PI3K Inhibitors Curtail MYC-Dependent Mutant P53 Gain

Published OnlineFirst January 22, 2020; DOI: 10.1158/1078-0432.CCR-19-2485

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

PI3K Inhibitors Curtail MYC-Dependent Mutant p53 Gain-of-Function in Head and Neck Squamous Cell Carcinoma Federica Ganci1, Claudio Pulito1, Sara Valsoni2, Andrea Sacconi1, Chiara Turco1, Mahrou Vahabi1, Valentina Manciocco3, Emilia Maria Cristina Mazza2, Jalna Meens4, Christina Karamboulas4, Anthony C. Nichols5, Renato Covello6, Raul Pellini3, Giuseppe Spriano3, Giuseppe Sanguineti7, Paola Muti8,9, Silvio Bicciato2, Laurie Ailles5, Sabrina Strano1, Giulia Fontemaggi1, and Giovanni Blandino1

ABSTRACT ◥ Purpose: Mutation of TP53 gene is a hallmark of head and neck Results: We identified a signature of transcripts directly squamous cell carcinoma (HNSCC) not yet exploited therapeuti- controlled by gain-of-function mutant p53 protein and prog- cally. TP53 mutation frequently leads to the synthesis of mutant p53 nostic in HNSCC, which is highly enriched of MYC targets. proteins with gain-of-function activity, associated with radioresis- Specifically, both in PDX and cell lines of HNSCC treated with tance and high incidence of local recurrences in HNSCC. the PI3Ka-selective inhibitor BYL719 (alpelisib) the down- Experimental Design: Mutant p53–associated functions were regulation of mutant p53/MYC-dependent signature correlates investigated through gene set enrichment analysis in the Cancer with response to this compound. Mechanistically, mutant p53 Genome Atlas cohort of HNSCC and in a panel of 22 HNSCC cell favors the binding of MYC to its target promoters and lines. Mutant p53–dependent transcripts were analyzed in HNSCC enhances MYC protein stability. Treatment with BYL719 dis- cell line Cal27, carrying mutant p53H193L; FaDu, carrying rupts the interaction of MYC, mutant p53, and YAP proteins p53R248L; and Detroit 562, carrying p53R175H. Drugs impinging with MYC target promoters. Of note, depletion of MYC, on mutant p53-MYC–dependent signature were identified inter- mutant p53, or YAP potentiates the effectiveness of BYL719 rogating Connectivity Map (https://clue.io) derived from the treatment. Library of Integrated Network–based Cellular Signatures (LINCS) Conclusions: Collectively, the blocking of this transcriptional database (http://lincs.hms.harvard.edu/) and analyzed in HNSCC network is an important determinant for the response to BYL719 cell lines and patient-derived xenografts (PDX) models. in HNSCC.

Introduction Head and neck squamous cell carcinoma (HNSCC) comprises 5.5% 1Oncogenomic and Epigenetic Unit, IRCCS Regina Elena National Cancer Insti- of all cancer incidence and is the sixth leading cancer worldwide (1, 2). tute, Rome, Italy. 2Department of Life Sciences, Center for Genome Research, It is typically characterized by locoregional diffusion with 60% of 3 University of Modena and Reggio Emilia, Modena, Italy. Otolaryngology Unit, patients affected by relapse. Unfortunately, advances in the surgical 4 IRCCS Regina Elena National Cancer Institute, Rome, Italy. Princess Margaret and medical treatments for HNSCC over the past two decades have not Cancer Centre, University Health Network, Toronto, Ontario, Canada. 5Depart- ment of Otolaryngology–Head and Neck Surgery, Western University, London, improved overall disease outcomes. As a consequence, locoregional Ontario, Canada. 6Department of Pathology, IRCCS Regina Elena National failure is the most common cause of death in patients with Cancer Institute, Rome, Italy. 7Radiation Oncology Department, IRCCS Regina HNSCC (1, 2). Elena National Cancer Institute, Rome, Italy. 8Department of Oncology, Jura- TP53 is the most frequently mutated gene in HNSCC (http://www- 9 vinski Cancer Center-McMaster University Hamilton, Ontario, Canada. Depart- p53.iarc.fr). Recently, whole-exome sequencing analyses reported that ment of Biomedical, Surgical and Dental Sciences, University of Milan La Statale, TP53 mutations occur in HNSCC with a frequency of 72% (3). The Milan, Italy. tetrameric transcription factor p53 is a stress-responsive protein that Note: Supplementary data for this article are available at Clinical Cancer suppresses cellular oncogenic transformation mainly by inducing Research Online (http://clincancerres.aacrjournals.org/). growth arrest, apoptosis, senescence, DNA repair, and differentiation F. Ganci and C. Pulito contributed equally to this article. in damaged cells (4–6). Missense mutations represent the majority of Current address for S. Valsoni: San Raffaele Telethon Institute for Gene Therapy TP53 mutations (7). It is becoming increasingly evident that many (SR-Tiget), IRCCS San Raffaele Scientiﬁc Institute, Milan, Italy; and current mutant p53 forms not only lose their tumor-suppressive function but address for E.M.C. Mazza, Laboratory of Translational Immunology, Humanitas also gain new oncogenic properties (8). This notion is termed “gain-of- Clinical and Research Center, Rozzano, Milan, Italy. function” (GOF). In vivo and in vitro studies have indeed demon- Corresponding Authors: Giovanni Blandino, Oncogenomic and Epigenetic Unit, strated that some mutant p53 proteins, usually expressed at markedly IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi, 53, Rome 00144, elevated levels in cancer cells, are associated with enhanced prolifer- Italy. Phone: 3906-5266-2911; Fax: 3906-5266-5523; E-mail: ative, invasive, and angiogenic abilities (9, 10). At the molecular level, [email protected]; and Giulia Fontemaggi, [email protected] mutant p53 proteins exert oncogenic functions through their ability to modulate gene expression. This occurs through the interaction with Clin Cancer Res 2020;XX:XX–XX other transcription factors, such as NF-Y, E2F1, NFkB, Sp1, Ets1, doi: 10.1158/1078-0432.CCR-19-2485 SREBP-2, the Vitamin D receptor (VDR), E2F4, and YAP (11–15). An 2020 American Association for Cancer Research. additional mechanism of GOF is based on the ability of mutant p53 to

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mutant p53-dependent gene expression in HNSCC counteracting Translational Relevance mutant p53 GOF. Head and neck squamous cell carcinoma (HNSCC) is typically characterized by mutation of TP53 gene, associated with therapy resistance and high incidence of local recurrences. However, drugs Materials and Methods specifically targeting mutant p53 proteins, frequently presenting Cell cultures and treatments gain-of-function activity associated with radioresistance, are not Cal27 (mutp53H193L), FaDu (mutp53R248L), and Detroit 562 available. We set out to identify mutant p53–associated functions (mutp53R175H) cell lines were obtained from ATCC. HNO91 cells that might be targeted with drugs currently used in HNSCC trials. (GeoDatabase GSM966874) were a kind gift from Elena Di Gennaro This study identifies MYC as a crucial mediator of mutant p53 (Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, Napoli, activity in HNSCC and PI3K inhibitors as compounds able to Italy). Sequencing of all coding exons of TP53 was performed to ensure impinge on mutant p53-MYC–dependent gene expression. Of the presence of wtp53 in HNO91 cells. These cells were cultured in note, downregulation of mutant p53-MYC–dependent genes is RPMI1640 (FaDu and Cal27) and DMEM (Detroit 562 and HNO91) associated with response to PI3Ka-selective inhibitor alpelisib medium (Invitrogen-GIBCO) supplemented with 10% FBS, penicillin (BYL719) in HNSCC. (100 U/mL), and streptomycin (100 mg/mL; Invitrogen-GIBCO). All cell lines were grown at 37C in a balanced air humidified incubator with 5% CO2.

bind to and sequester proteins with antitumor activity, such as p63 and Drug Treatments. MK-2206 (Selleckchem, #S1078), fostamatinib p73 (16). (Selleckchem, #S2625), BMS-754807 (Selleckchem, #S1124), PI-103 Several studies demonstrated that TP53 status has a prognostic role (Selleckchem, #S1038), everolimus (RAD001; Selleckchem, #S1120) in HNSCC; mutations in TP53 indeed predict shorter overall survival, and BYL719 (alpelisib; Selleckchem, #S2814), and cisplatin (Pfizer insurgence of local recurrence, and cancer treatment failure (17–22). Pharmaceuticals Group) were dissolved according to the manufac- In this study, we set out to define the transcriptional program turer's instructions. The cells were treated for 6 hours–24 hours–48 governed by mutant p53 proteins with GOF in HNSCC with the aim to hours–72 hours–96 hours according to the drug and the experiment highlight the most relevant mutant p53–dependent pathways that (see Results). could be exploited therapeutically. MYC emerged in our study as a major mediator of mutant p53 Irradiation Treatment. The cells were plated at subconfluent mono- GOF in HNSCC. MYC is a proto-oncogene with crucial role in layer and after 24 hours they were treated with g-ray irradiation from tumor initiation, progression, and maintenance (23). MYC onco- Varian Clinac 2100C source at 1, 2, and 4 Gy for 24 hours. proteins in solid tumors are frequently activated by copy gains but may be also induced by a variety of other mechanisms (24); Combined Treatments. The cells were plated at subconfluent mono- importantly, it is well established that even small changes in layer and after 24 hours they were pretreated with the drug (for the MYC levels can drive ectopic proliferation of somatic cells and doses, see Results) for 6 hours and finally irradiated or treated with oncogenesis (25–27). MYC regulates various cancer cellular func- cisplatin at different doses for additional 24 hours before performing tions, including cell cycle, cell survival, proliferation, and metabolic clonogenic or ATPlite assays. reprogramming; moreover, MYC-induced oncogenic and epigenetic reprogramming leads to the acquisition of cancer stem-like Cell transfection cell–associated properties and induction of the intratumoral The transfections were performed with Lipofectamine RNAiMax or heterogeneity (28). Lipofectamine 2000 (Life Technologies). All experiments were con- Also during HNSCC tumorigenesis, MYC plays an important role ducted according to the manufacturer's recommendations. siRNAs and its expression associates with poor overall survival (29). Impor- were purchased from Eurofins MWG and sequences are as follows: si- tantly, MYC depletion potentiates cisplatin-induced apoptosis in SCR: 50-AAGUUCAGCGUGUCCGGGGAG-30; si-MYC_1: 50- HNSCC cells (30), highlighting MYC protein as an important player GCCACAGCAUACAUCCUGU-30; si-MYC_2: 50-GGACUAUC- in chemoresistance. Of note, it has been shown that concomitant CUGCUGCCAAG-30; si-YAP: 50-GACAUCUUCUGGUCAGAGA- expression of mutant p53 and MYC proteins predicts clinical outcome 30; si-TEAD: 50- CGAUUUGUAUACCGAAUAA-30; Si-p53_1: 50- (DFS) in HNSCC more accurately than what these proteins do GACUCCAGUGGUAAUCUAC-30 (CDS); Si-p53_2: 50-GGUG- individually (31). AACCUUAGUACCUAA-30 (30UTR). The cells were transfected for MYC mRNA translation, MYC protein half-life, and MYC tran- 48–72 hours according to the cell line and the experiments (see scriptional activity are enhanced by mitogenic signaling through Results). receptor tyrosine kinases that activate PI3K–AKT–mTOR signaling in breast cancer (32–34). Accordingly, activation of this upstream RNA extraction and expression analysis kinase cascade is required for cell transformation by MYC in certain Total RNA was extracted using the TRIzol Reagents (GIBCO) cellular contexts (35). following the manufacturer's instructions. The concentration and Activation of PI3K–AKT–mTOR signaling in cancer occurs as the purity of total RNA was assessed using a Nanodrop TM1000 result of activating upstream stimuli, as, for example, those from RTKs, spectrophotometer (Nanodrop Technologies). Reverse transcrip- or by activating mutations in components of PI3K pathway. Sixty-six tion and qRT-PCR quantification were performed, respectively, by percent of HNSCC harbor genomic alterations in one of the major MMLV RT assay and SYBR Green or Taqman assays (Applied components of PI3K pathway, being PIK3CA, PTEN, and PIK3R1, the Biosystems) according to the manufacturer's protocol. H3, most frequently mutated genes of the pathway (3, 36). Here, we GAPDH,GUSB,RPL19,RNU2,andACTINwereusedasendog- characterized the ability of PI3K inhibitors to impinge on MYC- enous controls to standardize gene expression. Primers and

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Taqman assays used are indicated in Supplementary Table S1 (ser235/236; Cell Signaling Technology, #2211); anti-HA (Santa Cruz (sheet 6). Biotechnology, sc-57592). Secondary antibodies used were goat anti- For RNA-sequencing (RNA-seq) analysis through next-generation mouse and goat anti-rabbit conjugated to horseradish peroxidase sequencing, we extracted RNA from Cal27 cells transfected with si- (Amersham Biosciences). Immunostained bands were detected by SCR or si-p53 for 48 hours using miRNeasy kit (Qiagen) following the chemiluminescent method (Pierce). manufacturer's instructions. RNA-seq of total RNA from n ¼ 4 separate replicates of both TP53-silenced and control Cal27 cells was Coimmunoprecipitation performed on an Illumina HiSeq 2500 platform, according to the Cal27 cells were transfected with either a pcDNA3 or a pcDNA3- following parameters: Hiseq2500 4 plex run, 2 100 bp reads, about c-MYC expression vector. Cells were lysed at 4C in lysis buffer 60 M reads/sample (for mRNAs/lincRNAs) after preparing the librar- (HEPES pH 7.5 150 mmol/L, NaCl 300 mmol/L, 1% Triton X100, ies with the Truseq stranded with RiboZero Illumina kit. MgCl2 10 mmol/L, EGTA 5 mmol/L, KCl 5 mmol/L, EDTA 2 mmol/L) supplemented with protease and phosphatase inhibitors Ubiquitination assay (5 mmol/L phenylmethylsulfonylfluoride, 3 mmol/L NaF, 1 mmol/L 6 1.6 10 cells were transfected with a pCMV vector carrying DTT, 1 mmol/L NaVO4). One milligram of precleared cell lysate hemagglutinin (HA)-tagged ubiquitin (Ub-HA; ref. 37). After protein was incubated at 4C overnight with protein G-Agarose 18 hours, cells were treated with 25 mmol/L MG-132 for a further beads (Thermo Fisher Scientific) in lysis buffer containing 0.05% 6 hours. Protein extracts were immunoprecipitated as described below BSA and either human phospho c-Myc (ser62) antibody (Abcam, and subjected to Western blotting. #185656) or IgG rabbit (Santa Cruz Biotechnology, sc66931) antibody as control. Cell viability assay After incubation, agarose bead–bound immunocomplexes were Manual counting and Trypan blue dye exclusion assay were used to rinsed with lysis buffer and eluted in 50 mL of SDS sample buffer for assess cell growth. Viability of treated cells was assessed using ATPlite Western blotting. The following primary antibodies were used: assay (Perkin Elmer) at the indicated time points, accordingly to the anti-YAP1 (Invitrogen, # PA1-46189), anti-p53 (Santa Cruz Bio- manufacturer's instructions. Cells (8 102 cells) were seeded in technology, sc126), anti-c-Myc (Cell Signaling Technology, 96-well plates and cultured for 24–48–72–96 hours (see Results). Each #5605), anti-phospho-c-Myc (ser62; Abcam, #185656), anti- plate was evaluated immediately on a microplate reader (Expire GAPDH (Santa Cruz Biotechnology, sc32233). All the indicated Technology, Perkin Elmer). CalcuSyn software was used to calculate antibodies were used at the minimum dilutions suggested by the combination index (CI; ref. 38). manufacturer. Secondary horseradish peroxidase–conjugated was purchased from Santa Cruz Biotechnology. ECL reagent (Merck EnSpire cellular label-free platform Millipore) was employed for the chemoluminescence detection. Cal27 and FaDu cells were seeded in a specially designed 384-well The Uvitec Alliance software (Eppendorf) was used to quantify the plate with highly precise optical sensors able to measure changes in obtained data. light refraction resulting from dynamic mass redistribution (DMR) within the cell's monolayer. Change in the light refraction was indi- Pulse chase with cycloheximide cated by a shift in wavelength. Cycloheximide (Sigma Aldrich) is stored as a 100 mmol/L stock solution, dissolved in dimethylsulfoxide. Cal27 cells silenced or not for Lysate preparation and immunoblotting analysis p53 (48 hours) and treated or not with 5 nmol/L of BYL719 (24 hours) Cells were lysed in buffer with 50 mmol/L Tris–HCl pH 8, with 1% were treated with 70 mmol/L of cycloheximide for the indicated time. NP-40 (IgepalAC-630), 150 mmol/L NaCl, 5 mmol/L EDTA, and fresh protease inhibitors. Extracts were sonicated for 10 seconds and Wound healing assay centrifuged at 12,000 rpm for 10 minutes to remove cell debris. Cells were seeded in 6-well tissue culture plates, grown to 95% Protein concentrations were determined by colorimetric assay (Bio- confluence, and wounded with a sterile 10 mL pipette to remove cells in Rad). In the coimmunoprecipitation assay, the lysis buffer was mod- one perpendicular line. PBS 1 washing was used to remove loosely ified according to the protein isoelectric point. Protein concentrations attached cells. Digital micrographs were taken after scratching at the were determined by colorimetric assay (Bio-Rad). For each immuno- indicated times. precipitation, 1 mg of rabbit c-Myc (Cell Signaling Technology, #5605) and 1 mg of rabbit IgG (Santa Cruz Biotechnology, sc66931) as control Transwell migration assay were used. Precleared extracts were incubated with protein A/G- Migration assay was performed using a 24-well plate with a non- Agarose beads (Thermo Fisher Scientific) in lysis buffer containing coated 8-mm pore size filter in the insert chamber (Falcon). Cells were 0.05% BSA and antibodies, under constant shaking at 4C for 3 hours. transfected with siRNA-SCR as negative control or siRNA-p53 (Euro- After incubation, agarose bead–bound immunocomplexes were rinsed fins genomics). Forty-eight hours after transfection, HNSCC cells were with lysis buffer and eluted in 50 mL of SDS sample buffer for Western resuspended in RPMI media with 1% FBS and seeded into the insert blotting. The following primary antibodies were used: anti-c-Myc chamber. Cal27 cells were allowed to migrate for 48 hours or 72 hours (Santa Cruz Biotechnology, sc-40); anti-p53 (Santa Cruz Biotechnol- into the bottom chamber containing 0.7 mL RPMI media containing fi ogy, sc-6243); anti-phospho Akt (ser473; Cell Signaling Technology, 10% FBS in a humidi ed incubator at 37 Cin5%CO2. Migrated cells #4058); anti-Akt (Cell Signaling Technology, #4685); anti-phospho- that attached to the outside of the filter were visualized by staining with mTOR (Ser-2448; Cell Signaling Technology, #2971); anti-mTOR DAPI and counted. (Cell Signaling Technology, #4685); anti-GAPDH (Santa Cruz Bio- technology, sc-32233); anti-phospho p70 S6 Kinase (thr389; Cell Clonogenic assays Signaling Technology, #9234); anti-p70 S6 (Cell Signaling Technology, Cal27 and FaDu cell lines were grown to 70% confluence and pulse #9202); anti-S6 (Cell Signaling Technology, #2317); anti-phospho S6 treated with the indicated drugs or transfected with siRNA. Forty-eight

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hours after transfection or 16 hours later, cells were detached and Specifically, we determined the “Best Average Response” (BestAvg- – V t ¼ t seeded at 500 1,500 cells/well into 6-well dishes (COSTAR) in drug- Response) by calculating the average of D t from 0to , for each free media. Fresh media (25%) was added every five days. Colonies time point and then taking the minimum average found, where t > were stained with crystal violet and colonies (>50 cells) counted after 10 days. This metric uniquely captures the durability and strength 10–14 days. of response (39). To categorize responses, we used the modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria Chromatin immunoprecipitation and sequential ChIP (reChIP) established by Gao and colleagues, which is based on the RECIST— analysis a set of clinically established criteria defining when patients with Chromatin immunoprecipitation was performed as previously cancer “respond,” remain unchanged (“stable”), or “progress” dur- reported (12). Mouse monoclonal anti-MYC (Santa Cruz Biotechnol- ing the course of their treatment (40). The mRECIST criteria ogy, sc-40), anti-MYC-p(Ser62) (Abcam, #185656), anti-p53 (Santa considers both the BestAvgResponse, described above, as well as Cruz Biotechnology, DO1, sc-126), anti-YAP1 (Invitrogen, #PA1- the “Best Response” (BestResponse), which is the minimum value of V t 46189), and anti-H4 pan-acetylated (Millipore, 06-598) were used for D t for >10 (39). All models included in our PCT received a immunoprecipitations. Cal27 cells at <50% confluence were either minimum of 14 days of treatment with BYL719. treated with 5 nmol/L BYL719 for 24 hours or transfected with siRNAs directed to p53 or YAP (sequences listed above) for 48 hours before Bioinformatics analysis of Cal27 RNA-seq data crosslinking with 1% formaldehyde. SF3B3, CCT2, HPRT1, XPOT, Reads from Cal27 RNA-seq were quality controlled using FastQC and SERBP1 promoters were amplified using the primer pairs included (www.bioinformatics.babraham.ac.uk/projects/fastqc) and trimmed in Supplementary Table S1 (sheet 6). ReChIP experiments were using Trimmomatic v0.32 (41). Remaining paired reads were performed in proliferating Cal27 cells at <50% confluence and cross- aligned to the human genome using TopHat2 (42) and UCSC linked as above indicated. After first immunoprecipitation with the assembly (version hg19) and Gencode (version 19, GRCh37, antibody of interest, protein complexes were recovered by elution in Ensemble releases 74, 75) as reference genome and transcriptome, DTT 10 mmol/L at 37C for 30 minutes with agitation. Eluates were respectively. From the GTF file,weexcludedsomeannotation diluted 1:20 with reChIP buffer (665 mL; reChIP buffer: 1% Triton- features (pseudogenes, mitochondrial and ribosomal RNAs, small X100, 2 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L Tris-HCl pH RNA, and immunoglobulin coding genes) and retained only genes 8.0) and subjected to second immunoprecipitation. Negative control annotated as “known.” Expression levels were quantified using for second immunoprecipitation is represented by IgG as second HTSeq (43) with the “union mode” after sorting reads by name C antibody. ChIP experiments have been normalized by DD t method with samtools (44). Differential expression analysis was conducted over input sample (1:100 dilution) and subsequently over IgG. Data are with edgeR (45) after filtering genes with low expression level [<1 presented as folds over IgG or as percent of input signal. An intronic count per million (cpm) in at least half of the samples] and region of CCNB1 gene, previously reported by our group, has been considering samples as paired. Differentially expressed genes were used as negative control region (12). Primer sequences are available in identified setting the fold discovery rate (FDR) threshold to 0.05 Supplementary Table S1 (sheet 6). (Supplementary Table S1, sheet 1–2). Global unsupervised clustering was performed using the function hclust of R stats package with PDX models of HNSCC Pearson correlation as distance metric and average agglomeration All animal experiments were performed with the approval of the method. Gene expression heatmaps were generated using the University Health Network Animal Care Committee (protocol #1542) function heatmap.2 of R gplots package after row-wise standard- and adhered to the Canadian Council on Animal Care guidelines. ization of the expression values. Fresh surgical HNSCC specimens were received from consenting patients within 0.5 to 24 hours of surgery under a University Health Analysis of HNSCC tumor cohorts Network Research Ethics Board–approved protocol (REB# 12-5639). We downloaded gene expression data (raw counts and RSEM), PDX models were established by implanting small tissue fragments mutations, and clinical information of the The Cancer Genome Atlas (1mm3) under the skin on the flanks of NOD/SCID/IL2Rg / (TCGA)-HNSCC cohort from Firehose Broad Genome Data Analysis (NSG) mice. Once tumors reached 1–1.5 cm in diameter, mice were Centre (GDAC; https://gdac.broadinstitute.org/). Loss of heterozy- euthanized and tumors were dissociated in Media199 containing 1 gosity and processed copy-number variation (CNV) of the TP53 gene collagenase/hyaluronidase (StemCell Technologies) and DNASE 1 data were downloaded from Synapse and cBioPortal databases, respec- (125 U/mL; Worthington) and injected subcutaneously into 4 mice tively. Out of the original samples of the TCGA-HNSCC dataset, we per tumor model (minimum 100,000 cells/mouse) in 1:1 Matrigel/PBS. selected only the 202 primary tumors that had a single annotated When tumor volumes reached 80–120 mm3, mice were randomized mutation of TP53 (wt and missense, nonsense, frame-shift mutation) into BYL719 treatment (Novartis; 50 mg/kg) and vehicle control (corn and that received no neoadjuvant treatment. Raw counts were nor- oil) groups and treated by oral gavage 5 days per week. Mice were malized and gene expression levels quantified as cpm using functions evaluated for tumor size and body weight every 2–4 days. Individual of the edgeR R package. tumor volumes were calculated by the formula: [length (width)2] Gene expression data of the IRE cohort (n ¼ 22) were generated from 0.52. A PDX model was considered responsive to BYL719 treatment if raw .CEL files. Briefly, probe-level signals were converted to expression they had a statistically significant growth delay (a significant difference values using robust multi-array average procedure (RMA; ref. 46) of in slopes of growth curves using mixed model regression on repeated Bioconductor affy package and a custom definition file for human Gene measures). 1.0 ST arrays based on Entrez genes from BrainArray (http://brainarray. mbni.med.umich.edu/Brainarray/Database/CustomCDF/CDF_download. PDX Response Calls. The response of PDXs to BYL719 was determined asp; hugene10sthsentrezgcdf version 17.1.0). All analyses were per- by comparing the mean tumor volume change at time t to its baseline formed in R environment version 3.0.1 using BioC version 2.12 and ¼ ¼ V –V V size: % tumor volume change DVolt 100% (( t initial)/ initial). Bioconductor packages.

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Average signature (gene set) expression was calculated as the in treated cells to the query gene signature and characterized by a score standardized average expression of all signature genes in sample that ranges from 100 to 100 (based on the overlap with up- or subgroups (e.g., p53 mutant, p53 wt). downregulated genes after the perturbation and the strength of the To identify two groups of tumors with either high or low mutant p53 enrichment). In particular, a negative score indicates that the com- signature (mutp53-sig) and 22-gene MYC signature, we used the pound and the query signatures are opposing, that is, genes of query classifier described in ref. 47, that is, a classification rule based on signature are decreased by treatment with the compound. The mag- the signature score. Tumors were classified as signature “low” or nitude of the score corresponds to the magnitude of similarity or signature “high” if the combined score was negative or positive, dissimilarity. Drugs with negative score (i.e., drugs that revert the respectively. This classification was applied to expression values of signature expression levels) were filtered retaining those with a score the TCGA cohort. To evaluate the prognostic value of the mutp53-sig lower than the 90th percentile of all scores (97.6). Among the 243 and 22-gene MYC signatures, we estimated, using the Kaplan–Meier resulting compounds, we used the ClinicalTrials.gov registry to further method, the probabilities of patient overall survival. Kaplan–Meier select five drugs already in clinical trial for the treatment of HNSCC curves were compared using the log-rank (Mantel–Cox) test. P values tumors. were calculated according to the standard normal asymptotic distri- bution. Survival analysis was performed in GraphPad Prism. Ethics approval and consent to participate The study involved patients with histologically confirmed primary Gene set enrichment analysis HNSCC undergoing curative treatment at the Otolaryngology Head Functional over-representation was performed using gene set and Neck Surgery Department of IRCCS Regina Elena National enrichment analysis (GSEA; http://software.broadinstitute.org/gsea/ Cancer Institute (IRE, Rome). Enrollment of these patients was index.jsp), the curated gene sets of the Molecular Signatures Database approved by the scientific ethic committee of IRE (Rome) (protocols (MSigDB; ref. 48) derived from the Hallmark and Biocarta collections CE/379/08 and CE/868/16). (http://software.broadinstitute.org/gsea/msigdb/collections.jsp), and the gene sets listed in ref. 49. For Cal27 and TCGA-HNSC RNA- Availability of data and material seq data, GSEA was run in preranked mode using the log2 fold change The TCGA dataset analysed during the current study was obtained of all genes. Gene sets were considered significantly enriched at false from the GDAC repository (https://gdac.broadinstitute.org/). Cal27 discovery rate (FDR) ≤0.25 when using classic as metric and 1,000 RNA-seq raw data are available upon request. Affymetrix gene expres- permutations. The dot plot, showing the most significantly enriched sion raw data of HNSCC tumors and normal counterparts from the gene sets in si-mutp53 Cal27 cells in missense p53 TCGA-HNSC IRE cohort are available in GEO database (GSE107591). tumors and a panel of missense HNSCC cell lines (E-MTAB-3610; ref. 50) was generated using the ggplot function of the ggplot2 R package. Results MYC targets are enriched in mutant p53–dependent Identification of a mt-p53-myc–dependent HNSCC signature transcriptome in HNSCC We defined the 22-gene mutant p53-dependent and HNSCC- Mutations in TP53 gene frequently lead to the production of very specific MYC signature selecting MYC targets of the Hallmark col- stable mutant p53 proteins that may present GOF activity and induce lection (HALLMARK_MYC_TARGETS_V1) with a gene expression proliferation, invasion, and angiogenesis. These activities rely on the profile (i) significantly correlated to MYC expression levels in the ability of GOF mutant p53 proteins to modulate gene expression TCGA and IRE HNSCC cohorts, and (ii) modulated by mutant through the interaction with a variety of transcription factors (9). In p53 in the RNA-seq analysis of Cal27 cells. Briefly, the HNSCC, nearly 60% of tumors present a missense mutation in TP53, HALLMARK_MYC_TARGETS_V1 gene set was first merged to gene potentially displaying GOF activity. Here we investigated the mutant sets of the Biocarta collection and used as input in the GSEA analysis p53–associated transcriptome to highlight possible targets that could together with the expression profile of MYC gene in the TCGA and IRE be exploited to inhibit mutant p53 oncogenic activity. HNSCC tumors as continuous phenotype label. We then selected the GSEA was first performed to compare HNSCC tissues carrying core enrichment gene lists from the two analyses, obtaining two lists of missense TP53 mutation versus wt-p53 carrying tumors. To this end, MYC targets significantly correlated with the MYC mRNA profile in we considered 202 patients from the TCGA cohort of HNSCCs, the tumor samples. Merging these two lists (n ¼ 111 and n ¼ 108 genes adopting as inclusion criteria the absence of any chemo/radiother- using TCGA and IRE samples, respectively), we obtained a combined apeutic treatment before surgery. Tumors carrying TP53 missense list of 81 genes. Finally, we intersected these 81 genes with the 1,498 mutation presented higher expression of genes belonging to various downregulated genes obtained from the si-p53 versus scr comparison pathways, such as epithelial-to-mesenchymal transition, TGFb, MYC on Cal27 cells and generated the 22-gene mt-p53- and myc-dependent targets, and angiogenesis, compared to tumors with wt-p53 (Fig. 1A). signature (Supplementary Table S1; sheet 4). A parallel GSEA analysis comparing transcriptome from HNSCC cell lines carrying TP53 missense mutation (n ¼ 16) versus wt-p53 cell Identification of potential inhibitor of mt-p53-myc–dependent lines (n ¼ 6) confirmed the association between the presence of HNSCC activity signature missense TP53 mutation and the activation of MYC (Fig. 1B; Sup- The mt-p53-MYC–dependent signature was used to interrogate the plementary Table S1, sheet 3). MYC resulted as the most affected Library of Integrated Network-based Cellular Signatures (LINCS; pathway also from the comparison of transcriptome obtained by RNA- http://www.lincsproject.org/) through Connectivity Map (data version seq from Cal27 cell line, carrying endogenous mutant p53H193L, 1.1.1.2 and software version 1.1.1.38; https://clue.io/) to identify comparing si-SCR versus si-p53 counterpart in GSEA analysis (Fig. 1C compounds whose administration to cancer cells results in an opposite and D; Supplementary Table S1, sheet 1–2). Characterization of expression profile of the 22-gene signature. Briefly, Connectivity Map p53H193L GOF activity in Cal27 cells is shown in Supplementary generates a list of compounds rank-ordered by the similarity of DEGs Fig. S1.

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Figure 1. Identification of a prognostic mutant p53–dependent and HNSCC-specific 22-gene MYC signature. A–C, Dot plot of the gene sets obtained by preranked GSEA. A, Gene sets activated (normalized enrichment score NES > 0) in TCGA-HNSCC tumors carrying a missense TP53 mutation versus wt-p53 tumors; B, Gene sets activated (normalized enrichment score NES > 0) in HNSCC cell lines carrying a missense TP53 mutation (n ¼ 16) versus wt-p53 (n ¼ 6) cell lines. C, Gene sets deactivated (normalized enrichment score NES < 0) in si-mutp53 versus control si-scr Cal27 cells. Dot color indicates statistical significance of the enrichment; dot size represents the fraction of genes composing the core enrichment of gene set. Gene sets are ranked in increasing order based on the NES value. FDR, false discovery rate; NES, normalized enrichment score. D, Z-score heatmap of differentially expressed mRNAs after depletion of mt-p53 in Cal27 cells (RNA-seq). The mutant p53–dependent signature, composed of genes downregulated by si-p53 in Cal27 (Supplementary Table S1, sheet 1), has a significantly higher expression in TCGA-HNSCC tumors (n ¼ 202) carrying missense mutation in TP53 as compared with wild-type TP53 samples (E; , P < 0.0001). High level of mutp53-sig is associated with poor outcome in TCGA-HNSCC tumors (F). G, MYC-dependent genes associated with TP53 status specifically in HNSCC were identified starting from the MYC target gene set of the MSigDB Collection and selecting a subgroup of 81 MYC-dependent genes whose mRNAs were positively correlated to MYC mRNA in both IRE (52) and TCGA (3) HNSCC cohorts. This 81-gene list was further merged with the p53-signature obtained from Cal27 cells, obtaining a 22-gene mutant p53–associated, HNSCC-specific MYC signature (G; P < 1 10–5 in an hypergeometric test). The 22-gene MYC signature discriminates wt-p53–carrying tumors (WT) from those with TP53 missense mutations in TCGA HNSCCs cohort (H) and predicts survival (I).

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Table 1. Univariate Cox regression analysis from TCGA HNSCC patients considering mutant p53 signature (signature high) and 22-gene MYC signature (signature high) as clinical variables and multivariate Cox regression analysis from TCGA HNSCC patients considering 22- gene MYC signature (signature high).

Univariate Cox regression analysis Variable HR (95% CI) P

Primary site oral cavity (vs. primary site larynx) 0.7848 (0.4917–1.2530) 0.3098 Primary site pharynx (vs. primary site larynx) 0.4248 (0.1739–1.0380) 0.0604 HPV status positive (vs. HPV status negative) 0.2865 (0.1161–0.7074) 0.00671 Alcohol history yes (vs. alcohol history no) 0.91005 (0.5932–1.3960) 0.666 Tobacco smoking history yes (vs. tobacco history no) 1.7796 (0.9844–3.2170) 0.0564 Pathologic N ¼ 1 (vs. pathologic N ¼ 0) 1.4379 (0.9382–2.2040) 0.0955 Pathologic T ¼ 3/4 (vs. pathologic T ¼ 1/2) 1.7005 (1.0880–2.6580) 0.0198 Histologic grade G ¼ 3/4 (vs. histologic grade G ¼ 1/2) 0.8311 (0.5185–1.3320) 0.442 Clinical M ¼ 1 (vs. clinical M ¼ 0) 3.538 (0.4878–25.6600) 0.211 TP53 status mutated (vs. TP53 status WT) 2.4598 (1.4660–4.1280) 0.000657 TP53 copy number CNV (vs. TP53 copy number diploid) 1.6109 (1.0660–2.4330) 0.0235 mutp53 signature high (vs. signature low) 1.8924 (1.232–2.907) 0.0036 22-gene MYC signature high (vs. signature low) 2.2392 (1.445–3.471) 0.000312

Multivariate Cox regression analysis Variable HR (95% CI) P

HPV status positive (vs. HPV status negative) 0.5247 (0.1874–1.4690) 0.2196 Pathological T ¼ 3/4 (vs. Pathological T ¼ 1/2) 1.5081 (0.9589–2.3720) 0.0754 TP53 status mutated (vs. TP53 status WT) 1.6164 (0.8863–2.9480) 0.1173 22-genes MYC signature high (vs. signature low) 1.7070 (1.0729–2.7160) 0.0240 TP53 copy number CNV (vs. TP53 copy number diploid) 1.1510 (0.7420–1.7850) 0.5301

Note: , P ≤ 0.05.

On the basis of GSEA results, we analyzed tissues from a small and is prognostic in HNSCC (Fig. 1I). Moreover, expression of cohort of 23 PDX models of HNSCC, with previously characterized 14 genes of the MYC signature is still significantly higher in the TP53 status (51), to evaluate whether tumors with a mutated and missense mutant p53 versus wt-p53 group considering only the highly expressed p53 showed any enrichment of MYC protein. As HPV(–) cases of the TCGA cohort (Supplementary Fig. S3H). Eval- shown in Supplementary Fig. S3, Western blot analysis evidenced that uation of the expression of the 22-gene MYC signature in TCGA despite the low number of tissues with high p53 protein expression cohorts from additional tumors evidenced a higher expression in the (Supplementary Fig. S3A–S3C), MYC protein level was higher in mutant p53 versus wt-p53 group also in lung, colon, and breast cancer tumors with highly expressed mutated p53, compared with low-p53 (Supplementary Fig. S3J–S3L). tumors (Supplementary Fig. S3B and S3C). Analysis of MYC transcript Aimed at evaluating the prognostic value of the identified signa- evidenced a significantly higher expression of MYC in TP53 mutation tures, we first performed Univariate Cox regression analysis on the carrying versus wt-p53 cell lines and HNSCCs from the TCGA cohort TCGA HNSCC cohort. This revealed that expression level of both (Supplementary Fig. S3D and S3E). mutp53-sig and 22-gene MYC signature associates with overall sur- We further characterized the transcripts downregulated after vival, along with TP53 status, TP53 copy number, and other clinical p53H193L depletion in Cal27 cells, hereby indicated as mutp53-sig, variables, as HPV status and tumor size (T; Table 1). In a Multivariable by analyzing their expression in the 202 HNSCCs from TCGA also Cox proportional hazards regression model, adjusting for the signif- used for the GSEA analysis. As shown in Fig. 1E, mutp53-sig level is icant prognostic factors, we determined that a high expression level of higher in tumors carrying missense TP53 mutation, compared with the 22-gene MYC signature predicts overall survival independently wild-type TP53 tumors. Moreover, high expression of mutp53-sig is from the other variables, while mutp53-sig loses its prognostic value also associated with lower probability of survival, compared with low (Table 1). These results indicate that the 22-gene MYC-dependent mutp53-sig expression, as assessed by Kaplan–Meier analysis on the signature (part of the mutp53-sig) outperforms mutp53-sig prognostic TCGA cohort (Fig. 1F). power. To further characterize the activity of mutant p53 on MYC targets, we defined a mutant p53–dependent and HNSCC-specific MYC Mutant p53 controls MYC target promoters in HNSCC cells signature selecting those MYC targets of Hallmark ver.1 with a To further characterize the 22-gene MYC-dependent signature, we gene expression profile (i) significantly correlated to MYC expression transfected Cal27, FaDu, and Detroit 562 cell lines with siRNAs levels in two HNSCC cohorts (IRE and TCGA cohorts, see directed to MYC. We confirmed that the expression of the 22-gene Methods; Fig. 1G) and (ii) modulated by mutant p53 in the RNA- MYC signature decreases after the depletion of MYC (Fig. 2A–F). As seq analysis of Cal27 cells (Fig. 1G). This resulted in a 22-gene MYC also expected, mutant p53 depletion led to the signature downregula- signature (Supplementary Table S1, sheet 4) that, similar to the whole tion in Cal27, FaDu, and Detroit 562 cells, carrying, respectively, mutp53-sig, is differentially expressed between wt-p53 and mutant endogenous mutant p53H193L, p53R248L, and p53R175H with GOF p53–carrying tumors (Fig. 1H; Supplementary Fig. S3F, S3G, and S3I) (Fig. 2A–F; Supplementary Figs. S1 and S2).

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Figure 2. MYC and mutant p53 directly control the expression of the 22-gene signature. A–H, qRT-PCR (A, C, D,andG) analysis of the 22-gene MYC signature in Cal27, FaDu, and Detroit 562 cells depleted or not of MYC, mutant p53, YAP, or TEAD. Western blot (B, E,andF) and qRT-PCR (H) analyses were used to assess the depletion of the indicated proteins and transcripts after transfection of siRNAs. Results from three biological replicates are shown. I, qRT-PCR analysis of the indicated MYC target genes after depletion of MYC or p53 in Cal27 cells. Results from three biological replicates are shown. J–M, ChIP analysis of the indicated MYC target promoters,

performed in Cal27 cells using the indicated antibodies. Relative enrichment has been calculated by DDCt method as described in the Materials and Methods and is expressed as folds over IgG sample. Mean of at least two biological replicates is shown. N–Q, ChIP analysis of the indicated MYC target promoters, performed in Cal27 cells, depleted or not of mutant p53 expression as shown by Western blot analysis in N, using the indicated antibodies. Relative enrichment has been calculated by

DDCt method. Enrichment in control condition si-SCR was set to 1 to present result obtained with H4Ac Ab (O). Results are the mean of two biological replicates. P and Q, Folds over IgG are presented and results are the mean of three biological replicates. R–T, ReChIP experiments in Cal27 cells are the mean of three biological replicates. Negative region used as control is described in Materials and Methods. , P < 0.05; , P < 0.005; P values have been calculated by two-tailed t test.

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Interestingly, also the depletion of YAP (through the use of siRNAs catalog of gene expression profiles generated from several human targeting both YAP1 and YAP2), whose targets were significantly cancer cells in response to more than 27,900 perturbations. This enriched in GSEA analysis of mutant p53–associated transcriptomes approach highlighted a number of drugs potentially able to down- (Fig. 1A–C), impaired the expression of this signature (Fig. 2G and H). regulate the 22-gene MYC signature (Supplementary Table S1, sheet Of note, YAP protein was previously shown to cooperate transcrip- 5). Intersecting this list with the ClinicalTrials.gov registry to identify tionally with mutp53 (11, 52). No overlap between the 22-gene class of compounds exploited in clinical trials for patients with HNSCC signature and any published YAP signature was observed. Of note, led to the selection of five compounds that deserved further study depletion of TEAD proteins, canonically considered the mediators of (MKK-2206, PI-103, everolimus, fosfamatinib, and BMS-754807; YAP transcriptional activity, did not affect the expression of MYC Supplementary Fig. S4J). Interestingly, all these compounds impinge targets (Fig. 2G and H), suggesting that YAP might participate in the on pathways that have been linked to PI3K–Akt–mTOR signaling. transcriptional control of these genes by influencing MYC activity. Alteration of PI3K components in HNSCC and in cell lines used in our For ChIP experiments, we chose the promoters of three MYC experiments is included in Supplementary Fig. S4F–S4I. This finding targets of the 22-gene signature (SF3B3, CCT2, and HPRT1) and of particularly intrigued us because PI3K–Akt–mTOR signaling has been two MYC targets not modulated by mutp53 (XPOT and SERBP1). extensively shown to impact MYC activity in cancer (32–36). Expression of these genes after depletion of MYC or mutant p53 in We tested the ability of these compounds to sensitize cells to Cal27 cells is shown in Fig. 2I. We evidenced that MYC protein and conventional treatments using two HNSCC cell lines, Cal27 (carrying MYC phosphorylated in Serine 62 (MYC-p), previously reported as an p53H193L) and FaDu (carrying p53R248L) cells, suitable experimen- active form of MYC (53), are recruited onto these promoters (Fig. 2J tal systems for drug testing as they present GOF-mutant p53 proteins and K). Interestingly, also mutant p53 and YAP bind to the promoter conferring radioresistance (Supplementary Figs. S1 and S2A–S2E). We regions of SF3B3, CCT2, and HPRT1 (Fig. 2L and M). Mutant p53 also first administered increasing doses of the selected compounds and shows very low enrichment on XPOT and SERBP1 promoters. measured cell viability to identify nontoxic doses to be used in Recruitment of these factors onto MYC target genes is also present subsequent experiments for each drug (Supplementary Fig. S4K and in FaDu cells (Supplementary Fig. S4A–S4D). S4L). Capability of these drugs to overcome therapy resistance in We next performed ChIP assays in mutant p53–depleted Cal27 HNSCC cells was then evaluated by combination treatments with cells. Mutant p53 protein depletion significantly reduces the acetyla- conventional chemo- and radiotherapy. Specifically, cells were pre- tion degree of histone H4 on the promoters of mutant p53–dependent treated with targeted agents for 24 hours and subsequently treated with SF3B3, CCT2, and HPRT1, but not that of XPOT and SERBP1 (Fig. 2N cisplatin-based chemotherapy or with radiotherapy. We first evaluated and O). Depletion of mutant p53 also decreases the interaction of MYC the effect of pretreatment with the selected targeted drugs on the protein with these promoters (Fig. 2P and Q). expression of the 22-gene MYC signature and evidenced that a We next evaluated by ReChIP assays the existence of complexes significant decrease of these mRNAs occurs in both cell lines at the between MYC, mutant p53, and YAP proteins on chromatin. As low nontoxic doses employed (Supplementary Fig. S4M). By measur- shown in Fig. 2R,afirst immunoprecipitation of the phosphorylated ing cell viability and colony-forming ability, we observed that MK- form of MYC in Serine 62 (MYC-p), followed by a second immuno- 2206 and PI-103 synergize with radiotherapy (Supplementary precipitation of p53 or YAP proteins showed significant enrichment Fig. S5A–S5S) and everolimus synergizes with cisplatin treatment compared with second immunoprecipitation with control IgG, indi- (Supplementary Fig. S5C–S5F, S5K–S5N, S5Q–S5T) in Cal27 and cating the existence of MYC-p/mutant p53 and MYC-p/YAP com- FaDu cells. On the contrary, MK-2206 and PI-103 did not overcome plexes on SF3B3 promoter. No complexes were evidenced on SERBP1 cisplatin resistance and everolimus did not overcome radioresistance, promoter. Immunoprecipitation of YAP followed by a second immu- as shown in Supplementary Fig. S6A–S6F. noprecipitation of MYC-p confirmed the existence of MYC-p/YAP Altogether these results demonstrate that low doses of agents complex (Fig. 2S). Also the previously reported complex mutant p53/ targeting the PI3K–Akt–mTOR signaling potently favor the response YAP (11) was observed in Cal27 cells on SF3B3 promoter (Fig. 2T). to conventional treatments, as already indicated by the literature (54), Complexes MYC-p/mutant p53 and MYC-p/YAP were evidenced and downregulate the expression of MYC-mp53–dependent genes, also by coimmunoprecipitation assay in Cal27 cells (Supplementary indicating that PI3K inhibitors are compounds that should be kept in Fig. S4E). due consideration for the treatment of mutant p53–carrying HNSCC cells. Compounds targeting the 22-gene MYC signature sensitize HNSCC cells to conventional therapies BYL719 as a promising therapeutic tool in HNSCC As already mentioned, the presence of mutant p53 protein has been Chemical development of PI3K inhibitors has recently led to the related to poor outcome in many tumors, including HNSCC, as it may production of a very powerful compound, the BYL719 (alpelisib), favor proliferation, angiogenesis, and invasion. In HNSCC, the pres- which demonstrated effectiveness in various resistant cancers. BYL719 ence of mutant p53 is also associated with increased resistance to was designed as a specific inhibitor of PI3Ka (37), the product of therapies and treatment failure. frequently mutated PIK3CA, and should allow for a more favorable As the 22-gene MYC signature robustly predicts clinical outcome in side-effect profile because it does not affect other components of PI3K. HNSCC and is highly expressed in mutant p53–expressing cells and Of note, BYL719 has been recently shown to be active in HNSCC (38, tumor tissues, we hypothesized that the identification of compounds, 51, 55) even at low doses (56) and even in tumors not carrying PIK3CA which inhibit the expression of this signature, may represent a powerful mutation (57). tool to counteract mutant p53–associated oncogenic functions. To evaluate whether BYL719 impinges on the identified 22-gene To address this issue, we used the 22-gene MYC signature to MYC signature, we first treated Cal27 and FaDu cells with increasing interrogate Connectivity Map (https://clue.io) derived from the amounts of this drug. Western blotting analysis of phosphorylated and Library of Integrated Network-based Cellular Signatures (LINCS) nonphosphorylated components of the PI3K pathway evidenced that database (http://lincs.hms.harvard.edu/) and containing an extensive BYL719 is active in both cell lines (Fig. 3A and B). Dose–response

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Figure 3. 22-gene MYC-mp53 signature expression is affected by BYL719 treatment in HNSCC cells and correlates with response to BYL719 in PDX models of HNSCC. Western blotting showing the effect of BYL719 treatment at the indicated doses on components of the AKT/mTOR pathway in Cal27 (A) and FaDu (B) cells. C, Evaluation of the sensitivity to PI3K inhibitor BYL719 at different doses in Cal27 and FaDu cells using ATPlite assay, presented as percentage of decrease relatively to the untreated cells. D, Graph indicating the change in impedance of Cal27 cells treated BYL719 at the indicated doses for 0–2,400 minutes to evaluate early changes in cell ﬁtness. E– G, Box plot representing the average expression of the 22-gene MYC signature after treatment with 5 nmol/L BYL719 for 24 hours in Cal27 (E), FaDu (F), and Detroit 562 (G) cells (, P < 0.05; , P < 0.005; P values have been calculated by two-tailed t test). H, Box plot showing tumor weight in PDX mouse models of HNSCC responsive (left) and not responsive (right) to BYL719 treatment. Response details in this PDX models cohort have been previously reported in Ruicci and colleagues 2018 (51). I, 22-gene MYC-mp53 signature in BYL719-treated versus untreated tumors, evaluated by qRT-PCR in the groups of responsive and not responsive PDX tumors. J, Unsupervised hierarchical clustering of responsive and not responsive HNSCC PDX models based on the expression of the 22-gene MYC-mp53 signature in BYL719-treated versus untreated tumors. Collectively, 14 of 18 analyzed PDXs carry TP53 mutation while 1 of 18 exhibited MYC ampliﬁcation.

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Figure 4. Low-dose BYL719 treatment disrupts binding of MYC, mutant p53, and YAP to MYC target promoters. A, Western blot analysis of MYC, MYC-p(Ser62), p53, and YAP proteins in Cal27 cells treated with increasing amounts of BYL719 for 24 hours. B, Pulse-chase assay with 70 mmol/L cycloheximide to analyze MYC and MYC-p(Ser62) half-life in Cal27 cells treated or not with 5 nmol/L BYL719 for 24 hours. ChIP analysis of Cal27 cells treated or not with 5 nmol/L BYL719 using antibodies directed to MYC-p(Ser62; C), p53 (D), YAP (E), or H4Ac (F). ChIP data presented are the mean of at least two biological replicates. Ability of BYL719 to synergize with cisplatin, evaluated by analyzing cell viability through ATPlite assay in Cal27 (G), FaDu (I), and HNO91 (J) cells and by analyzing the change in impedance for 0–2,400 minutes to evaluate early changes in cell ﬁtness (cisplatin, CDDP 8 mmol/L) in Cal27 cells (H; , P < 0.05; , P < 0.001). Colony assay (K) and Western blot (L) analyses of Cal27 cells after treatment with cisplatin 8 mmol/L for 24 hours. Densitometry of MYC protein, normalized over GAPDH level, from three biological replicates is shown in M. N, Average expression of 14 genes belonging to the 22-gene MYC signature in Cal27 cells treated with CDDP 8 mmol/L and BYL719 5 nmol/L, single or combined, for 24 hours (, P < 0.05; , P < 0.005; , P < 0.0005; P values have been calculated by two-tailed t test).

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analysis defined 5 nmol/L as the lowest dose of BYL719 able to Depletion of mutant p53 decreases MYC protein stability upon impinge on PI3K signaling without affecting cell viability (viability treatment with BYL719 >80%; Fig. 3C and D). At this low dose, we also observed significant In addition to the reduction of MYC protein on target promoters, we downregulation of the 22-gene MYC signature in Cal27, FaDu, and interestingly observed that treatment with BYL719 decreases MYC Detroit 562 cells (Fig. 3E–G). protein level when mutant p53 expression is depleted by RNA We next used PDX models of HNSCC treated or not with BYL719 to interference in Cal27 and FaDu cells (Fig. 5A and B). This effect is evaluate whether the 22-gene MYC signature associates with response not observed on MYC mRNA expression (Fig. 5D). No MYC protein to single treatment with BYL719. Response to BYL719 treatment in modulation is observed in the same experimental conditions in this PDX cohort has been already described in detail in Ruicci and HNO91 cells, carrying wt-p53 (Fig. 5C), while BYL719 treatment per colleagues (51) and is summarized in Fig. 3H. Analysis of the 22-gene se causes MYC protein downregulation in Detroit 562 cells probably signature expression in treated versus untreated PDX showed a due to the presence in these cells of mutated hyperactive PIK3CA gene significant downregulation of the signature in tumors exhibiting (Supplementary Fig. S7L; Supplementary Fig. S4F). In Cal27 cells, we response to this agent compared with those not responsive (Fig. 3I; also observed MYC protein downregulation upon YAP depletion both Supplementary Fig. S6G). Moreover, hierarchical clustering analysis of in presence and absence of BYL719 (Fig. 5E). expression values of the 22-gene signature (treated vs. untreated) To investigate whether mutant p53 and YAP contribute to the separated responsive from not responsive tumors (Fig. 3J). stabilization of MYC protein, we evaluated the degree of ubiquitination of MYC after depletion of mutant p53 or YAP, or after treatment with BYL719 treatment impairs recruitment of MYC and mutant p53 BYL719. Specifically, Cal27 cells expressing a HA-tagged Ubiquitin proteins on MYC target promoters vector were transfected with siRNAs directed to p53 or YAP, or treated To investigate possible mechanisms through which BYL719 could with BYL719 5 nmol/L, in presence of proteasome inhibitor MG-132. lead to downregulation of the 22-gene MYC signature, we first Interestingly, we observed that depletion of mutant p53 or YAP leads analyzed MYC protein level upon treatment with increasing doses of to a striking increase of MYC ubiquitination (Fig. 5F), while BYL719 this PI3K inhibitor. We observed no major changes in MYC protein alone does not affect MYC ubiquitination. Pulse-chase assay with level (Fig. 4A; Supplementary Fig. S7A) and a dose-dependent down- cycloheximide evidenced that depletion of mutant p53 combined with regulation of phospho-MYC (Ser62; Fig. 4A). A pulse-chase assay with BYL719 treatment significantly reduced MYC protein stability cycloheximide evidenced that a low dose of BYL719 also decreased (Fig. 5G and H), while depletion of mutant p53 alone did not affect phospho-MYC (Ser62) half-life (Fig. 4B). Next, we performed ChIP MYC half-life (Supplementary Fig. S7M and S7N) as shown also for experiments in Cal27 cells upon treatment with low dose of BYL719. BYL719 alone (Fig. 4B). As shown in Fig. 4C–F and Supplementary Fig. S7B–S7D, treatment Altogether our results show that treatment of HNSCC cells with with BYL719 caused a decrease of MYC, phospho-MYC (Ser62), BYL719 is able per se to decrease MYC-p(Ser62) protein stability and to mutant p53, YAP, and H4Ac recruitment onto target promoters. We disrupt MYC binding to its target promoters, while in presence of observed a decrease of mutp53 recruitment on MYC targets upon reduced levels of mutant p53 (or YAP), BYL719 is also able to cause treatment with BYL719 also in FaDu cells (Supplementary Fig. S7E). reduction of MYC protein level (Fig. 5I). These results suggest that BYL719 is able to disrupt the binding of the transcriptional regulators MYC, mutant p53, and YAP on these target promoters, leading to decreased transcriptional activity. Discussion Combination treatments evidenced that pretreatment of cells with 5 The vast majority of HNSCCs are characterized by mutation of nmol/L BYL719 also sensitized response to conventional therapies, TP53 gene, which is associated with decreased probability of survival. such as cisplatin-based chemotherapy and radiotherapy (Fig. 4G–I; Mutations in TP53 frequently lead to the formation of mutant proteins Supplementary Fig. S5G and S5H) in Cal27 and FaDu cells, expressing with GOF activity positively contributing to the maintenance of mutant p53, but not in wtp53-carrying HNO91 (Fig. 4J). To evaluate malignant phenotype. We have recently shown that the presence of possible mechanisms at the basis of the synergy between BYL719 and TP53 mutations is particularly relevant for the outcome of advanced cisplatin, we analyzed whether cisplatin treatment affects MYC protein HNSCC (17). In this study, we aimed to identify mutant p53–asso- in HNSCC cells. As shown in Fig. 4K–M, treatment of Cal27 cells with ciated networks that could be targeted with molecular drugs currently 8 mmol/L cisplatin significantly reduced MYC-p(Ser62) protein level. used for HNSCC, due to the unavailability of compounds specifically Interestingly, combined treatment of cells with BYL719 (5 nmol/L) targeting mutant p53 itself. and cisplatin (8 mmol/L) led to a significant decrease of 12 genes of the The analysis of mutant p53–associated transcriptome in HNSCC MYC signature compared with the single treatments (Fig. 4N), sug- cells highlighted transcription factor MYC as a major mediator of gesting that impairment of MYC transcriptional activity might under- mutant p53 activity in this experimental system. A link between lie the synergistic effect of these compounds. mutant p53 GOF and MYC activation had been previously reported We next evaluated whether the depletion of mutant p53 favored in different cell context (58). the response to BYL719 in HNSCC cells. As shown in Supplemen- Importantly, analysis of mutant p53-associated transcriptome on tary Fig. S7F–S7H, p53 interference in Cal27, FaDu, and Detroit 562 the TCGA dataset of HNSCC confirmed the association between the cells, carrying mutant p53, increased the response to BYL719, while presence of a missense mutation in TP53 and the high expression of this effect was not observed in HNO91 cells, carrying wt-p53 MYC targets, strongly generalizing the findings obtained with mutants (Supplementary Fig. S7I). In Cal27 cells, we assessed that also the p53H193L, p53R248L, and p53R175H, respectively, in Cal27, FaDu, depletion of MYC or YAP increases the response to BYL719, and Detroit 562 cell lines. evaluated through a cell viability assay (Supplementary Fig. S7J Mutant p53 had been previously shown to exert its oncogenic and S7K). Collectively, our results show that depletion of any activity modulating gene expression through a plethora of transcrip- component of this transcriptional network potentiates BYL719- tional modulators, such as NF-Y and YAP (11, 12). In this study, we dependent effect on cell viability. show that mutants p53H193L and p53R248L, as well as MYC, reach

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Figure 5. Depletion of mutant p53 decreases MYC protein stability in presence of BYL719. Western blot analysis of the indicated proteins in Cal27 (A), FaDu (B), and HN091 (C) cells depleted or not of p53 expression and subsequently treated with the indicated doses of BYL719 for 24 hours. MYC mRNA level in the same experimental condition is shown in D. E, MYC protein levels evaluated by Western blot in Cal27 cells depleted of YAP and subsequently treated with the indicated doses of BYL719 for 24 hours. F, Ubiquitination of MYC protein was evaluated by immunoprecipitation of MYC followed by anti-HA Western blot in Cal27 cells transfected with an HA- tagged Ubiquitin expression vector. Cells treated or not with 5 nmol/L BYL719 and depleted or not of mutant p53 or YAP were analyzed. MG-132 was added to culture medium to inhibit proteasome-dependent degradation. G and H, Pulse-chase assay with 70 mmol/L cycloheximide to analyze MYC protein half-life in Cal27 cells depleted of mutant p53 expression and treated or not with 5 nmol/L BYL719 for 24 hours has been calculated by two-tailed t test (, P < 0.05). I, Mutant p53/MYC blocking as potential tool for the enhancement of PI3K inhibitor response. The presence of mutant p53 protein in HNSCC cells activates MYC-dependent gene expression by promoting MYC protein stability and favoring interaction of MYC with target promoters, ﬁnally leading to cell proliferation and resistance to anticancer agents (A, left). Inhibition of PI3K signaling and blocking of mutant p53 by RNAi leads to decreased interaction of MYC with its target promoters and to reduced levels of MYC protein, with consequent reduced cell viability and increased sensitivity to anticancer agents (B, right).

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the promoters of MYC target genes, modulating their expression. Myc double-transgenic, the BET inhibitor, JQ1, attenuates PI3K Interestingly, the presence of mutant p53 is required for an efficient pathway reactivation after treatment with the p110a/de–selectiv interaction of MYC with its binding sites. This suggests that in a inhibitor, pictilisib (60). mutant p53 cell context, a general increase of MYC activity could be The crosstalk between MYC and PI3K signaling in conferring sustained by mutant p53 protein recruitment at MYC promoters. This resistance to MYC or PI3K blocking has been extensively observed would represent an additional way to allow pervasive MYC hyper- in breast cancer (60) and other malignancies, and the efficacy of MYC activation, in addition to MYC gene amplification, which is observed in and PI3K blocking in favoring reciprocal response to selective inhi- only 14% of HNSCC and almost exclusively in HPV() cases. On the bitors supports further progression of this combination into clinical basis of our results, mutant p53 contributes to MYC hyperactivation testing (46). also by protecting MYC protein from degradation, as evidenced by the striking ubiquitination of MYC protein that is observed in the absence Conclusions of mutant p53, ubiquitination that probably participates in the MYC Head and neck squamous cell carcinoma (HNSCC) is typically protein decrease observed in presence of treatment with BYL719. characterized by mutation of TP53 gene, associated with therapy Through the interrogation of a pharmacogenomics database, we resistance and high incidence of local recurrences. However, drugs identified various classes of compounds able to potentially impact the specifically targeting mutant p53 proteins, frequently presenting GOF expression of mutant p53-MYC–dependent genes. Among other activity associated with radioresistance, are not available. We then set compounds, we validated the ability of PI3K/mTOR inhibitors to out to identify mutant p53–associated functions that might be targeted downregulate mutant p53-MYC–dependent genes and to sensitize with drugs currently used in HNSCC trials. This study identifies MYC HNSCC cells to conventional treatments. We particularly focused on as a crucial mediator of mutant p53 activity in HNSCC and PI3K PI3K inhibitor BYL719, designed as a specific inhibitor of PI3Ka, the inhibitors as compounds able to impinge on mutant p53-MYC– product of frequently mutated PIK3CA gene (37), as it is currently dependent gene expression. Of note, downregulation of mutant employed in clinical trials for HNSCC; being specific for PI3Ka, p53-MYC–dependent genes is associated with response to PI3Ka- it should associate with a more favorable side-effect profile (54, 59). selective inhibitor BYL719 in HNSCC. Analysis of HNSCC PDX models evidenced that downregulation of mutant p53-MYC–dependent genes correlates with response to this Disclosure of Potential Conflicts of Interest compound in vivo. A.C. Nichols reports receiving commercial research grants from Novartis Canada Purposely, for studies in HNSCC cells, we selected a very low dose of Ltd. No potential conflicts of interest were disclosed by the other authors. this compound, to avoid toxicity effects (cell viability >80%), in ’ addition to the biological effects, and to appreciate synergy in com- Authors Contributions bination treatments. At 5 nmol/L BYL719, we did not observe any Conception and design: F. Ganci, C. Pulito, V. Manciocco, S. Strano, G. Fontemaggi, G. Blandino effect on cell viability but we did observe modulation of downstream Development of methodology: F. Ganci, C. Pulito, R. Covello, P. Muti, effectors phosphor-Akt and phosphor-mTOR as well as sensitization G. Fontemaggi, G. Blandino to both radio- and chemotherapy treatments. Moreover, at 5 nmol/L Acquisition of data (provided animals, acquired and managed patients, provided BYL719, we observed reduction of MYC and mutant p53 recruitment facilities, etc.): C. Turco, M. Vahabi, V. Manciocco, J. Meens, A.C. Nichols, on MYC target promoters and parallel downregulation of mutant p53- R. Covello, R. Pellini, G. Spriano, L. Ailles MYC–dependent genes. Therefore, even in absence of striking effects Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Pulito, S. Valsoni, A. Sacconi, M. Vahabi, on cell viability, a very low dose of BYL719 is able to provoke a plethora E.M.C. Mazza, A.C. Nichols, R. Covello, P. Muti, S. Bicciato of molecular changes ranging from decreased phosphorylation of Writing, review, and/or revision of the manuscript: C. Pulito, R. Pellini, signal transducers to detachment of transcription factors from chro- G. Sanguineti, P. Muti, S. Bicciato, S. Strano, G. Fontemaggi, G. Blandino matin. These molecular changes (MYC and mutant p53 transcrip- Administrative, technical, or material support (i.e., reporting or organizing data, tional inactivation) most likely create a context that allows an effective constructing databases): V. Manciocco, C. Karamboulas, G. Fontemaggi action of conventional anticancer agents. These findings support the Study supervision: S. Strano, G. Fontemaggi, G. Blandino Other (performed Western blot and RT-PCR analyses): C. Turco already proposed concept of using low-dose–targeted drugs to sensitize to concomitant combined therapies. Acknowledgments Our data show that depletion of either MYC, mutant p53, or YAP The research leading to these results has received funding from AIRC under IG significantly potentiates the response to increasing doses of BYL719, 2017 - ID. 20613 (project principal investigator: G. Blandino) and from MIUR Epigen further underlining that blockage of MYC/mutant p53 activity is a (13/05/R/42; principal investigator: G. Blandino) and funding by Regina Elena prerequisite for response to BYL719 (Fig. 5I). These results suggest National Cancer Institute, Rome, Italy (to G. Fontemaggi). that therapeutic approaches aimed at blocking MYC activity could fi The costs of publication of this article were defrayed in part by the payment of page strongly contribute to the ef cacy of PI3K inhibitors in HNSCC. A charges. This article must therefore be hereby marked advertisement in accordance fi similar scenario is present in breast cancer, where MYC ampli cation with 18 U.S.C. Section 1734 solely to indicate this fact. is associated with resistance to PI3K/mTOR inhibitors and inhibition of BRD4 antagonizes MYC-dependent resistance to PI3K inhibitors. Received July 29, 2019; revised December 14, 2019; accepted January 15, 2020; Moreover, in breast cancer cell lines derived from mutant PIK3CA- published first January 22, 2020.

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OF16 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

PI3K Inhibitors Curtail MYC-dependent Mutant p53 Gain-of-function in Head and Neck Squamous Cell Carcinoma

Federica Ganci, Claudio Pulito, Sara Valsoni, et al.

Clin Cancer Res Published OnlineFirst January 22, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2485

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