Antitumor activity of the dual PI3K/MTOR inhibitor, PF-04691502, in combination with radiation in head and neck cancer

Nathan Tonlaar a, Sandra Galoforo a, Bryan J. Thibodeau b, Samreen Ahmed b, Thomas G. Wilson a, Paola Yumpo Cardenas a, Brian Marples a,1, George D. Wilson a,⇑
a Department of Radiation Oncology, William Beaumont Hospital; and b Beaumont BioBank, William Beaumont Hospital, Royal Oak, United States

Article history:
Received 18 April 2017
Received in revised form 27 July 2017 Accepted 2 August 2017
Available online xxxx

Head and neck cancer PI3K/MTOR inhibition Radiation
Next generation DNA sequencing
a b s t r a c t

Background and purpose: Head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge where new treatments are required to supplement the current-standard-of care of concurrent chemora- diation. The PI3K/AKT/MTOR pathway has been identified from several next generation DNA sequencing studies to be commonly altered and activated in HNSCC.
Material and methods: In this study we investigated the activity of PF-04691502, an orally active ATP- competitive, dual inhibitor of PI3K and mTOR, in combination with a clinically relevant fractionated radi- ation treatment in two contrasting, well characterized, low passage HNSCC models.
Results: We found that PF-04691502 combined synergistically with radiation in the UT-SCC-14 model derived from a primary cancer but was ineffective in the UT-SCC-15 model which was derived from a nodal recurrence. Further examination of the status of key signaling pathways combined with next gen- eration DNA sequencing of a panel of 160 cancer-associated genes revealed crucial differences between the two models that could account for the differential effect. The UT-SCC-15 cell line was characterized by a higher mutational burden, an excess of variants in the PI3K/AKT/MTOR pathway, increased constitutive activity of PI3K, AKT1 and 2 and MTOR and an inability to inhibit key phosphorylation events in response to the treatments.
Conclusion: This study clearly highlights the promise of agents such as PF-04691502 in selected HNSCCs but also emphasizes the need for molecular characterization and alternative treatment strategies in non- responsive HNSCCs.
ti 2017 Elsevier B.V. All rights reserved. Radiotherapy and Oncology xxx (2017) xxx–xxx

Background and significance
Head and neck squamous cell carcinoma (HNSCC) is a diverse disease which affects 560,000 people worldwide each year result- ing in almost 300,000 deaths [1]. Most HNSCCs are tobacco and alcohol related but infection with the human papillomavirus (HPV) is becoming more prevalent and is now a leading cause of HNSCC in non-smokers [2]. The distinction between HPV positive and negative cancers is important because HPV negative cancers have a worse prognosis [3] and emerging data indicate that geno- mic profiles differ between the two different tumor types [4,5]. HPV negative cancers represent a major challenge as treatment outcomes have improved very little during the last 20 years [6]despite significant advances in surgical and radiotherapeutic tech- niques and combined modality treatments.
The rapid development of molecular targeted therapeutics has opened up new opportunities to explore the interaction of radia- tion with inhibition of specific cancer-associated pathways. How- ever, identifying the most appropriate pathway to target still remains a challenge. In head and neck squamous cell carcinoma (HNSCC) early success was achieved by targeting the epidermal growth factor receptor (EGFR) pathway [7]. However, apart from the inhibitors of EGFR and vascular endothelial growth factor (VEGF), very few molecular targeted agents have advanced to phase III clinical trials in combination with radiation [8].
Full genomic analysis of HNSCC primary tumors has been reported in two seminal articles [4,5]. Results from these two stud- ies confirmed known HNSCC mutations including TP53, CDKN2A,CASP8 [9]. More recently, the largest data set of over 279 HNSCC primary tumors from the TGCA has been presented [10]. 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 PI3KCA which is a component of the PI3K/MTOR/PTEN/AKT signaling pathway involved in regulation of cell survival, growth, proliferation, metabolism and motility and an attractive drug target in cancer [11]. The mammalian target of rapamycin (MTOR) and the phosphoinositide 3-kinase (PI3K) sig- naling pathways have been found to have an important role in the pathogenesis of HNSCC [12] and there is evidence that PI3K antagonists are active against head and neck cancer cells [13,14]. PI3KCA, located on chromosome 3q26.32, encodes the catalytic subunit p110a of class IA PI3-kinase and has been implicated as an oncogene because of its genomic amplification in different can- cers [15,16]. High frequencies of mutations in PI3KCA have been reported in several cancer types including head and neck cancer [17,18]. The majority of mutations are clustered in the helical (exon 9) and kinase domains (exon 20) of PI3KCA with the most commonly reported hot spots identified as E542K, E545K and H1047R which result in increased lipid kinase activity and activa- tion of the downstream AKT pathway [19,20]. In head and neck cancer, the three common mutations outlined above were preva- lent but also a novel mutation Y343C in exon 4 nucleotide 1028 A ? G [20]. Interestingly, mutation status does seem to determine sensitivity to PI3KCA inhibitors [14,21] in head and neck cell lines. Indeed, the hot spot E545K and H1047R mutations sur- prisingly conferred increased rather than reduced sensitivity in engineered head and neck cancer cell lines when treated with PI3K inhibitors as well as MET and HSP90 inhibitors [22].
The PI3K/AKT/mTOR pathway is one of the principal signaling mechanisms regulating cell survival, growth, proliferation, meta- bolism, and motility [11]. The key components of this pathway are the class 1 phosphatidylinositol 3-kinases (PI3Ks). Of particular interest are the class 1A PI3Ks which are heterodimers that consist of a catalytic subunit (p110a, p110b and p110d) and a regulatory subunit (p85a, p55a, p50a, p85b, and p55c). The catalytic subunit p110a is encoded by PIK3CA, while the regulatory subunit p85a is encoded by PIK3R1 [23,24]. The PI3K/AKT/mTOR pathway is acti- vated with the involvement of growth factors binding to the recep- tor tyrosine kinases (RTK) and G-protein coupled receptors [11,25]or through direct activation of the catalytic subunit by Ras [26]
which generates phosphatidylinositol-3-phosphate (PIP3) by phos- phorylating phosphatidylinositol 4, 5-bisphosphate; this reaction is counter-balanced by phosphatase and tensin (PTEN) homolog [11]. PIP3 recruits AKT to the plasma membrane and phosphory- lates it to pAKT in a mechanism co-activated through phosphoryla- tion by phosphatidylinositol-dependent kinase 1 (PDK1) and the rictor-mTOR complex (mTORC2).
In malignancy, perturbation of the PI3K/AKT/mTOR pathway can occur through various mechanisms, including amplification or mutational activation of genes encoding receptor tyrosine kinases, RAS, and/or the p110a catalytic subunit of PI3K (PIK3CA) and inactivation of the tumor suppressor gene, PTEN. In head and neck cancer, PI3K/AKT/mTOR pathway genes, such as PTEN, TSC1 and PIK3CA, encompass over 30% of the mutations found in this cancer [4,12,18]. Additional gene mutations and amplifications also contribute to aberrant activation of the PI3K pathway. Increased RTK (EGFR, MET, etc.) signaling induces PI3K/PTEN path- way activation [27]. Mutations in HRAS and KRAS activate the PI3K pathway via p110a [28] and loss of p53 function promotes mTORC1 activation and regulation of PTEN transcription [29].
The PI3K/AKT/mTOR pathway has been investigated as an attractive target for anticancer drug development. The first PI3K/AKT/mTOR inhibitors were rapamycin analogs (e.g. everolimus, temsirolimus), which allosterically inhibited the raptor–mTOR complex (mTORC1) [30]. However these agents had limited clinical efficacy due to their inability to inhibit mTORC2, and the abroga- tion of negative feedback loops caused by inhibition of mTORC1 alone. Because of the structural similarity between the catalytic domains of the p110 subunit of PI3K and mTOR the opportunity for targeting the PI3K/AKT/mTOR pathway at multiple sites with a single pharmacologic agent has been explored. Dual PI3K/mTOR inhibitors have the theoretical advantage of offering more com- plete suppression of the PI3K/AKT/mTOR pathway by inhibiting all catalytic isoforms of PI3K as well as mTORC1 and mTORC2 [31]. PF-04691502 is an orally active, ATP-competitive, dual inhibi- tor of PI3K and mTOR [32].
Several studieshaveinvestigatedtheutilityofdifferentPI3Kinhi- bitors in various experimental settings in HNSCC both in vitro and in vivo [14,33–38]. However, only a few studies report the efficacy of the combination of PI3K inhibitors in combination with radiation [14,33,38]. In this study we explore the efficacy of PF-04691502 in combination with a clinically relevant fractionated radiation treat- ment in two contrasting, well characterized, low passage HNSCC models. PF-04691502 is an orally active ATP-competitive, dual inhi- bitor of PI3K and mTOR [32]. In the original pre-clinical studies, PF- 04691502 exposure results in inhibition of recombinant class I PI3K (Ki = 1.2–2 nM) and mTOR (Ki = 16 nM) kinase activity, AKT phos- phorylation (IC50 = 3.8–47 nM) and proliferation (IC50 = 170– 313 nM) of PIK3CA-mutant and PTEN-null cancer cell lines [32]. The same authors showed in vivo activity of the agent in established glioblastoma (U87]), ovarian (SKOV3) and various erlotinib/gefitinib-resistant non-small cell lung cancer cell lines (NCI-HI975, NCI-H460, NC-H-1650 and A549) with differing status of PTEN, PIK3CA and HRAS genes. Phase I studies in patients with advanced cancer have shown that PF-04691502 is well tolerated with a toxic- ity profile consistent with that of other PI3K/mTOR inhibitors [39].

Materials and methods
Cell lines and drug
The UT-SCC-14 and UT-SCC-15 cell lines were provided by Dr. Reidar Grénman (Turku University Hospital, Turku, Finland). The cell lines were derived from patients with oral tongue cancer with UT-SCC-14 being a primary tumor and UT-SCC-15 being recurrent. Both cell lines were negative for human papillomavirus (HPV). We have extensively studied these cell lines in vitro and in vivo [40– 44] and the radiosensitivity of these cell lines have been reported elsewhere [45–47].
Cells were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, peni- cillin (100 U/ml), and streptomycin (100 mg/ml) and maintained at low passage number. PF-04691502 was purchased from Selleck Chemicals (Houston, TX). A 10 mM solution was prepared in dimethyl sulfoxide and stored at ti70 tiC for in vitro experiments. For in vivo experiments PF-04691502 was prepared daily in 0.5% methylcellulose.

Cells were irradiated with a 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 tiC. 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-04691502 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 ti5 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 tiC, absorbance readings (at 560 nm and 670 nm) were taken on a Ver- samax multiplate reader (Molecular Devices, Sunnyvale, CA, USA). For the combination of PF-04691502 and 4 Gy of radiation at dif- ferent time intervals, it is not possible to keep exposure to radia- tion and drug constant. Therefore, the in these experiments the time interval after radiation exposure remained constant and a ser- ies of sham irradiated and drug exposures were set up for the dif- ferent time periods to as act as the controls.

Clonogenic survival assay
Cells were irradiated (0–4 Gy) and then plated into flasks con- taining growth media and 0.25 mM PF-04691502 either added con- currently or at 4 h 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 cal- culated. Data were normalized for plating efficiency and survival curves were fitted using the linear-quadratic equation.

Western immunoblot assay
Baseline levels of key proteins and the response of AKT, PI3K and mTOR to a single 4 Gy irradiation at 0.5, 1, 6 and 24 h was assessed by immunoblot assay. Cells were treated, washed with PBS and protein extracted with lysis buffer. Equal amounts of pro- tein (20 lg) were separated by 8% sodium dodecyl sulfate–poly- acrylamide gel electrophoresis and transferred onto a nitrocellulose membrane by electroblotting. After blocking, the membrane was incubated with antibodies EGFR (1:1000; Cell Sig- naling Technology, Inc.), phospho-EGFR (Tyr1068) (1:1000, Cell Signaling Technology, AKT (1:2000, Cell SignalingTechnology), phospho-AKT (Ser 473) (1:2000; Cell Signaling Technology, Inc), mTOR (1:1000, Cell Signaling Technology), phospho-mTOR (Ser2448) (1:1000, Cell Signaling Technology), PI3 Kinase p110a (1:1000, Cell Signaling Technology), phospho-PI3 Kinase p85
(Tyr458)/p55(Tyr199) (1:1000, Cell Signaling Technology), phospho-4E-BP1(Thr37/46) (1:1000, Cell Signaling Technology), phospho-S6 Ribosomal Protein(Ser240/244) (1:1000, Cell Signaling or actin (1:20,000; MP Biomedicals). The membrane was washed and the secondary antibody (IRDye 800CW, 1:20,000; Licor, Lin- coln, NB, USA) was applied for 1 h.
To extract protein from tumor tissue, a small piece of tumor was homogenized in T-PER(Thermo-Scientific) with Halt protease/phosphatase inhibitor(Thermo-Scientific) added. Unsolubilized tis- sue was centrifuged out and the supernatant was collected and stored at -70 tiC. Gel electrophoresis was the same as for protein samples from cells. Relative protein expression was analyzed with an Odyssey infra-red imaging system (Li-Cor).

Nucleic acid extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). SuperScript IV VILO Master Mix (Thermo Fisher Scientific Inc. Waltham, MA) was used for the synthesis of cDNA from a total of 2 mg RNA. The following pre-designed TaqMan gene specific pri- mers (Life Technologies, Carlsbard, CA) were used: AKT1 (Assay ID Hs00178289_m1), AKT2 (Assay ID Hs01086102_m1), AKT3 (Assay ID: Hs00987350_m1) EGFR (Assay ID: Hs01076078_m1), HER-2 (Assay ID: Hs01001580_m1), HER-3 (Assay ID: Hs00176538_m1), HER-4 (Assay ID: Hs00955525_m1), MTOR (Assay ID: Hs00234508_m1) PIK3CA (Assay ID: Hs00907957_m1), and GAPDH (Assay ID: Hs99999905_m1). Quantitative real-time PCR reaction mixture was prepared containing 2ul cDNA (10 ng), 1ti TaqMan Gene Expression Master Mix Life Technologies, Carlsbard, CA), and 1x Gene Expression Assay (Life Technologies, Carlsbard, CA). Gene expression levels were quantified using the ViiA 7 Real-Time PCR system (Life Technologies, Carlsbard, CA). The fol- lowing thermocycling condition was used: 50 tiC for 2 min, 95 tiC for 10 min, and 40 amplification cycles of 95 tiC for 15 s/60 tiC for 1min. The delta delta CT method was used for analysis. Gene expression data was normalized to GAPDH in these studies which does have limitations but was relatively consistent between the cell lines and normal tongue in this study.

DNA sequencing
Genomic DNA was isolated using QIAmp DNA Micro Kit (Qia- gen, Valencia, CA). Manufacturer’s protocol was followed with a modified condition that included an RNase A treatment immedi- ately following overnight incubation. Quality and amplifiable DNA material was assessed with the GeneRead DNA QuantiMIZE Kit (Qiagen, Valencia, CA). Targeted enrichment multiplex PCR of 160 genes was done using GeneRead DNAseq Comprehensive Can- cer Panel V2 in combination with GeneRead DNAseq Panel PCR Kit V2 (Qiagen, Valencia, CA) following manufacturer’s protocol. Start- ing amount of amplifiable DNA used and PCR cycles for initial library amplification were based on calculation results determined by GeneRead DNA QuantiMIZE kit. Sample pooling and purification was subsequently done using Agencourt AMPure XP Beads (Beck- man Coulter, Brea, CA). Library construction was done using Gene- Read Illumina based DNA Library Prep Kits with sample barcode multiplexing done using GeneRead Adapter I Set 12-plex (Qiagen, Valencia, CA). The resulting barcoded constructed libraries were then quantified using GeneRead DNAseq Quantification Kit. Sam- ples were then pooled and sequenced using the NextSeq 500 (Illu- mina, San Diego, CA) as paired-end 150 base reads (2 ti 150). Results of sequencing runs were uploaded to Illumina’s BaseSpace where they were de-mutliplexed and FASTQ files generated. The FASTQ files were converted into FASTA format for analysis in Next- GENe (SoftGenetics, State College, PA). In this conversion, reads were filtered to retain reads with median score threshold ti10, maximum number of uncalled bases ti3, and total bases called in a read ti20, as well as reject reads when ti3 bases with score ti10. Alignment to v37 of the whole human genome is then carried out in NextGENe. NextGENe used a Preloaded Index Alignment algorithm. This algorithm employs a suffix array that is repre- sented by the Burrows–Wheeler Transform (BWT). Mutations were called by NextGENe based upon mutation percentage ti5% and total coverage ti250. The coverage requirement was ignored for mutations that are homozygous. For variants occurring at a fre- quency ti80%, the mutation was excluded if the balance ratio of forward and reverse reads was less than 0.1. Variant files were imported into Ingenuity Variant Analysis (IVA; Qiagen, Redwood City, CA) software for further biological interpretation. The version of IVA used was the ‘‘2016 Winter Release”. ‘‘Common Variants” were filtered by excluding variants with an allele frequency ti1.0% in the 1000 genomes project, the NHLBI ESP exomes, the Allele Frequency Community, and the ExAC. Variants were then fil- tered to include only those experimentally observed to be: Patho- genic, Possibly Pathogenic, associated with gain of function (as established in the literature or gene fusion), or associated with loss of function of a gene (via frameshift, in-frame indel, or stop codon change, or a missense mutation).

Xenograft growth delay assay
All animal experiments complied with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the William Beaumont Hospital Research Institute Animal Care Committee (AL-12-06). Xenografts were established in 4- to 6-week-old female nude NIH III mice (Charles Rivers Lab- oratories, Wilmington, MA, USA) by injecting UT-SCC-14 subcuta- neously into the flank, at a density of 2 ti 106 cells per 100 ll of Matrigel (BD, Franklin Lakes, NJ, USA) or UT-SCC-15 at a density of 4 ti 106 cells per 100 ll Matrigel. Tumor volume was measured three times weekly by digital calipers and calculated using the for- mula (pab2)/6 (a = largest diameter, b = smallest diameter). When the tumor reached a volume of 200–300 mm3, animals were ran- domly assigned to the experimental groups. The endpoint of the experiment was when tumors grew to a volume of 3000 mm3.
PF-04691502 was given by oral gavage 4 hours post radiation treatment. Sham oral gavage was 0.5% methylcellulose only. Six mice were used in each experimental group.
The UT-14-SCC experiment consisted of four treatment groups: (1) control with sham oral gavage (0.5% methylcellulose), (2) PF- 04691502 (10.0 mg/kg) by oral gavage daily (five times per week) for 3 weeks, (3) radiation delivered as 2.0 Gy/day (five times per week) followed 4 h later by sham oral gavage for 3 weeks, and (4) radiation 2.0 Gy/day (five times per week) followed 4 h later by PF-04691502 (10.0 mg/kg) by oral gavage for 3 weeks. For UT-SCC- 15 tumors, the dose per fraction was increased to 3 Gy due to the rel- ative radioresistance of this cell line; a total dose of 45 Gy was deliv- ered in three weeks. The PF-04691502 concentration and dosing remained the same. Each treatment group consisted of 6 animals.

Statistical analysis
In vitro experiments were repeated three times and statistical analysis was carried out using Student’s t-test. Data are presented as the mean ± SE. A probability level of a p-value of <0.05 was consid- ered significant. Tumor growth data were analyzed by calculating, for each tumor, the time at which the tumor doubled in volume from the start of treatment and the differences analyzed by ANOVA. The analysis also derived the enhancement ratio as the quotient of the doubling time of the combined treatment versus the RT alone. Results Baseline levels and DNA copy number of key genes Fig. 1A shows representative immunoblots for baseline levels of EGFR, phosphorylated EGFR, PI3K, pan-AKT, phosphorylated AKT and mTOR in UT-SCC-14 and UT-SCC-15 cell lines. EGFR was heav- ily overexpressed in UT-SCC-14 as was phosphorylated AKT in comparison with UT-SCC-15 cells. However, PI3K levels were greater in UT-SCC-15. The gene expression data (Fig. 1B) generally agreed with the western analysis. EGFR levels were 5-fold higher in UT-SCC-14 compared to UT-SCC-15 while PI3KCA expression was 2.2-fold greater in UT-SCC-15 compared to UT-SCC-14. AKT 1 and 2were upregulated in UT-SCC-15 while AKT3 was upregulated in UT-SCC-14. The main discrepancy between the two analyses focused on mTOR where protein levels were similar but gene expression was 2.5-fold greater in UT-SCC-15. DNA copy number analysis only identified one major variation; EGFR had a CNV of 17.5 in UT-SCC-14 providing the basis for the observed overexpres- sion of the gene (data not shown). Temporal response in key proteins to 4 Gy irradiation UT-SCC-14 and UT-SCC-15 cells were irradiated with a single dose of 4 Gy and cells harvested for immunoblot assay at 0.5, 1, 6 and 24 h (Supplemental Fig. 1). The effects of radiation on AKT, PI3K and mTOR were subtle with the most pronounced changes associated with mTOR in the UT-SCC-14 cell line which showed an initial upregulation followed by repression at 24 h. AKT was upregulated at 0.5 h in UT-SCC-15 cells. None of the changes were significant when analyzed by near infra-red imaging. Response of cell lines to PF-04691502: Dose response Fig. 2 shows the growth inhibition data for PF-04691502 in the UT-SCC-14 and UT-SCC-15 cell lines. Both cell lines show a similar pattern of growth inhibition with the UT-SCC-14 cells being signif- icantly more sensitive (p = 0.02) with an IC50 of 0.098 lM ± 0.12 compared to an IC50 of 0.34 lM ± 0.14 in the UT-SCC-15 cell line. As the concentration increased from 1 to 10 lM, the drug became less effective and growth inhibition plateaued. Based on these data, we chose a concentration of 0.25 lM to study the temporal rela- tionship between drug and radiation exposure. Response of cell lines to PF-04691502: Timing with radiation The timing of drug exposure (at 0.25 lM) in combination with radiation was studied at 1 h before and 1 h after 4 Gy and at 4 and 24 h after 4 Gy using the MTT assay. Fig. 3 shows the data from these studies. 4 Gy of radiation reduced cell growth by 24% in the UT-SCC-14 cell line but only by 11% in the more radioresistant UT- SCC-15 cell line. In Fig. 4 the time from irradiation to the end of the experiment was kept constant while the drug exposure varied. Consequently, the statistical comparison was made between drug and RT and drug and sham RT at each timing variable. The effects of timing suggested that the optimal timing was obtained when the drug was administered 4 h after exposure to radiation. Clonogenic survival analysis To assess the effect of PF-04691502 on clonogenic survival, 0.25 lM of the drug was incubated with graded doses of X-rays administered concurrently or 4 h after each radiation dose level (Fig. 4). When the drug was delivered concurrently with radiation, there was no interaction between the two agents. When PF- 04691502 was administered 4 h after the irradiation there was a modest sensitization in the UT-SCC-14 cell line with a dose modi- fying factor (DMF) of 1.32 ± 0.15 (Fig. 4A). It was apparent from the colony assessment that although PF-04691502 did not appear to affect clonogenic survival (Fig. 4B), it was effective at reducing the growth of UT-SCC-15 cells causing smaller colonies compared to RT alone or control cultures (Fig. 4C) In vivo growth delay studies PF-04691502 had no effect on the growth of either UT-SCC-14 or UT-SCC-15 xenografts when administered daily (5 days per week) for three weeks (Fig. 5A and B). However, when combined with radiation there was a highly significant (p = 0.0148) enhance- ment of the radiation response in the UT-SCC-14 xenografts with a DMF of 1.47 ± 0.16 (Fig. 5A). The average survival of the UT-SCC-14 xenografts was 41.8 ± 5.1 days (controls), 53.5 ± 12.1 days (drug only), 94.0 ± 6.2 days (RT alone) and 112.2 ± 3.3 days (RT + drug). At the end of the observation period (120 days from implant), three of the combined treatment xenografts had failed to regrow and had no evidence of viable tumor in the excised specimens processed for histology. In contrast, PF-04691502 was ineffective in combination with radiation in the UT-SCC-15 xenografts. Indeed the combina- tion of PF-04691502 and radiation was less effective that radiation alone with a DMF of 0.88 ± 0.12 although this did not reach signif- icance (p = 0.36). Effect of treatments on protein expression during treatment in vivo The immunoblot analysis was studied in tumors which had undergone 2 weeks of treatment and were sacrificed after the last treatment. Fig. 6A shows the individual immunoblots for each tumor and each treatment and the data normalized to the control tumors for UT-SCC-14 tumors while Fig. 6B shows the same data and analysis for the UT-SCC-15 xenografts. In the UT-SCC-14 xeno- grafts, as expected PF-04691502 had no effect on EGFR or phospho- rylated EGFR (p-EGFR). RT reduced both EGFR and p-EGFR relative to the controls as did the combination of the drug and RT. Surpris- ingly, the inhibition of PI3K and p-PI3K was not significantly altered by PF-04691502 but p-AKT, mTOR, p-4EBP and p-S6 were all down regulated after treatment with the drug alone (Fig. 6A). Radiation treatment significantly increased several proteins in the PI3K/AKT/mTOR pathway including p-PI3K (p = 0.001), p-AKT (p = 0.004), mTOR (p = 0.01) and p-S6 (p = 0.008). Addition of the drug to radiation reduced p-PI3K although levels were still ele- vated compared to controls. However, the drug was able to down- regulate p-AKT (p = 0.04), p-mTOR (p = 0.01), p-4EBP (p = 0.005) and p-S6 (p = 0.02) compared to the untreated controls when com- bined with radiation treatment (Fig. 6A). The UT-SCC-15 xenografts showed a different pattern of protein expression during treatment (Fig. 6B). PF-04691502 had no effect on EGFR, PI3K, AKT or mTOR but did downregulate p-AKT, p- 4EBP and p-S6. Radiation treatment significantly upregulated EGFR (p = 0.001), p-PI3K (p = 0.001), AKT (p = 0.01), p-AKT (p = 0.001), p- mTOR (p = 0.001), p-4EBP (p = 0.02) and p-S6 (p = 0.006). Addition of the drug to radiation did diminish PI3K levels or p-mTOR but did reduce the downstream proteins p-4EBP and p-S6. Mutation analysis of UT-SCC-14 and UT-SCC-15 Mutation analysis was carried out with Ingenuity Variant Anal- ysis version 4.1.20160607. Variants were excluded that were observed with an allele frequency greater than or equal to 1.0% of the genomes in the 1000 genomes project OR greater than or equal to 1.0% of the NHLBI ESP exomes (All) OR greater than or equal to 1.0% of the AFC Frequency OR greater than or equal to 1.0% of the ExAC. Variants were kept that are experimentally observed to be associated with a phenotype: pathogenic, possibly pathogenic, unknown significance or established gain of function in the litera- ture or frameshift, 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 vari- ant. This resulted in 123 variants in 62 genes. The locations of the variants were 4 in the 30 UTR, 1 in the 50 UTR, 113 exonic and 5 splice site. 42 variants were found in both UT14 and UT15, 39 variants were only found in UT14 and 42 variants were only found in UT15. Supplemental Table 1 lists the variants excluding those resulting in a synonymous substitution. The more radioresistant 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 while 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 PI3K/AKT/MTOR pathway. Discussion In this study we have investigated the role of a dual PI3K/mTOR inhibitor, PF-04691502, in combination with fractionated irradia- tion in two contrasting models of head and neck cancer. PF-04691502 has been studied in the context of radiation enhancement in a previous preclinical study using head and neck cancer xenografts [38] which concluded that the agent produced significant radiosensitization in non-metastatic HNSCC. This study made several key observations including that dramatic radiosensi- tization was evident in all HNSCC cell lines tested regardless of p53 status and that radiosensitization in normal fibroblasts was signif- icantly reduced compared with malignant cells. Our study differs to Leiker et al. [38] in that we studied the combination of PF- 04691502 and fractionated radiation in the context of a primary and nodal recurrence model of HNSCC with widely differing radiosenstivities and with a detailed analysis of their mutational landscape. Like the previous study we were able show modest radiosensitization both in vitro (Fig. 4A) and in vivo (Fig. 5A) in a primary HNSCC xenograft model.Closer examination of their mutational status (Supplemen- tal Tables 1 and 2) and the activation (Fig. 1) and response (Fig. 6) of the key components on the PI3K/AKT/mTOR pathway reveals plausible reasons for the differential response of the two cell lines. First, UT-SCC-15 shows significantly higher baseline activity of PI3KCA, mTOR, AKT1 and AKT2 (Fig. 1). Second, in both cell lines radiation treatment alone caused a significant upregulation of sev- eral proteins in the PI3K/AKT/mTOR pathway. However, upregula- tion was more prominent in UT-SCC-15 tumors and, importantly, addition of PF-04691502 failed to downregulate several compo- nents of this pathway (Fig. 6B). In contrast, addition of PF- 04691502 to radiation in the UT-SCC-14 was effective in downreg- ulating p-AKT, p-mTOR, p-4EBP and p-S6 compared to the untreated controls (Fig. 6B). Thirdly, UT-SCC-15 harbors substan- tially more variants in PI3K/AKT/MTOR-associated genes as well as mutations in HRAS and KRAS (Supplemental Tables 1 and 2) and has a higher mutational burden than UT-SCC-14. Interestingly, neither cell line carried mutations in PI3KCA which has been shown to be one of the most commonly altered genes in HNSCC [12,42,48,49] with a frequency of 8–12%. Of inter- est were the deletions and alterations in PI3KR1 in UT-SSC-15. PIK3CA and PIK3R1 alterations have been shown to be mutually exclusive in breast cancer [50]. The protein p85a encoded by the PIK3R1 gene has been described to play an important role in PI3K pathway signaling by stabilizing p110a which is encoded by PIK3CA. Loss of the p85a tumor suppressor effect leads to down- stream PI3K pathway activation which was evident in UT-SCC- 15. The influence of PIK3R1 deregulation on pathway signaling has been attributed to the impaired ability of the two subunits to interact and loss of the inhibitory effect of p85a on p110a and PI3K activity [51]. The most common genetic alterations that have been reported for PI3KR1 are point mutations and deletions as we found in this study. PIK3R1 has been reported to play a tumor sup- pressor role in hepatocellular cancer [52] and is frequently altered in other cancer types such as endometrial cancer [53]. The data in this study question the optimism expressed in pre- vious publications concerning the combination of radiation and PF- 04691502 [38] and highlights the complex interplay between radi- ation and the pathways targeted by drugs such as PF-04691502. We showed that, although one HNSCC xenograft model showed significant radiosensitization resulting from the combination, another xenograft did not and there was even a hint of antagonism (Fig. 5B). This model was a later-stage cancer and may have become resistant to the targeted therapy due to the more complex molecular dependencies that had developed which led to the acti- vation of other survival pathways. This study, like others [54–56], showed that radiation activates the PI3K/AKT/mTOR survival path- way and PF-04691502 was only effective if it inhibited radiation- induced phosphorylation of genes in this pathway as demonstrated in UT-SCC-14 xenografts. Although there has been much progress in identifying mutated genes and proteins in HNSCC, many ques- tions remain regarding their physiological and pathological roles. It is clear that not every group of head and neck cancer patients will respond the same to therapy. Much research is still required to understand the contribution of mutations and deregulated path- ways in HNSCC and to determine the effects of inhibitors that tar- get molecules in these pathways. In some HNSCCs, a single targeted agent may be sufficient but others may require treat- ments that target several survival pathways. Conflict of interest statement The authors have no conflicts of interest associated with this manuscript. Acknowledgements We are grateful to Dr. Reidar Grénman, University of Turku, Fin- land for providing the UT-SCC-14 and UT-SCC-15 cell lines. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at 001. 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