrations generally resulted in higher levels of caspase 3/7 activation than lower concentrations. There was a trend towards a decrease in relative caspase 3/7 activation over time for most of the treatments. This effect might be due to the depletion of nutrients over time but also due to the fact that the caspase 3 and 7 activation is a relatively early event in apoptosis, which is likely to be induced shortly after treatment. Combination treatments The effects of BEZ235 and AZD4547 combination treatments on the relative induction of apoptosis in UT-SCC-60A cells are depicted in Figure 4B. Treatment with 1 µM BEZ235 and 25 µM AZD4547 seemed to increase the induction of apoptosis after 48 hours, although this effect was not significant. At all examined time points, significantly lower caspase 3/7 activation was observed after treatment with the remaining combinations than after treatment with PBS. A tendency towards lower relative caspase 3/7 activation over time was observed for all combinations. Nonetheless, the observed caspase 3/7 activation tended to be higher for higher concentrations. A benefit of the addition of AZD4547 to BEZ235 single treatments was observed only at the highest concentration. BEZ235 addition to AZD4547 single treatments did not result in obvious benefits. Effect of BEZ235 and AZD4547 on the induction of apoptosis in UPCI-SCC-154 cells Single treatments The effects of the BEZ235 and AZD4547 single treatments on the induction of apoptosis in UPCI-SCC- 154 cells are displayed in Figure 4C. All treatments with BEZ235 caused a significant decrease in the induction of apoptosis at all time points. The treatments with AZD4547, however, seemed to increase the caspase 3/7 activation after 48 hours but not at later time points. Higher AZD4547 concentrations tended to cause higher levels of apoptosis induction. A trend towards a decrease in relative caspase 3/7 activation over time was observed. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 17 (31)

Figure 4. Induction of apoptosis after treatment of the cell lines UT-SCC-60A and UPCI-SCC-154 with the PI3K and mTOR inhibitor BEZ235 and the FGFR inhibitor AZD4547. The apoptosis induction was determined with the Caspase- Glo 3/7 assay 48, 72, 96 and 120 hours after treatment. (A) Relative caspase 3/7 activation in UT-SCC-60A cells after single treatments with BEZ235 and AZD4547. (B) Relative caspase 3/7 activation in UT-SCC-60A cells after treatment with combinations of BEZ235 and AZD4547. (C) Relative caspase 3/7 activation in UPCI-SCC-154 cells after single treatments with BEZ235 and AZD4547. (D) Relative caspase 3/7 activation in UPCI-SCC-154 cells after treatment with combinations of BEZ235 and AZD4547. The bar heights represent the means after normalisation to PBS of three independent experiments, while the error bars indicate the standard deviations. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with PBS at the same time point. Combination treatments The effects of BEZ235 and AZD4547 combination treatments on the induction of apoptosis in UPCI- SCC-154 cells are summarised in Figure 4D. After 48 hours of treatment with all three combinations, significantly higher levels of caspase 3/7 activation were observed than with treatment with PBS, while this was not observed at later time points. Higher inhibitor concentrations tended to cause higher levels of caspase 3/7 activation for most time points. AZD4547 addition to BEZ235 single treatments resulted in an increase in apoptosis induction. However, BEZ235 addition to AZD4547 single treatments resulted in no clear trend towards an increase or decrease. A decrease in relative caspase 3/7 activation over time was observed for all combinations. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 18 (31)

Summary of the data for UT-SCC-60A and UPCI-SCC-154 cells In summary, the HPV- cell line UT-SCC-60A was less sensitive than the HPV+ cell line UPCI-SCC-154 to the induction of apoptosis by BEZ235 and AZD4547. In UT-SCC-60A cells all treatments, apart from 25 µM AZD4547 at 48 and 72 hours, lowered the relative caspase 3/7 activation compared with PBS. No clear benefits were observed with the combination treatments compared with the AZD4547 single treatments. This reflects what was observed with the CellTox Green Cytotoxicity assay. UPCI-SCC-154 cells were more sensitive than UT-SCC-60A cells to the AZD4547 single and combination treatments. While BEZ235 caused a reduction in apoptosis induction, AZD4547 caused an increase at early time points with all tested concentrations. Similar to UT-SCC-60A cells, no clear benefit of the combination treatment was observed compared with the AZD4547 single treatment with regard to the induction of apoptosis in UPCI-SCC-154 cells. This is compatible with the hypothesis that BEZ235 causes mainly cytostatic effects, while AZD4547 has cytostatic and cytotoxic properties. Effects of BEZ235 and AZD4547 single and combination treatments on the proliferation of UT- SCC-60A and UPCI-SCC-154 cells To test the effects of BEZ235 and AZD4547 single and combination treatments on the cell proliferation of UT-SCC-60A and UPCI-SCC-154 cells, the xCELLigence RTCA DP instrument was used to follow cell proliferation over time. The measurements were started 24 hours before the treatment and continued until 84 hours after treatment. Due to space constraints on the plates the intermediate concentrations of the inhibitors (0.5 µM BEZ235 and 10 µM AZD4547) were not used as single treatments. The same three combinations as in the CellTox Green Cytotoxicity assay and the Caspase-Glo 3/7 assay were used for these experiments. The results are presented in Figure 5 after normalisation to the end value of PBS for each experiment. Effects of BEZ235 and AZD4547 on the proliferation of UT-SCC-60A cells Single treatments The effects of BEZ235 and AZD4547 single treatments on the cell index of UT-SCC-60A cells are depicted in Figure 5A. Treatment with PBS resulted in a continuous increase of the cell index. Towards the end, the proliferation slowed down and a plateau seemed to be reached. The treatments with 1 µM BEZ235 as well as 25 µM AZD4547 resulted in a continuous decrease of the cell index and were the two most effective single treatments. Treatment with 5 µM AZD4547 did not influence the cell index until approximately 20 hours after treatment but thereafter the cell index decreased until the end. After treatment with 0.25 µM BEZ235 the cell index decreased slightly for roughly 34 hours, after which it increased again until the end of the experiment. Combination treatments The relative effects on the cell index of UT-SCC-60A cells before and after combination treatments with BEZ235 and AZD4547 are displayed in Figure 5B. Treatment with PBS resulted in a continuous increase of the cell index. A plateau was observed towards the end of the experiment. The most effective treatment, 1 µM BEZ235 and 25 µM AZD4547, caused a rapid decrease of the cell index almost to the baseline within 20 hours after treatment. Treatment with 0.25 µM BEZ235 and 5 µM AZD4547, resulted in a continued initial increase of the relative cell index, after which the cell index decreased until the end of the experiment. A generally similar but slightly stronger effect on the relative cell index was observed after treatment with 0.5 µM BEZ235 and 10 µM AZD4547. In this experiment, all the combinations resulted in a strong decrease of the cell index. In UT-SCC-60A cells, both the lowest and the highest combinations seemed to reduce the cell index more than the two corresponding single treatments. As expected, the effect of the intermediate concentration of the combinations was higher than that of the low but lower than that of high concentration. This experiment UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 19 (31)

confirmed that UT-SCC-60A cells were relatively sensitive to 25 µM AZD4547 and that the combination treatments resulted in beneficial effects.

Figure 5. Proliferation after treatment of the cell lines UT-SCC-60A and UPCI-SCC-154 with the PI3K and mTOR inhibitor BEZ235 and the FGFR inhibitor AZD4547. The proliferation was measured with the xCELLigence RTCA DP instrument from cell seeding until 84 hours after treatment. (A) Relative cell index before and after single treatments of UT- SCC-60A cells with BEZ235 and AZD4547. (B) Relative cell index before and after treatment of UT-SCC-60A cells with combinations of BEZ235 and AZD4547. (C) Relative cell index before and after single treatments of UPCI-SCC-154 cells with BEZ235 and AZD4547. (D) Relative cell index before and after treatment of UPCI-SCC-154 cells with combinations of BEZ235 and AZD4547. All values were normalised to the end point value of PBS. Every graph is representative of three independent experiments. Effects of BEZ235 and AZD4547 on the proliferation of UPCI-SCC-154 cells Single treatments In Figure 5C, the effects of BEZ235 and AZD4547 single treatments on the cell index of UPCI-SCC-154 cells are presented. Treatment with PBS resulted in a continuous increase of the cell index. After treatment with 25 µM AZD4547 a rapid decrease of the relative cell to the baseline was observed within 2 hours, which was the largest observed effect. Treatment with 5 µM AZD4547 resulted in a continuous decrease of the cell index to the baseline until the end of the experiment. After treatment with 1 µM BEZ235 the cell index initially decreased, after which it remained stable at approximately 20 % of the maximum value for PBS until the end of the experiment. Treatment with 0.25 µM BEZ235 resulted in a slow decrease of the cell index during the first 30 hours, before it increased again until the end of the experiment. Combination treatments The effects of BEZ235 and AZD4547 combination treatments on the cell index of UPCI-SCC-154 are displayed in Figure 5D. In the PBS control a continuous increase was observed. All three combination treatments caused very effective inhibitions of cell proliferation and decreases of the cell index. The UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 20 (31)

decreases were faster with higher treatment concentrations. After treatment with 1 µM BEZ235 and 25 µM AZD4547 the relative cell index decreased rapidly to the baseline within 2 hours. Treatment with 0.5 µM BEZ235 and 10 µM AZD4547 resulted in a decrease to the baseline within 32 hours, while a similar decrease was observed after treatment with 0.25 µM BEZ235 and 5 µM AZD4547 within 56 hours. Treatment of UPCI-SCC-154 cells with AZD4547 resulted in larger effects on the cell index than treatment with BEZ235. Additional beneficial effects, compared with the single treatments, could be observed with the lower two combination treatments. For the highest combination, the effect was approximately comparable with that of the treatment with 25 µM AZD4547. Summary of the data for UT-SCC-60A and UPCI-SCC-154 cells The HPV- cell line UT-SCC-60A was generally less sensitive to a BEZ235- and AZD4547-induced decrease of the cell index than the HPV+ UPCI-SCC-154 cell line. This confirms the results of the previous three assays. Treatment of UT-SCC-60A cells with the lowest single concentrations (0.25 µM BEZ235, 5 µM AZD4547) did not cause as pronounced effects as the higher concentrations (1 µM BEZ235, 25 µM AZD4547). The combination treatments resulted in beneficial effects compared with the single treatments. The UPCI-SCC-154 cells were sensitive to all tested treatments, which is in agreement with the observations from the viability assay. However, the effects of the BEZ235 treatments (especially 0.25 µM) were temporary. In UPCI-SCC-154 cells, treatments with the combinations resulted in some, but rather small, beneficial effects compared with the single treatments. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 21 (31)

Discussion To investigate the potential of targeted therapies with PI3K and FGFR inhibitors for patients with TSCC or BOTSCC, the inhibitors BEZ235 and AZD4547 were tested as single and combination treatments in vitro on the HPV- TSCC cell line UT-SCC-60A and the HPV+ BOTSCC cell line UPCI-SCC-154. The treatment effects were evaluated with the WST-1 viability assay, the CellTox Green Cytotoxicity assay, the Caspase-Glo 3/7 assay and the xCELLigence RTCA DP instrument. Notably, the HPV+ cell line UPCI-SCC-154 was more sensitive to both inhibitors than the HPV- cell line UT-SCC-60A in all assays. Across the assays, 25 µM and, if used, 50 µM AZD4547, were the most effective single treatments on UT-SCC-60A cells. UPCI-SCC-154 cells were sensitive to all the tested BEZ235 and AZD4547 single treatments. These AZD4547 treatments reduced cell viability and proliferation and trends towards increases in cytotoxicity and early apoptosis of both cell lines were also observed. This suggests a cytotoxic and, possibly, an additional cytostatic role of the AZD4547 single treatments. The treatments with BEZ235 decreased cell viability and proliferation but also cytotoxicity and apoptosis in UPCI-SCC-154 cells. This proposes a cytostatic rather than a cytotoxic effect of BEZ235. These general sensitivity trends were also reflected in the calculated IC50 values. A previous study on nasopharyngeal squamous cell carcinoma found that BEZ235 induced G1 arrest and apoptosis in vitro (103). The same was observed for AZD4547 in a study on colorectal cancer cell lines (104). Since these studies do not conclude a pure cytostatic or cytotoxic nature of any of the two drugs, it would be interesting to perform more experiments, e.g., cell cycle analyses to further investigate this in the TSCC and BOTSCC setting. HPV+ tumours are generally more sensitive to treatments than HPV- tumours (11, 12). However, it is not clear if this also applies to targeted therapies. In these experiments, the HPV+ BOTSCC cell line UPCI- SCC-154 (105) was indeed more sensitive than the HPV- TSCC cell line UT-SCC-60A (106). However, it is not really possible to conclude a sensitivity dependence on the HPV status from these data, since only two cell lines were tested. Furthermore, the two cell lines originated from different anatomical sites, although both sites belong to the Waldeyer’s ring in the pharynx and are composed of similar lymphoid tissue. To further pursue this issue, additional cell lines with different qualities (HPV+/HPV-, TSCC/BOTSCC) need to be tested. The combination treatments generally caused beneficial effects compared with the corresponding single treatments. These favourable effects seemed to be more pronounced for UT-SCC-60A than for UPCI- SCC-154 cells, possibly because the latter cell line was more sensitive to the single treatments. All combination treatments reduced the viability and proliferation of UT-SCC-60A cells, while only the treatment with the highest combination (1 µM BEZ235 and 25 µM AZD4547) seemed to induce cytotoxicity and apoptosis. For UPCI-SCC-154 cells, however, all combinations reduced cell viability, increased early apoptosis and reduced proliferation, while only the two higher combinations tended to increase cytotoxicity. Calculations according to the “Highest Single Agent” approach confirmed the positive effects of most of the combinations at least at one of the time points. For the UT-SCC-60A cell line, the effects of all combinations, apart from 1 µM BEZ235 and 10 µM AZD4547 as well as 1 µM BEZ235 and 25 µM AZD4547, were higher than the added effects of the two corresponding single treatments after 48 hours. It was especially promising that the relative reduction in cell viability with two of the lowest combinations (0.25 µM BEZ235 and 5 µM AZD4547 as well as 0.5 µM BEZ235 and 5 µM AZD4547) was pronounced. The corresponding single treatments did not cause a significant decrease or even an increase in cell viability, especially at 72 hours. As the treatment effects were large for the UPCI-SCC-154 cells, it could have been of interest to also test lower inhibitor concentrations. In this case, the combinations may have resulted in similar beneficial effects as they did for UT-SCC-60A cells at the concentrations that were tested here. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 22 (31)

No research has been performed on the combination of the PI3K inhibitor BEZ235 and FGFR inhibitor AZD4547 in TSCC and BOTSCC so far. However, there was one study that tested combinations of AZD4547 and mTOR inhibitors on a nasopharyngeal and a laryngeal SCC cell line and found beneficial effects of the combination treatments (107). Similarly, another study on bladder cancer found that combination treatments with the PI3K inhibitor BKM120 and AZD4547 resulted in beneficial effects in vitro and significantly reduced tumour growth in vivo, compared with single treatments (108). Since FGFR3 mutations frequently occur in bladder cancer (109) and since many studies have been performed in this setting, these studies can provide some additional insight into what the situation in HNSCC might look like. Other studies on HNSCC and bladder cancer cell lines found EGFR signalling to limit the response to FGFR inhibitors and observed beneficial effects when combining FGFR and EGFR inhibitors (110, 111). In summary, the data presented here agree with observations from the literature that combinations of different targeted therapies can have beneficial effects and extend the knowledge on PI3K and FGFR inhibitors to TSCC and BOTSCC. There were some limitations with the assays that were used here and the data that was obtained. One main concern is that the agreement of the different replicates was limited for some assays, time points and concentrations. This affected for example the single treatments in the viability assay, especially after 24 and 72 hours, as well as the cytotoxicity assay and the apoptosis assay at 48 hours after treatments with 25 µM AZD4547 and combination treatment with 1 µM BEZ235 and 25 µM AZD4547. In these cases a repetition of the experiment may increase the precision of the estimates and improve the power to reveal even more significant differences. The differences between the replicates also affected the calculations of the IC50 and the “Highest Single Agent” analysis. In the IC50 calculations, the values for 72 hours were sometimes higher than for 48 hours. In these cases, it could not be resolved whether this reflected actual effects or was attributable to the large variations. For the calculations of reliable IC50 values, a repetition of the single treatment experiments is essential. Additionally, more inhibitor concentrations should have been tested as some of the IC50 values had to be extrapolated from the nonlinear model. The results of the calculations according to the “Highest Single Agent” approach agreed very poorly across the different time points. It is likely that this does not reflect actual differences but was due to the high standard deviations of the values of the single treatments. However, since almost all combinations showed significant positive effects compared with the single treatments, at least at one of the examined time points, it can be assumed that the combination treatments were generally favourable compared with the single treatments for both cell lines. The CellTox Green Cytotoxicity assay showed very high deviations even of the triplicates of one replicate and therefore seemed to be relatively unstable. The large standard deviations prohibited the statistical detection of small treatment effects. It would be important to confirm the results with another method, e.g., a flow cytometry experiment utilising propidium iodide and annexin V stainings to be able to detect dead cells and differentiate between apoptotic and necrotic cells. Furthermore, an issue that has to be viewed with caution is that only one run of the proliferation experiment is presented here. The cells grew slightly differently every time the experiment was performed, so that no experimental run is completely representative for all. In general, however, the observed effects were in good agreement. An important consideration regarding the CellTox Green Cytotoxicity assay and the Caspase-Glo 3/7 assay is that the amount of cell death in the PBS control increased over time. These effects occurred probably due to high cell numbers and a depletion of nutrients. After the normalisation, this led to that the treatment effects appeared to be lower at later time points. Since a decrease in cytotoxicity and induction of apoptosis was observed over time, it would be important to repeat these experiments with lower cell numbers at seeding to evaluate the relevance of these observations. It would have also been UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 23 (31)

interesting to examine the time point at 24 hours after treatment to see even earlier treatment effects. Generally, it can be emphasised that the optimisation of cell density and time is very important in these kinds of assays. Moreover, the time span that can be investigated with these types of assays is relatively short. In future research it will be important to perform other types of experiments, e.g., clonogenic assays that are able to show the treatment effect over a longer period. Other studies that investigated BEZ235 and AZD4547 used lower concentrations of the two drugs for their in vitro tests in the HNSCC setting. For both drugs, concentrations in the one- or two-digit nanomolar range were commonly used (103, 107, 112–114). Some of these studies also reported IC50 values in the same concentration range (107, 113, 114). Compared with these values, the IC50 values that were obtained here were approximately 101- to 103-fold higher for BEZ235 and 103- to 104-fold higher for AZD4547. This may of course reflect differences between the different cell lines but could also be due to that the standard deviations of the single treatments were very high and few concentrations were tested here. Therefore, more concentrations in a wider range should be tested to determine the IC50 values of UT-SCC-60A and UPCI-SCC-154 more precisely. In summary, the four assays complemented each other relatively well. They revealed sensitivities of the UT-SCC-60A cells especially to 5 µM BEZ235 as well as 25 µM and 50 µM AZD4547 and additional beneficial effects of the combination treatments in this cell line. UPCI-SCC-154 cells were sensitive to all the tested single treatments and the combinations caused some, but compared with UT-SCC-60A cells less pronounced, beneficial effects. The data suggest that BEZ235 caused mainly cytostatic effects, while AZD4547 was both cytostatic and cytotoxic. Since the effects of the BEZ235 and AZD4547 combination treatments were beneficial compared with the single treatments, it is promising to continue with this line of research. The side effects of targeted therapies with PI3K inhibitors are relatively severe (115). One hypothesis is that by administering combination treatments, it will be possible to lower the concentrations of each drug, thus reducing the side effects. This might actually be true for combinations of BEZ235 and AZD4547, since synergistic effects on UT-SCC-60A cells were observed for some of the combinations 48 hours after treatment. However, this remains to be investigated further in preclinical models or eventually in clinical trials. Another expectation is that it could be possible to prevent or delay resistance development with targeted drug combinations. Studies on HNSCC and bladder cancer have investigated the switch in signalling pathways during resistance development and the residual sensitivity to other therapies once resistance development has occurred (114, 116). More studies like these will be necessary in the future to fully understand the potentials and risks of specific targeted therapies. Furthermore, two studies on HNSCC cell lines concluded beneficial effects of the combination of BEZ235 with chemotherapy and radiation (103, 112). All of these studies indicate that with an intelligent selection of two targeted therapies for combination, the sensitivities can be enhanced and the development of acquired resistances can be delayed. Moreover, it is notable and promising that beneficial effects have also been observed together with the conventional therapeutic regimes. In the future, it will be interesting to test whether TSCC and BOTSCC cell lines with PIK3CA and FGFR3 mutations are more sensitive to these inhibitors. However, it has been shown in several studies on HNSCC that the relationship between PIK3CA and FGFR3 mutations and the sensitivities to the corresponding inhibitors is not always as straightforward as one might assume. Several studies in the HNSCC setting reported PIK3CA and FGFR3 mutations to influence the sensitivity to BEZ235 or the FGFR inhibitor BGJ398 (113, 117, 118). However, in two of these studies the effect size was largely dependent on the specific mutation and some mutations were observed to decrease instead of increase sensitivities (113, 118). Furthermore, the report of a phase I study with the PI3K inhibitor GSK2126458 disclosed no obvious relationship between PIK3CA mutations and the treatment response (119). In UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 24 (31)

summary, more research is needed to transfer this knowledge, which was mostly obtained in other HNSCC types, to TSCC and BOTSCC. Additionally and more importantly, it remains to be established which specific mutations influence the response to these inhibitors and in what way. This will then help to determine specific genetic signatures that can potentially predict the treatment responses. To conclude, this thesis investigated targeted therapies with the PI3K and mTOR inhibitor BEZ235 as well as the FGFR inhibitor AZD4547 on TSCC and BOTSCC cell lines. Both cell lines showed some sensitivity to single treatments with the two inhibitors and even larger effects were observed when the two drugs were combined. By combining this kind of approach with the current standard treatments, an additional step towards individualised cancer medicine can be taken. This way, TSCC and BOTSCC patients that do not receive sufficient treatment today could be offered additional combined targeted therapy, while those with tumour biomarkers that predict a good therapy response could potentially be treated with less intensive therapy. Thereby, the survival of patients that are currently undertreated could increase, while patients with responsive tumours would encounter lower side effects and have a better quality of life. This approach is ethically desirable because it would reduce the numbers of patients that are treated with either too harsh or too weak therapies.

Acknowledgements I would like to thank Tina Dalianis for the possibility to conduct this thesis in her laboratory as well as her guidance. I would also like to express my gratitude towards my supervisors Stefan Holzhauser and Ourania Kostopoulou for their practical guidance and support. I am also grateful to Staffan Johansson for being allowed to be a part of the Medical Research programme and for always answering my questions. UPPSALA UNIVERSITET FGFR and PI3K inhibitors in oropharyngeal cancer 25 (31)

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Fighting head and neck cancers with two weapons at the same time Imagine that the best treatment could be given to every cancer patient – an efficient one with little or no side effects. Unfortunately, we are not there yet. Nevertheless, one step in this direction is to investigate the relatively new, currently developing targeted therapies. These therapies target the dependence of cancer cells on certain cellular signalling pathways. Sometimes the molecules involved in these pathways are mutated in cancer cells, so that these therapies may be very specific to cancer cells and harmless to normal cells. If these pathways are inhibited, the cells can either not divide anymore or die. One disadvantage is the development of drug resistance. To increase the therapeutic effect, we investigated combinations of targeted therapies that target two cellular pathways simultaneously. We work on head and neck cancer; more specifically squamous cell carcinomas, that develop in the tonsils (TSCC) or base of the tongue (BOTSCC). Some of these tumours are the consequence of infections with human papillomaviruses (HPV). Others develop due to smoking and alcohol consumption. Patients with HPV-positive (HPV+) TSCC or BOTSCC generally respond better to therapies. Current therapy is too harsh for patients with sensitive tumours, but not sufficient enough for the group of patients with more resistant tumours and comes with severe side effects. There is, therefore, a great need for the development of new therapies. Our group and others have observed that fractions of patients with TSCC and BOTSCC, especially of those with HPV+ tumours, carry mutations in two genes with important roles in cellular signalling pathways, namely PIK3CA and FGFR3. This suggests the importance of the corresponding signalling pathways for the tumours of these patients. Here, inhibitors against PIK3CA and FGFR3 were tested as single and combination treatments on two cell lines which were obtained from patients with HPV+ BOTSCC and HPV-negative (HPV-) TSCC and have no PIK3CA and FGFR3 mutations. Different assays were used to evaluate the effect of the drugs. A viability assay, which detects cells with an active metabolism, and proliferation tests revealed that the combination treatments affected the cells more than the single treatments. Other assays that measure cytotoxicity and apoptosis, a form of induced cell death, showed effects of the FGFR3 but not the PIK3CA inhibitor. This indicates that the treatment with the PIK3CA inhibitor affected the cellular metabolism rather than causing cell death, while the FGFR3 inhibitor gave rise to both effects. HPV+ cells were more sensitive to both inhibitors than the HPV- ones and were affected at lower concentrations. Combination treatments increased the effects of the drugs on both cell lines. To conclude, combination treatments with PIK3CA and FGFR3 inhibitors showed better effects than single treatments on HPV+ BOTSCC and HPV- TSCC cells. The HPV+ cell line was generally more sensitive to treatment than the HPV- cell line. These results are promising and bring us closer to providing personalised cancer treatments to patients. Nevertheless, more experiments on cell lines with relevant mutations are necessary.