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Cite this article: Wilson GD, Galoforo S, Blas K, Tonlaar N, Wilson TG, et al. (2018) Comparison of Inhibitors of PIK3 and MTOR in Combination with Radia-tion in Head and Neck Cancer. JSM Biochem Mol Biol 5(1): 1034.

*Corresponding author

George D. Wilson, Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073, USA, Tel: 1-248-551-0214; Fax: 1-248-551-2443; Email:

Submitted: 21 April 2018

Accepted: 25 June 2018

Published: 27 June 2018

ISSN: 2333-7109

Copyright© 2018 Wilson et al.

OPEN ACCESS

Keywords•Head and neck cancer; Radiation PIK3; MTOR;

Xenografts

Research Article

Comparison of Inhibitors of PIK3 and MTOR in Combination with Radiation in Head and Neck CancerGeorge D. Wilson1*, Sandra Galoforo1, Kevin Blas1, Nathan Tonlaar1, Thomas G. Wilson1, Arth Patel1, Alaa Hana1, Mohamad Dabjan1, Katie Buelow1, and Brian Marples2

1Department of Radiation Oncology, William Beaumont Hospital, USA2Department of Radiation Oncology, University of Miami, USA

Abstract

In this study we have combined fractionated radiation with three different drugs targeting the PIK3/AKT/MTOR pathway in pre-clinical models of head and neck cancer. Three drugs were studied; two were dual PIK3/MTOR inhibitors, PF-04691502 and gedatolisib, whilst buparlisib was a pan-PIK3 inhibitor without effect on MTOR. The effect of the drugs was studied in vitro and in vivo on tumor growth and on phosphorylation status of key signaling proteins. The drugs had differential sensitivity in vitro with gedatolisib being the most active. None of the agents significantly modified radiation sensitivity in clonogenic assays. The agents had variable effects in vivo both alone and in combination with radiation. Buparlisib was effective against UT-SCC-15 xenografts both alone (p= 0.001) and combined with RT (p= 0.008) but was without any activity against UT-SCC-14 xenografts. The dual PIK3/MTOR inhibitors exhibited mixed activity. PF-04691502 alone had some activity although not quite significant in UT-SSC-14 but was inactive in UT-SCC-15. Gedatolisib showed inhibition of growth in both xenograft models. When combined with RT, PF-04691502 maintained a significant enhancement in UT-SSC-14 but had no effect in UT-SCC-15.Gedatolisib proved less effective when combined with radiation. Alterations in the phosphorylation status of key proteins mirrored the effect on tumor growth inhibition. The effectiveness of drugs targeting the PIK3/MTOR axis was difficult to predict as was their combination with radiation. The complex genomic landscape of HNSCC and the substantial cross-talk between signaling pathways present a significant challenge to predict and prescribe the optimal treatment for individual patients.

ABBREVIATIONSHNSCC: Head and Neck Squamous Cell Cancer; HPV: Human

Papillomavirus; RT: Radiation Treatment; EGFR: Epidermal Growth Factor Receptor; TCGA; The Cancer Genome Atlas; PIK3: Phosphoinositide 3-kinase; MTOR: Mammalian Target of Rapamycin; PIK3CA: Catalytic Subunit p110α of Class IA PI3-Kinase; IRS: Insulin Receptor Substrate; GAB; GRB2-Associated Binder; PIP3: Phosphatidyl Inositol 3,4,5 Tri-Phosphate; PDK1: 3-Phosphoinositide-Dependent Kinase; mTORC1: mTOR Complex 1; mTORC2: mTOR Complex 2; RAPTOR: Regulatory-Associated Protein of mTOR; mLST8: Mammalian Lethal with Sec13 Protein 8; S6K1: Ribosomal Protein S6 Kinase 1; 4EBP1: Eukaryotic Initiation Factor 4E (eIF4E)-Binding Protein 1; RICTOR: Rapamycin-Insensitive Companion of mTOR; PKCα: Protein Kinase Cα; SGK1: Glucocorticoid-Induced Protein Kinase 1; PIKK: PIK3-Related Kinases; MTT: 3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyltetrazolium Bromide; DMF: Dose Modifying Factor; DEF: Dose Enhancement Factor; DDR: DNA Damage Response; PARP: poly ADP Ribose Polymerase; HR: Homologous Recombination

INTRODUCTIONHNSCC is the sixth most common cancer worldwide and it is

estimated that over $3.6 billion is spent on HNSCC cancer care each year in the United States. Although the past three decades have witnessed significantly improved outcomes for locally advanced HNSCC, it is important to appreciate that much of this improvement has been driven by the emergence of human papillomavirus (HPV) associated disease characterized by good prognosis [1]. In contrast, patients with HPV-negative disease carry a poor prognosis despite optimal treatment with chemo radiation, with a 5 year survival of less than 50% [2]. The future for HPV-negative disease must lie in further escalation of therapy which represents a difficult task given the fact that treatment already approaches the limit of tolerance. The emergence of a wide array of molecularly targeted agents over the last decade with the unique capacity of modulating cancer-specific cellular signaling and response to chemo radiation has garnered significant enthusiasm in the radiation oncology community based on the potential of these compounds to serve as radiation (RT) sensitizers. Early success was achieved by targeting the EGFR pathway [3] but since then very few targeted agents have advanced to phase III trials with radiotherapy [4].

The search for alternative pathways to treat HNSCC has been illuminated through full genomic analysis of HNSCC primary

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tumors [5,6]. Results from initial studies confirmed known HNSCC mutations including TP53, CDKN2A, PIK3CA, HRAS, PTEN and found NOTCH1 and FBXW7 as novel mutations. The largest data set of over 279 HNSCC primary tumors was reported by the Cancer Genome Atlas (TGCA) [7]. This study identified at least 15 significantly mutated genes including: CDKN2A, TP53, PIK3CA, FAT1, MLL2, TGFBR2, HLA-A, NOTCH1, HRAS, NFE2L2, and CASP8.

From these studies, common actionable mutations are present in HNSCC. One of these is PIK3CA which is a component of the PIK3/MTOR/PTEN/AKT signaling pathway involved in regulation of cell survival, growth, proliferation, metabolism and motility and an attractive drug target in cancer [8]. The phosphoinositide 3-kinase (PIK3) and mammalian target of rapamycin (MTOR) signaling pathways have been found to have an important role in the pathogenesis of HNSCC [9] and there is evidence PIK3/MTOR antagonists are active against head and neck cancer cells [10,11].

PIK3CA, located on chromosome 3q26.32, encodes the catalytic subunit p110α of class IA PI3-kinase (PIK3) and has been implicated as an oncogene because of its genomic amplification in different cancers [12,13]. High frequencies of mutations in PIK3CA have been reported in several cancer types including head and neck cancer [14,15]. Growth factors activate the lipid kinase PIK3 through either direct PIK3 recruitment to the growth factor receptor or indirectly through recruitment of IRS (insulin receptor substrate) or GAB (GRB2-associated binder) docking proteins. PIK3 generates PIP3 (phosphatidyl inositol 3,4,5 tri-phosphate), which recruits the protein kinase AKT to the plasma membrane where it is activated by 3-phosphoinositide-dependent kinase 1(PDK1) and mTORC2. AKT phosphorylates many survival, proliferation, and motility factors. The majority of mutations in PIK3CA are clustered in the helical (exon 9) and kinase domains (exon 20) with the most commonly reported hot spots identified as E542K, E545K and H1047R which result in increased lipid kinase activity and activation of the downstream AKT pathway [16,17]. Interestingly, mutation status does not seem to determine sensitivity to PIK3CA inhibitors [11,18] in head and neck cell lines. Isoform-specific PIK3 inhibitors have been developed with the aim of targeting specific alterations in the PIK3 pathway, while avoiding the cumulative toxicity of inhibiting multiple isoforms and offering the possibility of combining these agents with inhibitors of other pathway components. A key discussion point is whether inhibiting multiple pathway components with a combination of separate agents may offer greater opportunities to customize therapy than single agents with multiple targets such as dual PIK3/MTOR inhibitors.

MTOR is a key downstream signal of PIK3/AKT and responds to growth factors, energy status, amino acid levels, and cellular stress [19,20]. There are two distinct MTOR complexes designated as MTOR complex 1 (MTORC1) and MTOR complex 2 (MTORC2). MTORC1 is composed of MTOR, regulatory-associated protein of MTOR (RAPTOR) and mLST8 (mammalian lethal with Sec13 protein 8) [21]. MTORC1 promotes protein synthesis, proliferation, cell survival, ribosome biogenesis, angiogenesis, migration, invasion, and metastasis by phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). The activation

of S6K1 promotes ribosome biogenesis via upregulation of ribosomal protein S6. MTORC2 is comprised of MTOR, rapamycin-insensitive companion of MTOR (RICTOR), mLST8 and mammalian stress-activated protein kinase interacting protein (mSIN1) [20]. MTORC2 functions in actin remodeling, cell-cycle progression, and cell survival through the regulation of protein kinase Cα (PKCα) and glucocorticoid-induced protein kinase 1 (SGK1). MTORC1 is a nutrition- and rapamycin-sensitive complex, while MTORC2 is insensitive to rapamycin [22].

Targeting mTOR alone has been studied in HNSCC using both first-generation (rapamycin and its derivatives temsirolimus, everolimus, and ridaforolimus) and second-generation ATP-competitive MTOR inhibitors [23]. Although some studies have demonstrated the inhibitory effects of MTOR inhibitors on head and neck cancer using in vitro cell line models and in vivo xenograft models, little evidence of efficacy has been obtained from clinical trials. These failures have been attributed to compensatory AKT activation as a result of the S6K/IRS-1 feedback loop driven by unregulated MTORC2 signaling [24]. This has led to the conclusion that combining MTOR inhibitors with other modalities such as radiation, chemotherapeutic agents, or other targeted therapeutic agents may provide the best opportunity for synergistic repression on head and neck cancer.

PIK3 and MTOR both belong to the PIK3-related kinases (PIKK) superfamily and share structural domains, and as a consequence, certain inhibitory compounds target both kinases [25]. Dual inhibitors of PIK3 and MTOR target the active sites of both holoenzymes, inhibiting the pathway both upstream and downstream of AKT, thus avoiding the problem of AKT activation following abolition of the MTORC1–S6K–IRS1 negative feedback loop mentioned above. Preclinical in vitro cell screenings with dual PIK3/MTOR inhibitors suggest a broader efficacy across more genotypes compared with agents targeting only one component of the pathway [26,27].

The question arises whether targeting PIK3 alone is sufficient or whether dual inhibition of PIK3 and MTOR is more effective in suppressing the signaling pathway. In addition, which approach is more optimal in combination with radiation? The addition of radiation adds another level of complexity as the radiation-induced DNA damage response can activate multiple signaling pathways within cells [28]. There are several reports of radiation activation of the EGFR/RAS/RAF/MEK/ERK and PIK3/AKT/MTOR pathways in cell lines [29-32].

We have previously reported on the combination of radiation and PF-04691502 which is an orally active ATP-competitive, dual inhibitor of PIK3 and MTOR [33]. Moreover, in a recent study we have investigated of dual inhibition of the EGFR pathway and the PIK3 pathway using binimetinib and buparlisib [34].Buparlisib is a specific oral inhibitor of the pan-class I PIK3 family currently under investigation in clinical trials [35-37]. Finally we are currently studying gedatolisib (PF-05212384, PKI-587) which is a highly potent dual inhibitor of PIK3α, PIK3γ and MTOR [38]. In this manuscript, we have brought the data for all three agents together to specifically compare the efficacy of the three agents targeting the PIK3/MTOR pathway in combination with radiation in our well established models of HNSCC.

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MATERIALS AND METHODS

Cell lines and drugs

The UT-SCC-14 and UT-SCC-15 cell lines were provided by Dr. Reidar Grénman (Turku University Hospital, Turku, Finland). Both are low passage HPV-negative cell lines which we have previously described [33]. Cells were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) and maintained at low passage number. PF-04691502 was purchased from Selleck Chemicals (Houston, TX), buparlisib was kindly provided by Novartis Pharma (Basel, Switzerland) and gedatolisib was kindly provided by Pfizer (NY, USA). A 10mM solution of each was prepared in dimethyl sulfoxide and stored at -70°C for in vitro experiments.

Irradiation

Cells were irradiated with an Xstrahl X-ray System, Model RS225 (Xstrahl, UK) at a dose rate of 0.29 Gy/min, tube voltage of 160 kVp, current of 4 mA and filtration with 0.5 mM Al and 0.5 mM Cu. Cells were irradiated (0.5-4 Gy) in 25 cm2 flasks at 37°C. Animals were irradiated with a Faxitron Cabinet X-ray System, Model 43855F (Faxitron X-Ray, Wheeling, IL, USA) at a dose rate of 0.69 Gy/min, tube voltage of 160 kVp and current of 4 mA.

3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay

For the MTT assay, cells were plated into 96-well plates and allowed to attach overnight. The next day, media were exchanged for media containing different concentrations of PF-03691502, buparlisib and gedatolisib and the plates returned to the incubator. After an additional 3 days, MTT (5 mg/ml in PBS) was added to each well and the plate returned to the CO2 incubator for ~5 h. The media containing MTT were then aspirated from the wells, and DMSO was added to dissolve the purple formazan. After 5 min incubation at 37°C, absorbance readings (at 560 nm and 670 nm) were taken on a Versamaxmultiplate reader (Molecular Devices, Sunnyvale, CA, USA).

Clonogenic survival assay

Cells were irradiated (0-4Gy) and then plated into flasks containing growth media and 0.25uM PF-04691502, 0.5μM buparlisib or 0.01μM gedatolisib at 4 hours post irradiation. Colonies were allowed to develop for 10-14 days. Colonies were then stained with crystal violet and colonies were counted, and surviving fractions were calculated. Data was normalized for plating efficiency and survival curves were fitted using the linear-quadratic equation.

Immunoblotting

Two tumors from each treatment group were collected at the end of treatment for protein analysis as previously described [33]. After blocking, the membrane was incubated with antibodies. All antibodies except anti- actin were obtained from Cell Signaling Technology, Danvers, MA and used at the following dilutions pan-AKT (1:2000), phospho-AKT (Ser 473) (1:2000),PI3 Kinase p110α (1:1000), phospho-PI3 Kinase p85(Tyr458)/p55(Tyr199) (1:1000), phospho-4E-BP1(Thr37/46) (1:1000), phospho-S6

Ribosomal Protein(Ser240/244) (1:1000) and actin (1:20,000; MP Biomedicals, Solon, OH). Membranes were incubated with IRDye 800CW (1:20,000; Licor, Lincoln, NB, USA) and analyzed with an Odyssey infra-red imaging system (Li-Cor).

Xenograft growth delay

After approval by the Animal Care Committee (AL-15-07), xenografts were established in 4-to-6-week old female nude NIH III mice (Charles Rivers Laboratories, Wilmington, MA, USA) as previously described [33]. Tumor volume was measured twice weekly by digital calipers and calculated using the formula (πab2)/6 (a= largest diameter, b= smallest diameter). When the tumors reached a volume of 200-300 mm3, animals were randomly assigned to the experimental groups. Experiment endpoint was a tumor volume of 2,000 mm3. Seven mice were used in each experimental group.

PF-04691502 (10mg/kg) and buparlisib (10mg/kg) were given by oral gavage 4 hours post radiation treatment five times per week for 3 weeks. Sham oral gavage was 0.5% methylcellulose only. Gedatolisib was administered at 6mg/kg/day by i.v. tail vein injection in 0.15 ml of 5% dextrose and 0.25% lactic acid (pH 3.3) given on two days of the week for the three week schedule.

Radiation was delivered using a Faxitron Cabinet X-ray System at a dose rate of 0.69 Gy/min, tube voltage of 160 KVp, current of 4mA and filtration with 0.5 mm Al and 0.5 mm Cu. Unanesthetized animals were restrained and protected in custom-made lead jigs with only the right flank exposed and irradiated in groups of 4 in a cross-shape configuration with the flank tumors positioned to the center of the radiation field. Radiation treatment (RT) consisted of 2 Gy/day, five times/ week for three weeks for UT-SCC-14. For UT-SCC-15 tumors, the dose per fraction was increased to 3 Gy due to the relative radio resistance of this cell line. For each tumor there were four treatment groups: (1) control with sham oral gavage (2) RT and sham oral gavage (3) inhibitor (4) RT followed by inhibitor.

Statistical analysis

In vitro experiments were repeated three times and statistical analysis was carried out using a two-way t-test or one-way analysis of variance. Data are presented as the mean ± SE. A probability level of a p-value of <0.05 was considered significant. In vivo growth delay data was analyzed based on a time-to-event analysis, i.e. the time to reach 3 times initial volume. Differences between treatment groups were analyzed using a one-way ANOVA and a Tukey post hoc test was then performed between each group comparison, p <0.05 was considered statistically significant. Animals sacrificed prior to reaching tumor volume endpoint due to predetermined animal welfare criteria (as per protocol) were censored at the time of euthanasia.

RESULTS AND DISCUSSION

Sensitivity of the cell lines to PF-04691502, buparlisib and gedatolisib

Figure (1) shows the sensitivity of the cell lines to the different drugs using the MTT growth inhibition/viability assay. The cell lines were equally sensitive to the specific PIK3 inhibitor, buparlisib, with IC50 values of 0.59 ± 0.15μM and 0.61 ± 0.12 μM

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for UT-SCC-14 and UT-SCC-15 respectively. In contrast the cell lines showed differential sensitivity to the dual PIK3/MTOR inhibitors with UT-SSC-14 (Figure 1A) being more sensitive to both inhibitors than UT-SCC-15 (Figure 1B). IC50 values for PF-04691502were 0.098 ± 0.01μM for UT-SCC-14 and 0.34±0.14μM for UT-SCC-15 (p=0.02). Gedatolisib was the most active of the agents studied with IC50 values of 0.0062 ± 0.001μM for UT-SCC-14 and 0.019 ± 0.001μM for UT-SCC-15 (p=0.001).

Modification of radio sensitivity Using classical clonogenic assays, we investigated whether

the drugs affected radiation sensitivity of the cell lines. None of the drugs, at the concentrations used caused a reduction in clonogenic survival (data not shown). We investigated both concurrent administration of the drugs with radiation and also incubating with the drugs 4 hours after radiation. When combined with graded doses of radiation, only PF-04691502 appeared to increase the sensitivity of UT-SCC-14 (Figure 2B) with a dose modifying factor (DMF) of 1.32 ± 0.15. None of the agents affected radio sensitivity in UT-SCC-15.

Tumor growth in vivo

Figure (3) displays the growth curves for UT-SCC-14 and UT-SCC-15 whilst Figure (4) shows the dose enhancement factors (DEF) for the agents. The DEF was calculated for both the drug alone and the drug plus RT combination by taking the ratio of the time to triple the initial starting volume of the drug versus the untreated control and by RT plus drug combination divided by the time taken by radiation alone. Buparlisib, was effective against UT-SCC-15 xenografts (Figure 3D,4B) both alone (p=0.001) and combined with RT (p=0.008) but was without any activity against UT-SCC-14 xenografts (Figure 3A,4A). The dual PIK3/MTOR inhibitors exhibited mixed activity. PF-04691502 alone had some activity although not quite significant in UT-SSC-14 (Figure 4A) but was inactive in UT-SCC-15 (Figure 4B). Gedatolisib showed inhibition of growth in both xenograft models. When combined with RT, PF-04691502 maintained a significant enhancement in UT-SSC-14 but had no effect in UT-SCC-15. Gedatolisib proved less effective when combined with radiation and significantly reduced the effect of radiation in UT-SCC-15 (Figure 4B).

Cell signaling in vivo

We explored whether the drugs were effective at inhibiting the PIK3/AKT/MTOR signaling pathway in vivo and whether RT altered this activity. Tumors were harvested at the end of two weeks of treatment for the PF-04691502 studies and at the end of 3 weeks of treatment for buparlisib and gedatolisib. Figure (5) shows the data for phosphorylated PIK3, AKT, S6 and 4EBP1 as a ratio of the sham-treated controls in 5A and C for the drugs and the RT alone, whilst Figure (5B,D) shows the combined treatments as a ratio of the RT alone. The drugs had little effect on phosphorylated PIK3 but effectively inhibited phosphorylation of AKT and S6 in both xenograft models (Figure 5A,C); p4EBP1 was not inhibited to the same extent as AKT and S6. Radiation enhanced phosphorylation of PIK3, AKT and S6 in both tumor models but showed less activation of 4EBP1. Against this background of RT activation, PF-04691502 maintained strong inhibition of AKT, S6 and 4EBP1 when combined with RT (Figure 5B,D) but buparlisib and gedatolisib were not as effective at inhibiting the phosphorylation of the PIK3/AKT/MTOR pathway when combined with RT.

DISCUSSIONThe PIK3/AKT/MTOR signaling axis is of great significance

to cancer cell growth, survival, motility, and metabolism. This pathway is activated by several different mechanisms including somatic mutation and amplification of genes encoding key components. However, the central role of PIK3/AKT/MTOR signaling in a large array of diverse biologic processes in normal cells has raised concerns about its utility as a therapeutic target. Nevertheless, therapeutics targeting this pathway are being developed and preclinical and clinical studies are demonstrating activity in several cancers [8]. The mammalian target of rapamycin (MTOR) and the phosphoinositide 3-kinase (PIK3) signaling pathways have been found to have an important role in the pathogenesis of HNSCC [9] and there is evidence PIK3 antagonists are active against head and neck cancer cells [10,11]. A number of potential therapeutics targeting this signaling cascade have been developed which can be classified as 1) PIK3

Figure 1 The effect of the drugs on growth/viability of UT-SCC-14 cells (A) and UT-SCC-15 cells (B) using a MTT assay.

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Figure 2 The effect of the drugs on radiosensitivity. (A) The effect of buparlisib on radiation sensitivity of UT-SCC-14 cells and (D) UT-SCC-15 cells. (C) The effect of PF-04691502 on UT-SCC-14 cells and (E) UT-SCC-15 cells.(E) The effect of gedatolisib on UT-SCC-14 cells (E) and UT-SCC-15 cells (F).

inhibitors, 2) dual PIK3-MTOR inhibitors, 3) AKT inhibitors and 4) MTOR catalytic site inhibitors. In this study we have investigated the pre-clinical therapeutic activity of a pan-PIK3 inhibitor and two dual PIK3-MTOR inhibitors in combination with radiation in head and neck cancer.

As mentioned previously, PIK3 signaling is activated in human cancers via several different mechanisms. These include amplification or mutational activation of genes encoding receptor tyrosine kinases, RAS, and/or the p110α catalytic subunit of PIK3 (PIK3CA) and inactivation of the tumor suppressor gene, PTEN. In

head and neck cancer, PIK3/AKT/mTOR pathway genes, such as PTEN, TSC1 and PIK3CA, encompass over 30% of the mutations found in this cancer [5,9,15]. Additional gene mutations and amplifications also contribute to aberrant activation of the PIK3 pathway. Increased RTK (EGFR, MET, etc.) signaling induces PIK3/PTEN pathway activation [39]. Mutations in HRAS and KRAS activate the PIK3 pathway via p110α [40] and loss of p53 function promotes MTORC1 activation and regulation of PTEN transcription [41]. In a previous study we described the mutational landscape of the UT-SCC-14 and UT-SCC-15 cell lines [33]. We identified 123 variants in 62 genes that were classified

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Figure 3 The effect of the drugs and radiation on tumor growth in vivo. (A) The effect of buparlisib and radiation on the growth of UT-SCC-14 xenografts and (D) UT-SCC-15 xenografts. (B) The effect of PF-04691502 on UT-SCC-14 xenografts and (E) UT-SCC-15 xenografts.(C) The effect of gedatolisib on UT-SCC-14 xenografts and (F) UT-SCC-15 xenografts.

as pathogenic, possibly pathogenic, unknown significance or established gain of function in the literature or frame shift, in-frame indel, or stop codon change or missense unless predicted to be innocuous by SIFT or disrupt splice site up to 2.0 bases into intron or deleterious to a microRNA or structural variant. 42 variants were found in both UT-SCC-14 and UT-SCC-15, 39 variants were only found in UT-SCC-14 and 42 variants were only found in UT-SCC-15. The more radio resistant UT-SCC-15 cell line harbored mutations in both HRAS and KRAS as well as several DNA repair genes including ATM, FANCD2, MSH2 and PMS2 that were not found in UT-SCC-14 whilst UT-SCC-14 carried mutations

in EGFR, ERBB4, MTOR, RB1 and TP53 that were not found in UT-SCC-15. Of particular interest in this study are mutations in PIK3/AKT/MTOR pathway. The UT-SCC-15 cell line harbored more variants in AKT1, AKT2, MTOR, PIK3CA, PIK3R1, PTEN and TP53 than the UT-SCC-14 cell line [33].

The agents used in this study showed a spectrum of activity in vitro with gedatolisib being the most active drug by an order of magnitude and the buparlisib being the least active. The PIK3 inhibitor buparlisib was equally active in both cell lines whereas the dual PIK3/MTOR inhibitors showed greater sensitivity in UT-SCC-14 compared to UT-SCC-15. This may reflect the activating

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Figure 4 Dose enhancement factors (DEF) for the drugs alone or in combination with radiation. The DEFs for drugs alone are expressed as a ratio of the untreated animals and calculated from the time to triple the initial volume for each tumor. The DEFs of the drugs plus RT are expressed as a ratio of the RT only animals and calculated from the time to triple the initial volume for each tumor. (A) data from UT-SCC-14 and (B) data from UT-SCC-15.

mutation in HRAS in UT-SCC-15 as well as its greater mutational burden in PIK3-associated genes. PF-04691502 and gedatolisib have been compared in the setting of advanced cancer [42,43] with gedatolisib emerging as the preferred agent due to a more favorable toxicity profile. Buparlisib has advanced the most in clinical studies particularly in the setting of breast cancer [44]. None of the agents are currently under investigation in combination with radiation.

None of the agents significantly modified radio sensitivity in vitro even though there is some evidence linking PIK3/AKT/MTOR pathway to the DNA damage response (DDR) [45]. Suppression of PIK3 has been demonstrated to sensitize breast cancer cells to poly ADP ribose polymerase (PARP) inhibitors by down regulating homologous recombination (HR) [46] and inhibition of the PIK3/AKT/MTOR pathway sensitized endometrial cells to PARP inhibitors [47].

The in vivo data differed to the in vitro findings. Despite all of the drugs showing ability to reduce phosphorylation of key

components of the PIK3/AKT/MTOR signaling pathway in both tumor models, buparlisib alone was the most active agent in the UT-SCC-15 tumor model followed by gedatolisib whereas PF-04691502 was inactive. In the UT-SCC-14 tumor model, buparlisib was inactive whereas the two dual PIK3/MTOR inhibitors showed modest activity at the doses used.

The main innovation and central focus of this study was how these agents interact with radiation treatment. Although radiation is an effective treatment for head and neck cancer, its combination with cisplatin has reached clinical tolerance and there is an urgent need to introduce other agents into the treatment paradigm. As previously mentioned, radiation-induced DNA damage response can activate multiple signaling pathways within cells [28] such as the EGFR/RAS/RAF/MEK/ERK and PIK3/AKT/mTOR pathways [29-32]. Our previous studies [33,34]and this current study emphasize the radiation-induced phosphorylation of the PIK3/AKT/MTOR survival pathway. This presents a challenge to the targeted agents to inhibit higher levels of phosphorylated proteins than present in non-radiation treated cells. In the context of radiation treatment only PF-04691502 was able to significantly enhance the radiation response in UT-SCC-14 whilst buparlisib was the only active radiation enhancer in UT-SCC-15 with gedatolisib diminishing the radiation effect in this tumor model. The growth delay data mirrored the pathway inhibition analysis where PF-04691502 was the most effective agent at reducing phosphorylation AKT, S6 and 4EBP1 in UT-SCC-14 in combination with radiation and gedatolisib was the least effective agent at inhibiting the PIK3 pathway during radiation treatment.

To date there is no doubt that PIK3/AKT/MTOR pathway inhibitors may have significant single-agent activity in some cancers dependent on the genetic status of key genes such as PIK3CA, PTEN and RAS [8]. In addition, there is also evidence that drugs targeting this pathway may enhance radiation treatment [48-50]. However, there is a complex interplay between the PIK3/AKT/MTOR pathway and the EGFR/RAS/RAF/MEK/ERK and pathway. These pathways extensively cross-talk [51] to both positively and negatively regulate each other that is dependent on the activation status of key genes such as KRAS, RAF, PTEN and PIK3CA. Consequently, it may be necessary to combine PIK3 pathway inhibitors with agents targeting to induce dramatic responses. It has been suggested that there are downstream proteins that integrate the oncogenic functions of these two pathways [52]. 4EBP1 has been identified as the convergence between the two pathways and this may explain our data in Figure (5) where the PIK3/MTOR inhibitors were effective at reducing phosphorylation of AKT and S6 but not 4EBP1. Combined inhibition of both pathways may be required to inhibit tumor growth. Indeed, inhibition of PIK3 signaling with NVP-BEZ235 failed to shrink established Kras G12D-driven lung tumors [53] whereas combined PIK3 and MAPK pathway inhibition by treatment with NVP-BEZ235 and the MEK inhibitor ARRY-142886 led to marked tumor regression in this model.

CONCLUSIONIn summary, the effectiveness of drugs targeting the PIK3/

MTOR axis was difficult to predict in two contrasting models of HNSCC as was their combination with radiation. The complex

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Figure 5 Analysis of the effect of the drugs on the PIK3/AKT/MTOR signaling pathway derived from immunoblots of tumors removed during or at the end of treatment and analyzed using image analysis. In (A) and (C) the effects of buparlisib (), PF-04691502 (), gedatolisib () and RT () are presented for UT-SCC-14 and UT-SCC-15 respectively. The expression data for each treatment was normalized to the untreated control. In (B) and (D) the effect of the drugs plus radiation is present for UT-SCC-14 and UT-SCC-15 respectively. In this analysis the drugs plus RT have been normalized to RT alone.

genomic landscape of HNSCC and the substantial cross-talk between signaling pathways dependent on the genomic landscape still present a significant challenge to predict and prescribe the optimal treatment for individual patients.

ACKNOWLEDGEMENTSWe acknowledge Dr. Reidar Grénman from the University

of Turku, Finland for providing the UT-SCC-14 and UT-SCC-15 cell lines. We acknowledge Novartis for providing buparlisib and Pfizer for providing gedatolisib. Some financial support was provided by the Herb and Betty Fisher Foundation Seed Grant.

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