Frequent Mutation of the PI3K Pathway in Head and Neck Cancer Deﬁ Nes Predictive Biomarkers

Published OnlineFirst April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103

RESEARCH BRIEF

Frequent Mutation of the PI3K Pathway in Head and Neck Cancer Deﬁ nes Predictive Biomarkers

Vivian W.Y. Lui 1 , Matthew L. Hedberg 1 , 2, Hua Li 1 , Bhavana S. Vangara 1 , Kelsey Pendleton1 , Yan Zeng 1 , Yiling Lu 6 , Qiuhong Zhang 1 , Yu D u 1, Breean R. Gilbert 1 , Maria Freilino 1 , Sam Sauerwein1 , Noah D. Peyser1 , Dong Xiao1 , Brenda Diergaarde5 , Lin Wang3 , Simion Chiosea 3 , Raja Seethala 3 , Jonas T. Johnson 1 , Seungwon Kim 1, Umamaheswar Duvvuri1 , Robert L. Ferris1 , Marjorie Romkes4 , Tomoko Nukui4 , Patrick Kwok-Shing Ng6 , Levi A. Garraway 7 , Peter S. Hammerman 7 , Gordon B. Mills 6 , and Jennifer R. Grandis 1 , 2

ABSTRACT Genomic fi ndings underscore the heterogeneity of head and neck squamous cell carcinoma (HNSCC ). Identifi cation of mutations that predict therapeutic response would be a major advance. We determined the mutationally altered, targetable mitogenic pathways in a large HNSCC cohort. Analysis of whole-exome sequencing data from 151 tumors revealed the phosphoinositide 3-kinase (PI3K) pathway to be the most frequently mutated oncogenic pathway (30.5%). PI3K pathway–mutated HNSCC tumors harbored a signifi cantly higher rate of mutations in known cancer genes. In a subset of human papillomavirus-positive tumors, PIK3CA or PIK3R1 was the only mutated cancer gene. Strikingly, all tumors with concurrent mutation of multiple PI3K pathway genes were advanced (stage IV), implicating concerted PI3K pathway aberrations in HNSCC progression. Patient- derived tumorgrafts with canonical and noncanonical PIK3CA mutations were sensitive to an mTOR/ PI3K inhibitor (BEZ-235), in contrast to PIK3CA –wild-type tumorgrafts. These results suggest that PI3K pathway mutations may serve as predictive biomarkers for treatment selection.

SIGNIFICANCE: Treatment options for HNSCC are limited, in part, because of an incomplete under- standing of the targetable mutations that “drive” tumor growth. Here, we deﬁ ne a subgroup of HNSCC harboring activating mutations of genes in the PI3K pathway where targeting the pathway shows antitumor efﬁ cacy. These results suggest that PI3K pathway mutation assessment may be used to guide HNSCC therapy. Cancer Discov; 3(7); 761–9. ©2013 AACR. See related commentary by Iglesias-Bartolome et al., p. 722.

INTRODUCTION wide spectrum of unexpected genetic aberrations ( 1, 2 ). This genomic heterogeneity of HNSCC tumors underscores an Head and neck squamous cell carcinoma (HNSCC) is a obstacle to the identifi cation of effective molecular targeting frequently lethal cancer with few effective therapeutic options. agents likely to benefi t the majority of patients with HNSCC. Recent genomic fi ndings in head and neck cancer revealed a To date, there is a translational gap between genomics and

Authors’ Afﬁ liations: Departments of 1 Otolaryngology, 2 Pharmacology & Corresponding Author: Jennifer R. Grandis, University of Pittsburgh Chemical Biology, 3 Pathology, and 4Medicine, University of Pittsburgh School School of Medicine, 200 Lothrop Street, Suite 500, Pittsburgh, of Medicine; 5Department of Epidemiology, University of Pittsburgh, Pitts- PA 15213. Phone: 412-647-5280; Fax: 412-647-2080; E-mail: jgrandis@ burgh, Pennsylvania; 6 Department of Systems Biology, The University of pitt.edu 7 Texas MD Anderson Cancer Center, Houston, Texas; and Dana-Farber Cancer doi: 10.1158/2159-8290.CD-13-0103 Institute, Harvard Medical School, Boston, Massachusetts ©2013 American Association for Cancer Research. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

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RESEARCH BRIEF Lui et al. treatment selection for patients with HNSCC. TP53 mutation primarily by HRAS mutations, which accounted for seven is the single most common mutational event. Yet the loss-of- of 12 pathway mutations identifi ed. PIK3CA is the most function of this tumor suppressor gene has remained challeng- commonly mutated gene in the HNSCC PI3K mutational ing to exploit therapeutically. Mitogenic pathways are crucial profi le (Fig. 1B). These results show that despite the genomic for cancer development and progression. In other malignan- heterogeneity of HNSCC tumors, the PI3K pathway is the cies, mutations of growth pathway genes have been shown most frequently somatically mutated mitogenic pathway in to result in pathway activation, enhanced tumor growth, and HNSCC tumors, found in 30.5% of cases, providing a poten- increased sensitivity to agents targeting the mutated pathway. tial approach to treat a substantial subset of patients. However, the potential of genomics-based therapy selection A detailed analysis of the PI3K pathway mutational events has not been widely investigated in HNSCC. showed that 19 of 151 tumors (12.6%) harbor a PIK3CA muta- We and others recently reported genomic mutational pro- tion (Fig. 1B). This mutation rate is similar to that detected in fi les of more than 100 HNSCC tumors (1, 2). Here, we prior reports of HNSCC tumors [7.4% and 10.8% rate ( 6, 7 )]. analyzed an additional 45 HNSCC tumors by whole-exome Furthermore, we found PIK3CG and PTEN mutations in 4.0% sequencing using the Illumina platform. In an effort to (six of 151) of HNSCC tumors, whereas PIK3R1 (also known identify mutationally altered, targetable mitogenic pathways as p85), PIK3R5 , and PIK3AP1 were mutated in 2.7% tumors in HNSCC, we combined all currently available mutational (four of 151). Other components of the PI3K pathway were data (from whole-exome sequencing) of 151 HNSCC primary mutated in less than 2% of cases ( Fig. 1B ). Major downstream tumors and evaluated the mutational events of genes in effectors of the PI3K pathway, including PDK1 and AKT1 , were three major mitogenic pathways that have been previously not mutated, whereas AKT2 and MTOR were only mutated in implicated in HNSCC pathophysiology, namely the mitogen- 1.3% (two mutations) of HNSCC tumors. Although PIK3CA activated protein kinase (MAPK; ref. 3 ), Janus-activated gene amplifi cation data were not available for the previously kinase (JAK)/signal transducer and activator of transcrip- sequenced tumors, in the newly added cohort, PIK3CA was tion (STAT) (4 ), and the phosphoinositide 3-kinase (PI3K) amplifi ed in 24.4% (11 of 45) of the cases. pathways ( 3 ). These key mitogenic pathways are targetable in Previous reports noted loss of heterozygosity of the tumor human cancers with a variety of agents currently in various suppressor PTEN in HNSCC by PCR-based microsatellite analy- stages of clinical development. sis primarily using D10S215 and/or D10S541 probes in relatively small HNSCC cohorts (e.g., 17 and 39 tumors, respectively; refs. RESULTS 8, 9). Although comprehensive analysis of PTEN copy number was not available in the published genomic HNSCC studies ( 1, 2 ), Nearly One Third of HNSCC Tumors Harbor PI3K PTEN gene copy number change was analyzed using a highly Pathway Mutations sensitive Affymetrix Genome-Wide Human SNP Array 6.0 plat- To date, whole-exome sequencing data of 106 HNSCC form in our 45 newly sequenced HNSCC tumors. Our results tumors are available. Here, we reported the whole-exome showed that PTEN gene copy loss (≥1 copy loss) was found in sequencing data of an additional 45 HNSCC tumors col- only 8.16% of cases (four of 45), indicating that PTEN loss is lected at the University of Pittsburgh (Pittsburgh, PA; Supple- not likely to be the primary mediator of PI3K pathway altera- mentary Table S1). Our results, similar to previous reports, tion in this cohort of 45 HNSCC (unlike other cancers such showed a high degree of intertumor mutation heterogeneity, as glioblastoma, where PTEN loss can be as high as 20%–60%; further confi rming the complexity of HNSCC biology and refs. 10, 11). However, all four tumors with PTEN gene copy the associated challenges in defi ning subsets of patients likely loss expressed relatively low levels of PTEN protein when com- to respond to specifi c targeted therapies. With the aim of pared with HNSCC tumors without PTEN gene copy alteration identifying mutationally altered, targetable mitogenic path- (P < 0.001; Supplementary Fig. S1). ways in HNSCC, we assessed the mutational events of genes comprising three major mitogenic and targetable pathways in PI3K Pathway–Mutated HNSCC Tumors Show an HNSCC: the JAK/STAT, MAPK, and PI3K pathways. Pathway Increased Rate of Cancer Gene Mutations component genes were defi ned as follows: JAK/STAT pathway To determine whether HNSCC tumors harboring mutations ( JAK1, JAK2 , JAK3 , STAT1 , STAT2 , STAT3 , STAT4 , STAT5A , in PI3K pathway genes contained a higher number of muta- STAT5B, STAT6 , SOCS3 , SHP2 , IL6ST , IL6R , and IL6 ), MAPK tions in other cancer-associated genes, we compared the muta- pathway (ERK1 , ERK2 , MEK1 , MEK2 , RAF1 , ARAF , BRAF , tion rates of PI3K pathway–mutated tumors with non-PI3K HRAS, KRAS , NRAS , SHC1 , SHC2 , SHC3 , and GRB2 ), and PI3K pathway–mutated tumors. We found that tumors harboring pathway (PIK3CA , PIK3AP1 , PIK3C2A , PIK3C2B , PIK3C2G , PI3K pathway mutations have higher rates of mutation than PIK3CB, PIK3CD , PIK3CG , PIK3IP1 , PIK3R1/2/3/4/5/6 , non-PI3K–mutated HNSCC tumors. As shown in Fig. 1C , PI3K AKT1/2/3 , MTOR , PTEN , PDK1 , TSC1/2 , RICTOR , and RPTOR ). pathway–mutated HNSCC tumors harbored 2.3 times more Strikingly, almost one third of all HNSCC tumors analyzed nonsynonymous mutations (165.5 ± 24.1 vs. 72.1 ± 6.6 muta- in our cohort (30.5%; 46 of 151 tumors) harbored PI3K path- tions; P < 0.0001) than tumors without PI3K mutations, indi- way mutations, whereas only 9.3% (14 of 151) and 8.0% (12 cating increased genomic instability in tumors harboring PI3K of 151) of tumors contained mutations in the JAK/STAT or pathway mutations. Furthermore, cancer gene fi ltering analy- the MAPK pathways, respectively (Fig. 1A and Supplemen- sis showed that these PI3K pathway–mutated HNSCC tumors tary Table S2A–S2E). In contrast to other smoking-related harbored twice as many cancer gene mutations than those aerodigestive tract cancers where KRAS is frequently mutated without PI3K pathway mutations [ Fig. 1D ; 7.2 ± 0.8 vs. 3.6 ± (5 ), the HNSCC MAPK mutational profi le is characterized 0.3; P < 0.0001: defi ned by the Cancer Gene Census, Catalogue

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Mutation of PI3K Pathway in Head and Neck Cancer RESEARCH BRIEF

A C Non-syn. mutations P PI3K pathway JAK/STAT pathway MAPK pathway 200 < 0.0001

150 30.5% 9.3% 8.0% (46/151) (14/151) (12/151) 100 69.5% (105/151) mutations/tumor 50 90.7% (137/151) 92.0% of non-syn. no. Average (139/151) 0 PI3K-mutated Tumors with tumors no PI3K mutation

PIK3CA PIK3R1/2/3/4/5/6 JAK1 SOCS3 ERK1 HRAS Tumors with D Cancer gene mutations mutated pathway 10 PIK3AP1 AKT1/2/3 JAK2 SHP2 ERK2 KRAS component(s) P < 0.0001 PIK3C2A MTOR JAK3 IL6 MEK1 NRAS MEK2 SHC1 8 PIK3C2B PTEN STAT1 IL6R Tumors with no PIK3C2G PDK1 STAT2 IL6ST RAF1 SHC2 mutations 6 PIK3CB TSC1/2 STAT3 ARAF SHC3 in pathway PIK3CD RICTOR STAT4 BRAF GRB2 component(s) PIK3CG RPTOR STAT5A 4

PIK3IP1 STAT5B mutations/tumor STAT6 2 Average no. of cancer gene no. Average

B 20 20 0 No.of mutations PI3K-mutated Tumors with

18 18 % Tumors with mutation tumors no PI3K mutation % Tumors with mutation E 16 16 12 14 14 10 12 12 10 10 8

8 8 6 6 6 No. of mutations No. 4 4 4

2 2 components In pathway 2 0 0 No. of tumors with multiple mutations of tumors with multiple No. 0 TSC2 PTEN TSC1 MTOR AKT2 PIK3R1 RPTOR PIK3R6 PIK3R5 PIK3CA PIK3CD PIK3IP1 RICTOR PIK3CG PIK3AP1 PIK3C2A PIK3C2B PIK3C2G PI3K pathway MAPK pathway JAK/STAT pathway

Figure 1. Mutations in oncogenic signaling pathways in HNSCC. A, mutation rates of the major mitogenic pathways (the PI3K pathway, the MAPK pathway, and the JAK/STAT pathway) in 151 HNSCC patient tumors determined by whole-exome sequencing. Components of each pathway examined are displayed underneath each pie chart. B, bar graph detailing the number of mutations (dark bars) of each particular component of the PI3K pathway as well as the percentage of HNSCC tumors harboring these mutations (gray bars). C, PI3K pathway–mutated HNSCC tumors have higher rates of nonsynonymous (non-syn.) mutations and (D) cancer gene mutations when compared with HNSCC tumors without any PI3K pathway mutations. Bar graph representing the average number of nonsynonymous mutations per tumor (C) and the average number of cancer gene mutations per tumor (D) in 151 HNSCC tumors. Statis- tical signiﬁ cance was calculated by Fisher’s exact test; P < 0.0001 (N = 151). E, graphical representation of the number of HNSCC tumors with mutation of multiple components of the PI3K, MAPK, and the JAK/STAT pathways, respectively.

of Somatic Mutations in Cancer (COSMIC ) Database; ref. 12 ]. (186.3 ± 27.1; n = 32; data not shown) are signifi cantly higher These results suggest that PI3K pathway mutations in HNSCC than the rates of mutation in tumors from the other anatomic may facilitate the expansion or selection of tumor cells that are locations (78.6 ± 8.3; n = 116; P = 0.0005; data not shown). In already genetically unstable, and thus harbor more genomic addition, the prevalence of PI3K pathway mutations is higher aberrations, including aberrations in known cancer genes. This in laryngeal tumors (53.1% ± 9.0%; n = 32; data not shown) as contention is supported by further analysis showing that DNA compared with tumors from the other anatomic locations damage/repair genes [based on the DNA damage gene list (25.0% ± 4.0%; n = 116; P = 0.0005; data not shown). in the cBio portal database ( 13 ), which includes ATM , ATR, Comutation analysis showed that tumors with PI3K path- CHEK1/2 , BRCA1/2 , FANCF , MLH1 , MSH2 , MDC1 , PARP1 , and way mutation(s) are also associated with mutations of several RAD51] were found to be mutated at a signifi cantly higher known tumor suppressor genes including ARID1A , MLL , and frequency in the PI3K-mutated tumors (average mutation rate MLL3 (P < 0.05; Supplementary Table S3), which contribute to of 37.0%; 17 mutations in 46 tumors) compared with tumors chromatin remodeling and transcriptional regulation in can- without PI3K pathway mutations (average mutation rate of cers ( 14–17 ). Intriguingly, ARID1A has been shown to infl uence 15.2%; 16 mutations in 105 tumors; P = 0.033). signaling through the PI3K pathway, suggesting that ARID1A The association between PI3K pathway mutations and may regulate the PI3K pathway and expand the number of genomic instability is observed in HNSCC derived from all tumors susceptible to targeting the PI3K pathway ( 18 ). Of note, anatomic sites in our cohort (e.g., oral cavity, pharynx, and PI3K pathway–mutated tumors are not associated with TP53 larynx; data not shown). Mutation rates in laryngeal tumors (P = 1.0) or NOTCH1 mutations ( P = 0.34; data not shown).

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Interestingly, we also identifi ed three tumors where tional consequences of these mutations, we stably expressed, PIK3CA or PIK3R1 was the only known mutated cancer gene by retroviral infection, each of the novel mutations and [HN_00361, HN_63027, and HN41PT with the respective PI3K a hotspot mutation (H1047R) in a representative HNSCC mutations of PIK3R1 (453_454insN), PIK3CA (E542K), and cell line that is wild-type (WT) for all PI3K pathway compo- PIK3CA(H1047L)]. Strikingly, all three tumors were associated nents. Overexpression of WT PIK3CA (mimicking PIK3CA with infection by the human papillomavirus (HPV), suggesting gene amplifi cation), and expression of all the engineered that, although the number of HPV-positive HNSCC tumors in PIK3CA mutants individually, resulted in enhanced growth this cohort is relatively small [15 cases, fi ve of which (33%) har- compared with infection by enhanced GFP (EGFP) control. bored PI3K pathway mutations; see Supplementary Table S4], a Furthermore, the canonical hotspot mutation showed sig- subset of HPV-positive HNSCC tumors (20%; three of 15 cases) nifi cantly enhanced growth compared with overexpression of may be driven by PI3K pathway mutation(s) alone, without an WT PIK3CA (P = 0.0001). The novel mutations were found to associated increase in underlying genomic instability. confer moderate growth advantage compared with simulated WT amplifi cation (R115L, P = 0.1174; G363A, P = 0.9637; Only Advanced-Stage HNSCC Tumors Harbor C971R, P = 0.6503; R975S, P = 0.0958). Immunoblotting of Multiple PI3K Pathway Mutations cell lysates revealed that enhanced HNSCC growth, conferred In HNSCC tumors containing PI3K pathway mutations, by the introduction of the novel mutations, was associ- 21.7% (10 of 46) harbored mutations in more than one ated with increased PI3K pathway activation as refl ected by PI3K pathway member gene, indicating that genetic altera- elevated expression of phosphorylated AKT ( Fig. 2B and C ). tions at multiple levels in the PI3K pathway are relatively In the absence of complete functional characterization of common in HNSCC ( Table 1 ). In contrast, HNSCC tumors these novel mutations, these fi ndings should be considered rarely, if ever, harbored multiple mutations in the MAPK supportive but not defi nitive evidence of oncogenic function. pathway (0 tumors), or the JAK/STAT pathway (only one contained both JAK3 and STAT1 mutations; HN_63080; Fig. HNSCC Patient Tumorgrafts with PIK3CA 1E). Strikingly, all of these HNSCC tumors (100%; 10 of 10 Mutations Are Exquisitely Sensitive to BEZ-235 cases) with multiple PI3K pathway mutations were advanced Reports in other cancers suggest that tumors with PI3K (stage IV; Table 1 ). None of these tumors was associated with pathway activation may be more sensitive to PI3K pathway HPV infection. These fi ndings suggest that concerted PI3K inhibitors ( 19 ). To determine the predictive value of PIK3CA pathway aberrations may contribute to HNSCC progression. mutational status in HNSCC, we examined the sensitivity of This fi nding seems to be unique to HNSCC. Examination of HNSCC cell lines that did and did not harbor intrinsic acti- recently published tumor datasets including breast, colon, vating driver PIK3CA (H1047R) hotspot mutations to PI3K and lung squamous cell carcinoma (SCC) showed that only pathway inhibitors. As shown in Fig. 3A , HNSCC cell lines one of 25 breast tumors, one of 27 colon carcinomas, and 0 containing endogenous PIK3CA (H1047R) mutations (CAL-33 of 31 lung SCC tumors that harbored multiple PI3K pathway and Detroit 562; ref. 20 ) showed increased sensitivity to PI3K mutations were stage IV [data not shown; cBio portal (13 )]. pathway inhibition by the mTOR/PI3K inhibitor BEZ-235 Although all 10 tumors with multiple PI3K pathway muta- compared with representative HNSCC cells with WT PIK3CA tions were advanced (stage IV), there is no signifi cant associa- [SCC-9 and PE/CA-PJ34(clone C12)]. Next, mice bearing CAL- tion between advanced disease and individual PI3K pathway 33 xenografts were found to be sensitive to BEZ-235 treatment mutations (data not shown). In addition, mutation rates do in vivo when compared with vehicle control (Fig. 3B). Because of not vary signifi cantly between stage IV and earlier stage (I–III) the lack of HPV-positive HNSCC cell line models that contain HNSCC (data not shown). In the absence of models assessing PIK3CA mutations, we developed an HPV-positive PIK3CA - the specifi c contribution of each mutation to cell growth or mutated HNSCC patient tumorgraft model (E542K; Fig. 3C) survival, it is not possible to determine the precise biologic to determine the sensitivity of HPV-positive PIK3CA -mutated effect(s) of individual mutations in tumors that harbor more HNSCC tumors to PI3K pathway targeting. As shown in than one mutation in the PI3K pathway. Fig. 3D , BEZ-235 treatment (at 25 mg/kg/day by oral gavage) signifi cantly inhibited the growth of an HPV-positive PIK3CA - PIK3CA Canonical and Novel Mutations Increase mutated patient tumorgraft in vivo ( P < 0.0001). Inhibition of Survival and Pathway Activation in HNSCC Tumors tumor growth was accompanied by decreased PI3K signaling PIK3CA is a critical gene in the PI3K signaling pathway. In as evidenced by downregulation of p-AKT(S473) (P = 0.0124), HNSCC tumors, the most common sites of PIK3CA muta- and p-S6(S235/236) (P < 0.0001) in the BEZ-235–treated tions included H1047R/L (eight mutations total), E545K/G tumors (Fig. 3E). Another HNSCC patient-derived tumorgraft (four mutations), and E542K (three mutations; Fig. 2A ), all model (HPV-negative) harboring a PIK3CA mutation (E110K) of which represent previously reported canonical (“hotspot”) was also found to be sensitive to BEZ-235 treatment (Sup- mutation sites. This HNSCC PIK3CA mutation pattern (∼90% plementary Fig. S2). In contrast, patient tumorgrafts with WT of mutations found in the helical/kinase domains) is similar PIK3CA and low baseline p-AKT levels were not sensitive to the to that observed in cervical and breast cancers, as well as lung growth-inhibitory effects of BEZ-235 ( Fig. 3F and Supplemen- SCC, but is distinct from other tumors such as endometrial tary Fig. S3). These results indicate that activating mutations cancer, lung adenocarcinoma, glioblastoma multiforme, and of the PI3K pathway have the potential to serve as biomark- prostate carcinoma (Supplementary Table S5). In addition, we ers for treatment selection in HNSCC. Xenografts developed detected four previously unreported, novel PIK3CA mutations from an HNSCC cell line harboring a PIK3CA mutation (R115L, G363A, C971R, and R975S). To determine the func- (H1047R) were more sensitive to the combination of BEZ-235

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Mutation of PI3K Pathway in Head and Neck Cancer RESEARCH BRIEF

Table 1. HNSCC tumors with multiple mutations in a single mitogenic pathway

Pathway Tumor Annotated genes Amino acid change TNM stage/overall staging PI3K pathway HN_00190 PIK3C2G p.V656L T1N2bM0/stage IV PTEN p.D92E HN_62421PIK3R1 p.D560H T4N0M0/stage IV MTOR p.L2260H HN_62469PIK3CA p.H1047R T2N2bM0/stage IV MTOR p.R1161Q HN_63039PIK3CA p.H1047L T4N2bM0/stage IV PTEN p.R335* HN_22PTPIK3CG p.G491E T4aN2bM0/stage IV PIK3AP1 p.G313R HNPTS_1 PTEN p.R14S T4N0M0/stage IV PIK3IP1 p.A144S PIK3CA p.E545K HNPTS_29PIK3C2G p.S1272L T4N2bM0/stage IV PIK3R5 p.E322K PIK3CA p.E542K HNPTS_38PIK3R5 p.E60* T4N2M0/stage IV PIK3CA p.H1047R HNPTS_42TSC2 p.S1514* T4N2BM0/stage IV PIK3CA p.E545K PIK3CG p.A156V HNPTS_45AKT2 p.Y351C T4N1M0/stage IV PIK3CA p.H1047R JAK/STAT HN_63080JAK3 p.R948C T4aN2bM0/stage IV pathway STAT1 p.Q330K MAPK pathway None

NOTE: The table describes all tumors in our cohort harboring mutations in more than one gene in a deﬁ ned mitogenic pathway by mutation type and pathologic stage. *, nonsense mutation (mutation to a stop codon) Abbreviation: TNM, tumor–node–metastasis.

plus cetuximab [the only U.S. Food and Drug Administra- exome sequencing data (a total of 151 primary HNSCC tion (FDA)-approved molecular targeting agent in HNSCC] tumors) revealed several key ﬁ ndings with important impli- compared with cetuximab alone (Supplemental Fig. S4), sug- cations for HNSCC pathobiology and treatment. The PI3K gesting that targeting PI3K in the setting of PIK3CA -mutant pathway is the most frequently mutated oncogenic path- tumors can enhance treatment responses to cetuximab. way in HNSCC, with the relative number of PI3K-mutated tumors compared with RAS/MAPK and JAK/STAT–mutated tumors being approximately threefold greater. Similar ratios DISCUSSION of PI3K pathway mutations (relative to RAS/MAPK or JAK/ The increasing number of targeted agents for cancer treat- STAT) are seen in SCC of the lung and in cervical cancer, ment results in an unprecedented opportunity for personal- both of which share common risk factors with HNSCC, ized cancer medicine. Selection of therapies based on mutation including tobacco and HPV infection, respectively. In con- status of molecular targets has transformed clinical manage- trast, the RAS/MAPK pathway is more frequently mutated ment and survival of several human malignancies. The EGF than the PI3K pathway in colon and thyroid cancers, and receptor (EGFR) monoclonal antibody cetuximab is the only both the PI3K and RAS/MAPK pathways are mutated at targeted therapy that is FDA-approved to date for HNSCC comparable rates in lung adenocarcinomas ( 13 ). The per- treatment, yet there are no biomarkers that can be assessed centage of HNSCC tumors harboring multiple mutations in the primary tumor to predict clinical responses to this agent. in the PI3K pathway is similar to that observed in breast The recent elucidation of HNSCC genomics offers an opportu- cancers (4.9%; 25 of 507 tumors) and glioblastomas (9.1%; nity to identify genetic subgroups of HNSCC tumors to guide 25 of 276 tumors), higher than in thyroid cancer (0.3%; one treatment decisions. of 323 tumors), and much lower than in most other cancers, In this report, we used a bioinformatic approach to iden- including uterine carcinoma (65.7%; 163 of 248 tumors), tify mutationally altered, targetable mitogenic pathways in melanoma (24.9%; 63 of 253 tumors), and, interestingly, HNSCC. Analyses of all currently available HNSCC whole- lung SCC (17.4%; 31 of 178 tumors), which otherwise shares

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5 Mutation event

PIK3CA G363A (1) E542K (3) E545K/G/(3/1) C971R (1) R975S (1) H1047L/R (3/5) R115L (1)

ABD RBD C2 Helical Kinase a.a.1 108 191 291 330 480 525 696 1068

PE/CA-PJ34 (clone C12) stable cells BCEGFP PIK3CA PIK3CA PIK3CA PIK3CA PIK3CA PIK3CA 2.5 Ctrl WT (G363A) (C971R) (R975S) (R115L) (H1047R)

2.0 *** pAKT(T308) *** *** 0.06 0.62 0.14 0.09 0.39 0.13 1.03 *** *** *** 1.5 pAKT(S473) 0.20 1.43 0.79 0.37 0.74 0.60 1.46 1.0

AKT 2.26 2.25 1.59 1.69 1.51 1.65 1.60 to EGFP relative Growth 0.5

β -Tubulin 0.0 EGFP WT PIK3CA PIK3CA PIK3CA PIK3CA PIK3CA Ctrl PIK3CA (G363A) (C971R) (R975S) (R115L) (H1047R)

Figure 2. PIK3CA mutations in HNSCC tumors. A, schematic of all PIK3CA mutations found in 151 HNSCC tumors by whole-exome sequencing. The amino acid (a.a.) positions of each domain are shown in gray below each domain. The number of mutational events at each site is indicated by a fi lled trian- gle (▲) in the graph above. Blue triangles indicate mutations found in HPV-positive HNSCC tumors. ABD, p85-binding domain; RBD, Ras-binding domain; C2, superfamily; Helical, PIK domain; Kinase, kinase domain of PIK3CA. B, effects of PIK3CA mutations on PI3K signaling in HNSCC cells. WT PIK3CA , hotspot mutant H1047R, and novel mutants R115L, G363A, C971R, and R975S were stably expressed in an HNSCC cell line harboring no endogenous mutations in the PI3K pathway, PE/CA-PJ34 (clone C12) cells, by retroviral infection. Shown here is a representative Western blot analysis with densitometry values normalized to β-tubulin loading controls for each engineered cell line. Increased phosphorylation of AKT at the T308 and/or S473 residue was generally observed in HNSCC cells stably expressing WT or mutant PIK3CA constructs relative to the EGFP-expressing HNSCC cells, indicating enhanced activation of the PI3K signaling pathway. C, effects of PIK3CA mutations on HNSCC cell growth. HNSCC cells stably expressing WT or mutant PIK3CA constructs showed enhanced growth at 72 hours in media with 2% FBS by MTT assay compared with cells expressing EGFP vector control (***, P < 0.0001). PIK3CA (H1047R)-expressing cells further showed enhanced growth when compared with simulated WT PIK3CA amplifi cation (P = 0.001). Data shown here represent growth studies from three sets of independent replicate cell lines (separate infections, n = 18 for each group). common risk factors and similar relative rates of pathway tion, as well as other non-hotspot mutations (such as E110K), mutation with HNSCC ( 13 ). may also identify an HNSCC subgroup potentially responsive Using novel patient-derived tumorgraft models with an to PI3K pathway inhibitors. In particular, our results using oncogenic PIK3CA (E542K) mutation, we showed that these HNSCC patient-derived tumorgrafts suggest that HNSCC tumors are exquisitely sensitive to a PI3K pathway inhibitor tumors with activating PIK3CA mutations may be more (BEZ-235). Similar results were shown in another HNSCC sensitive to a dual PI3K/mTOR inhibitor (such as BEZ-235) patient-derived tumorgraft model with a PIK3CA (E110K) compared with tumors with WT PIK3CA ( Fig. 3E and Sup- mutation, previously reported in breast cancer (21 ). In con- plementary Fig. S3), as indicated by signifi cant inhibition of trast, treatment of human-derived heterotopic tumorgrafts p-S6 expression in the PIK3CA mutated, but not in the WT with WT PIK3CA and low basal expression levels of phospho- tumorgrafts. In fact, a recent report of fi ve HNSCC cases AKT with a PI3K pathway inhibitor was ineffective. These found that mTOR-based targeted therapy may be more effec- fi ndings suggest that (i) PI3K pathway inhibitors can be tive in HNSCC tumors harboring PIK3CA mutation and/or effective for treating HNSCC tumors with PI3K mutations; PTEN loss (23 ). and (ii) mutation-guided treatment responses can be evaluated/ PI3K pathway–mutated HNSCC tumors were found to monitored using patient-derived HNSCC tumorgraft models have a higher rate of nonsynonymous mutations, including in vivo . In fact, early-phase clinical trial results showed that an increased number of defi ned cancer gene mutations, com- patients with solid tumors harboring a PIK3CA hotspot pared with tumors without PI3K pathway mutations. This mutation (H1047R) were found to be responsive to PI3K observation implies that the PI3K pathway–mutated HNSCC pathway inhibitors (22 ). However, the effects of other PIK3CA tumors have an “oncogenic” advantage even with genomic mutations on mediating drug sensitivity in HNSCC preclini- instability, and/or that PI3K-mutated HNSCC tumors cal models or clinical trials have not been previously reported. intrinsically display a “mutator” phenotype rendering them Findings from our study indicate that PIK3CA (E542K) muta- more prone to mutation. The oncogenic advantage of PI3K

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Mutation of PI3K Pathway in Head and Neck Cancer RESEARCH BRIEF

A B 900 CAL-33 xenografts C 90 Veh CAL-33 800 PIK3CA 80 Detroit 562 BEZ-235 (E542K)–mutated tumor PE/CA-PJ34(clone C12) 1,400 1,450 1,500 1,550 700 70 SCC-9 ) 3 600 T C T CCTTG A AAT C A C T G 60 115 120 125 130 500 50 PIK3CA(E542K) GAA>AAA 40 400 **** *** 300 30 ** 20 200

Tumor volume (mm volume Tumor * 10 100

% Growth inhibition (vs. DMSO) inhibition (vs. % Growth 0 0 2 46 8 0 123456789101112 –10 Log [BEZ-235] (pmol/L) Days

PIK3CA PIK3CA (E542K) patient tumorgrafts PIK3CA WT patient tumorgrafts D 7 (E542K) patient tumorgrafts E F 16 VEH BEZ-235 15 6 Veh 1 2 3456 7 1 2 345 14 p-AKT 13 Veh BEZ-235 (S473) 5 12 BEZ-235 11 AKT 10 4 9 P = 0.8121 8 3 p-S6 7 (S235/236) 6 2 5 4 P S6 < 0.0001*** 3 Fractional tumor volume Fractional 1 tumor volume Fractional 2 β-Tubulin 1 0 0 0246810121416182022 0 2 4 6 8 10 12 14 16 18 20 Days Days

Figure 3. PIK3CA mutation enhances sensitivity to PI3K pathway inhibition. A, HNSCC cells containing endogenous PIK3CA mutation (H1047R, CAL-33, and Detroit 562) and cells containing WT PIK3CA [SCC-9, PE/CA-PJ34 (clone C12)] were treated with a PI3K/mTOR inhibitor, BEZ-235, followed by growth measurements at 48 hours (n = 4). Experiments were repeated three times with similar results. DMSO, dimethyl sulfoxide. B, BEZ-235 inhibited growth of CAL-33 xenografts [with endogenous PIK3CA (H1047R) mutation]. CAL-33 cells (0.5 × 10 6 cells) were inoculated into the fl anks of nude mice. Treatment was started when the tumors became palpable 8 days after tumor cell inoculation. BEZ-235 (25 mg/kg/day; n = 9) or vehicle (Veh; n = 10) was given by oral gavage daily. Treatment with BEZ-235 signifi cantly reduced tumor size when compared with vehicle control. *, P = 0.01; **, P < 0.05; ***, P = 0.0002; ****, P < 0.0001. C, Sanger sequencing results showing PIK3CA (E542K) mutation in the HPV-positive HNSCC patient tumors that were implanted into the fl anks of NOD/SCIDγ mice. D, PIK3CA -mutated tumorgrafts are sensitive to BEZ-235 treatment. HNSCC patient tumorgrafts were implanted into the fl anks of NOD/SCIDγ mice. BEZ-235 (25 mg/kg) or vehicle control was given daily by oral gavage. Mice were given vehicle (n = 7) or BEZ-235 (n = 5) when tumors became palpable. Treatment with BEZ-235 signifi cantly reduced the tumor size when compared with vehicle control (P < 0.0001). E, Western blot analyses showing the effects of BEZ-235 (vs. vehicle control) on expression of PI3K signaling components in the PIK3CA -mutated tumorgrafts. Tumors were harvested for Western blotting at the end of the experiment on day 22. Densitometry values of band intensity are shown below each band (normalized to total β-tubulin level). Phospho-AKT (S473) and phospho-S6 (S235/236) levels were signifi cantly reduced upon BEZ-235 treatment when compared with the vehicle-treated tumors (P = 0.0124 and P < 0.0001, respectively). F, HNSCC patient tumorgrafts from a WT PIK3CA tumor expressing low levels of pAKT were implanted into the fl anks of NOD/SCIDγ mice, and treatment was started when tumors became palpable. BEZ-235 (25 mg/kg; n = 6) or vehicle control ( n = 6) was given daily by oral gavage. Treatment with BEZ-235 failed to signifi cantly reduce the tumor size when compared with vehicle control (P = 0.300).

pathway–mutated tumors can be partly explained by PIK3CA that PIK3CA mutations were signifi cantly more common in “driver” mutations’ growth-promoting activity (ref. 24 ; Fig. TP53–WT tumors (26 ). Hence, PI3K pathway mutations may 2C), whereas the “mutator” phenotype of these tumors is sup- mediate tumor progression in the absence of TP53 genetic ported by our fi nding that PI3K pathway–mutated tumors alteration. are associated with higher rates of mutation in DNA dam- Our fi nding that all 10 HNSCC tumors with concurrent age/repair genes, and with ARID1A and MLL3 mutations, mutations of multiple PI3K pathway genes were advanced- which are important tumor suppressor genes ( 15, 16 , 25 ). It stage cancers (stage IV) suggests the potential involvement is possible that both the “oncogenic/growth” advantage and of concurrent alterations of multiple nodes of the PI3K “mutator” phenotype associated with PI3K pathway–mutated pathway in HNSCC progression. This agrees with the recent HNSCC tumors are necessary for HNSCC progression; espe- report that in addition to PIK3CA mutation, other pathway cially because PI3K pathway mutations in these tumors are components such as PIK3R1 and PIK3R2 , when mutated, can not associated with TP53 mutation, a previously recognized also serve to drive cell growth/survival (27 ). Although the tumor suppressor alteration that contributes to HNSCC effects of multiple PI3K pathway mutations on cancer cell carcinogenesis. Although the relationship of PI3K pathway growth or progression have not been previously investigated, mutations and TP53 mutation has not been carefully exam- our results support the possibility that genetic alterations at ined in most cancers, a recent study in bladder cancer showed multiple nodes in this oncogenic pathway, a common feature

JULY 2013CANCER DISCOVERY | 767

RESEARCH BRIEF Lui et al. of many solid tumors, may identify a subgroup of patients Disclosure of Potential Confl icts of Interest with cancer most likely to respond to PI3K pathway inhibi- L.A. Garraway has received a commercial research grant from tors. These cumulative fi ndings identify the PI3K pathway Novartis; has ownership interest (including patents) in Foundation as the most frequently mutated mitogenic pathway in Medicine; and is a consultant/advisory board member of Foundation HNSCC tumors. Prospective identifi cation of patients whose Medicine, Novartis, Millennium, and Boehringer Ingelheim. G.B. tumors harbor these mutations is likely to identify a sub- Mills has received commercial research grants from AstraZeneca, Cel- group of individuals who may benefi t from treatment with gene, CeMines, Exelixis/Sanofi , GlaxoSmithKline, Roche, Wyeth, and PI3K pathway inhibitors. Pfi zer/Puma; has ownership interest (including patents) in Catena Pharmaceuticals, PTV Ventures, and Spindle Top Ventures; and is a consultant/advisory board member of AstraZeneca, Catena Pharma- METHODS ceuticals, Critical Outcome Technologies, Daiichi Pharmaceuticals, Additional methods are detailed in the Supplementary Data. Targeted Molecular, Foundation Medicine, Han AlBio Korea, Komen Foundation, Novartis, Symphogen, and Tau Therapeutics. J.R. Gran- Cell Cultures dis receives research support from Bristol-Myers Squibb, Novartis, and Atellas (previously OSI Pharmaceuticals). The HNSCC cell lines Detroit 562 and SCC-9 were obtained from the American Type Culture Collection, and the PE/CA-PJ34(clone Authors’ Contributions C12) cells were obtained from Sigma-Aldrich. CAL-33 was a kind Conception and design: V.W.Y. Lui, M.L. Hedberg, J.R. Grandis gift from Dr. Gerard Milano (University of Nice, Nice, France). All Development of methodology: V.W.Y. Lui, M.L. Hedberg, Y. Zeng, cell lines were genotypically verifi ed. The HNSCC cell lines were S. Sauerwein, N.D. Peyser, S. Chiosea, G.B. Mills cultured in the respective culture medium containing 10% fetal calf Acquisition of data (provided animals, acquired and managed × serum, 1 penicillin/streptomycin solution (Invitrogen): CAL-33 and patients, provided facilities, etc.): M.L. Hedberg, H. Li, Y. Zeng, Y. Lu, Detroit 562 in Dulbecco’s Modifi ed Eagle Medium (DMEM), SCC-9 M. Freilino, D. Xiao, B. Diergaarde, L. Wang, S. Chiosea, R. Seethala, μ in DMEM/F12 with 0.4 g/mL hydrocortisone, and the PE/CA- J.T. Johnson, S. Kim, U. Duvvuri, M. Romkes, T. Nukui, P.K.-S. Ng, PJ34(clone C12) cells in Iscove’s Modifi ed Dulbecco Minimum Essen- J.R. Grandis tial Medium with 2 mmol/L glutamine (Mediatech, Inc.). All cell lines Analysis and interpretation of data (e.g., statistical analysis, were maintained in a humidifi ed cell incubator at 37°C, 5% CO2 . biostatistics, computational analysis): V.W.Y. Lui, M.L. Hedberg, H. Li, B.S. Vangara, K. Pendleton, Y. Zeng, Y. Lu, Q. Zhang, Y. Du, Cancer Gene Census Comparison and Comutation Analysis B.R. Gilbert, S. Sauerwein, B. Diergaarde, L. Wang, S. Chiosea, R. Seethala, A mutation comparison program was written in Visual Basic for R.L. Ferris, L.A. Garraway, P.S. Hammerman, G.B. Mills Microsoft Excel to compare the existence of HNSCC mutations Writing, review, and/or revision of the manuscript: V.W.Y. Lui, versus a reference list of mutations of interest (in this case, cancer M.L. Hedberg, B.S. Vangara, B. Diergaarde, S. Chiosea, J.T. Johnson, genes). The program allows side-by-side comparison between multi- R.L. Ferris, G.B. Mills, J.R. Grandis ple groups (two or more) to discover common mutational events, as Administrative, technical, or material support (i.e., reporting or organ- well as the number of common events in multiple groups. A cancer izing data, constructing databases): V.W.Y. Lui, M.L. Hedberg, Y. Zeng, gene list was generated in each subgroup of tumors by comparing Q. Zhang, S. Sauerwein, B.S. Vangara, D. Xiao, B. Diergaarde, L. Wang, the Cancer Gene Census list (COSMIC Database) with a nonsynony- S. Chiosea, R. Seethala, U. Duvvuri, G.B. Mills mous mutation gene list of each tumor subgroup (the PI3K-mutated Study Supervision: V.W.Y. Lui, J.R. Grandis tumors, tumors without PI3K mutation, PIK3CA -mutated tumors, and PIK3CA –WT tumors) using this comparison program. This Grant Support analysis allows us to discover the number of cancer genes mutated The study is supported by P50 CA097190 and the American in each subgroup. Cancer Society (to J.R. Grandis), the Patricia L. Knebel Fund of the Pittsburgh Foundation (to V.W.Y. Lui), National Cancer Institute Mutation Validation by Sanger Sequencing K08 1K08CA163677 (to P.S. Hammerman), University of Pittsburgh Sanger sequencing was conducted on patient tumors that were Cancer Institute Cancer Center Support Grant P30CA047904ALL grafted for tumorgraft studies. About 25 to 50 mg of tumor tis- (to M. Romkes and T. Nukui), and funding support for MLH sue (pathologically confi rmed HNSCC with >70% tumor cell con- (5T32DC000066-10 to J.R. Grandis, Research Training in Otolaryn- tent) was used for extraction of DNA by QIAamp DNA Mini Kit gology, University of Pittsburgh). (Qiagen, Inc.). Sequencing primers for HNSCC-associated PIK3CA hotspot mutations were synthesized (Sigma-Aldrich) and used for Received March 8, 2013; revised April 23, 2013; accepted April 24, Sanger sequencing. The primer sequences for E542 site mutation 2013; published OnlineFirst April 25, 2013. are: 5′-cacgagatcctctctctaaaatcactgagcaggag-3′ (forward) and 5′-ctc ctgctcagtgattttagagagaggatctcgtg-3′ (reverse). 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Frequent Mutation of the PI3K Pathway in Head and Neck Cancer Defines Predictive Biomarkers

Vivian W.Y. Lui, Matthew L. Hedberg, Hua Li, et al.

Cancer Discovery 2013;3:761-769. Published OnlineFirst April 25, 2013.

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