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Somatic PIK3CA mutations as a driver of sporadicvenous malformationsPau Castel,1 F. Javier Carmona,1 Joaquim Grego-Bessa,2 Michael F. Berger,1,3 Agnès Viale,4

Kathryn V. Anderson,2 Silvia Bague,5 Maurizio Scaltriti,1,3 Cristina R. Antonescu,3

Eulàlia Baselga,6 José Baselga1,7*

Venous malformations (VM) are vascular malformations characterized by enlarged and distorted blood vessel channels.VM grow over time and cause substantial morbidity because of disfigurement, bleeding, and pain, representing a clinicalchallenge in theabsenceofeffective treatments (Nguyenetal., 2014;Uebelhoeretal., 2012). Somaticmutationsmayactasdrivers of these lesions, as suggestedby the identificationof TEKmutations in a proportionof VM (Limaye et al., 2009).Wereport that activating PIK3CA mutations gives rise to sporadic VM in mice, which closely resemble the histology of thehumandisease. Furthermore, we identifiedmutations in PIK3CA and related genes of the PI3K (phosphatidylinositol3-kinase)/AKT pathway in about 30% of human VM that lack TEK alterations. PIK3CAmutations promote downstreamsignaling and proliferation in endothelial cells and impair normal vasculogenesis in embryonic development. We suc-cessfully treated VM in mouse models using pharmacological inhibitors of PI3Ka administered either systemically ortopically. This study elucidates the etiology of a proportion of VM and proposes a therapeutic approach for this disease.

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INTRODUCTION

Venous malformations (VM) [Online Mendelian Inheritance in Man(OMIM) #600195] are the most frequent form of vascular malformationand are characterized by the presence of a single endothelial layer formingdistended blood vessels of variable diameter that are surrounded by adisorganized mural cell layer containing both smooth muscle cells andpericytes (1, 2). Sporadic VM are particularly evident when they involveskin or mucosae. These lesions grow over time, causing substantial mor-bidity such as disfigurement, bleeding, and pain. They may also affectother tissues including muscles, joints, or the intestine (3). Furthermore,they represent a clinical challenge because of the lack of effective treat-ments, although some patients derive limited benefits from surgery orsclerotherapy (4, 5). Currently, there are no approved pharmacological treat-ments for these lesions that often tend to recur after conventional therapy.

Previous studies in sporadic VM have identified activating somaticmutations in the gene coding for the endothelial-specific tyrosine kinasereceptor TEK (TIE2) in almost half of the cases analyzed (6, 7). Activatingmutations in TEK result in enhanced activation of the downstream PI3K(phosphatidylinositol 3-kinase)/AKT andMAPK (mitogen-activated proteinkinase) pathways (8–11) and have been shown to promote the growth ofhuman umbilical vein endothelial cells (HUVECs) in xenograft assays(11), deregulate the expression of genes involved in vascular develop-ment (12), compromise the endothelial cell (EC) monolayer as a result ofthe loss of fibronectin (13), and decrease the expression of the mural cellattractant PDGFB (platelet-derived growth factor, b polypeptide) (12).However, in a large proportion of VM that do not harbor mutations inTEK, the underlying pathogenesis remains unknown.

The PI3K pathway is a central regulator of cell survival, growth,and metabolism (14), and its deregulation as a result of genetic or epi-

1Human Oncology and Pathogenesis Program (HOPP), Memorial Sloan Kettering CancerCenter, 1275 York Avenue, New York, NY 10065, USA. 2Developmental Biology Program, SloanKettering Institute, New York, NY 10065, USA. 3Department of Pathology, Memorial Sloan Ket-tering Cancer Center, New York, NY 10065, USA. 4Genomics Core Laboratory, Memorial SloanKettering Cancer Center, New York, NY 10065, USA. 5Department of Pathology, Hospital dela Santa Creu i Sant Pau, 167 Sant Antoni M. Claret, Barcelona 08025, Spain. 6Department ofDermatology, Hospital de la Santa Creu i Sant Pau, Barcelona 08025, Spain. 7Department ofMedicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.*Corresponding author. E-mail: baselgaj@mskcc.org

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genetic perturbations is observed in a variety of human tumors andovergrowth syndromes (15, 16). PI3K is regulated by virtually all receptortyrosine kinases, including TIE2, and mediates the phosphorylation ofphosphatidylinositol 4,5-biphosphate, giving rise to the second mes-senger phosphatidylinositol 3,4,5-trisphosphate that triggers down-stream signaling. AKT and mTOR (mammalian target of rapamycin)are two well-studied effectors of the PI3K pathway and are responsiblefor the cellular phenotypes such as cell cycle progression, proliferation,anabolism, and others (14). PI3K also plays a critical role in vascularhomeostasis, and this pathway is essential for angiogenesis and main-tenance of the mature endothelium (17, 18).

Hotspot mutations in PIK3CA, the gene encoding for the catalyticp110a subunit of PI3K, cause pathway hyperactivation resulting incellular transformation and uncontrolled growth in epithelial andmesenchymal cells (15). This aberrant PI3K/AKT signaling is asso-ciated with overgrowth syndromes that are often accompanied by dif-ferent types of vascular malformations including lymphatic, venous,and arteriovenous malformations (19), as well as isolated lymphaticmalformations (20).

We report the existence of gain-of-function mutations in the PI3K/AKT pathway in clinical specimens of isolated sporadic VM and providea genetically engineered mouse model (GEMM) of this disease. Thepresence of PIK3CAmutations in sporadic VM, together with the obser-vation that these mutations are mutually exclusive with TEK mutations,suggests that VM may be defined as a disease state characterized by thepresence of somatic activating mutations in the TIE2-PI3K-AKT axis.Together, these findings could result in the development of efficacioustherapies for this challenging disease and could contribute to a genomi-cally based classification of vascular lesions.

RESULTS

PIK3CASprr2f-Cre mice develop spinal and cutaneous VMAn unexpected finding pointed us toward the critical role of PI3K in thepathogenesis of VM. We were originally interested in studying the roleof PIK3CA, the gene encoding the catalytic p110a subunit of PI3K

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(PI3Ka), in uterine cancer, which is char-acterized by the presence of these muta-tions in approximately half of the cases(21). To investigate the role of PIK3CAoncogenicity in this disease, we took ad-vantage of the previously reported trans-genic mouse strain LoxP-STOP-LoxP(LSL)–PIK3CAH1047R, which allows theexpression of the activating PIK3CA mu-tation H1047R in a tissue-specific mannerusing the Cre-loxP technology upon re-moval of the floxed synthetic transcription-al/translational STOP cassette (22). Theseanimals were crossed with the Sprr2f-Crestrain, shown to drive Cre recombinaseexpression in both luminal and glandularuterine epithelial cells (fig. S1A) (23). Un-expectedly, although PIK3CASprr2f-WT micewere viable and normal, PIK3CASprr2f-Cre

littermates exhibited hindlimb paralysisat an early age (4 to 10 weeks) (Fig. 1A).Because this phenotype was observed inboth males and females, we decided tofurther explore the pathologic events under-lying this phenotype. Histologic exami-nation revealed lesions in the spinal cordresembling human vascular malforma-tions that were not present in wild-typeanimals (Fig. 1B). Specifically, these ab-normalities showed dilated “cavernous”vascular spaces with extensive blood poolsand hemorrhage involving both white andgray matter.

We further examined the spinal lesionsin PIK3CASprr2f-Cre mice injected intra-venously with gold nanoparticles, usingthe in vivo x-ray computed tomographyimaging to confirm the presence of hy-perdense lesions in the spine. These vas-cular lesions were present in animals withboth advanced and milder phenotypes butnot in wild-type littermates (Fig. 1C) andshowed slow blood flow and extravasa-tion, radiological features of vascularmalformations of the spine (24). The ab-normal vascular channels, represented bycavernous spaces and capillary prolifera-tion, were consistent with a diagnosis ofVM according to the current classifica-tion of the International Society for theStudy of Vascular Anomalies (25). Amongthe observed alterations, cutaneous VMwere the most frequent, exhibiting highpenetrance in the PIK3CASprr2f-Cre mice

(about 90%) (Fig. 1D). Microscopically, the skin lesions resembled hu-man VM with positivity for CD31 (Fig. 1E and fig. S1B) and Perl’sPrussian blue staining (Fig. 1F), indicative of EC lining and hemosid-erin deposition, respectively.

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To further characterize the observed lesions, we stained our mouseVM for both glucose transporter 1 (GLUT-1) andWilms tumor 1 (WT-1),which are immunophenotyping markers of infantile hemangioma (IH),a different vascular disease with a distinct natural history that responds

Fig. 1. PIK3CASprr2fCRE mice develop spinal vascular malformations. (A) Hindlimb paresis phenotypeobserved in PIK3CASprr2fCRE mice. WT, wild type. (B) Gross and detailed histology of the spinal cord of

PIK3CASprr2fCRE mice compared to a normal WT spine. Arrows indicate the multiple focal hemorrhages foundin the spinal cord. (C) microCT (micro-computed tomography) scan of a WTmouse compared to PIK3CASprr2fCRE

mice littermates showing an early and an advanced phenotype. Arrows indicate the slow flow and extrav-asation lesions observed in the spinal cord. (D) Hematoxylin and eosin (H&E) histology from normal skin andcutaneous VM. Dashed line delimits the dermis (lower) from the epidermis (upper). Arrows indicate normalblood vessels. (E) CD31 immunohistochemistry (IHC) of the skin VM lesions. Arrows indicate normal bloodvessels. (F) Prussian blue staining. Dashed line delimits the dermis (lower) from the epidermis (upper). Arrowindicates normal blood vessel.

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to the b-blocker propranolol (26–28). Both types of staining were negativein mouse VM samples as compared to positive controls from human IHspecimens and mouse tissue (fig. S1, C and D). Lymphatic malforma-tions, which can histologically resemble VM, can also harbor PIK3R1and PIK3CA mutations (29). In fact, mice with a knockout of PIK3R1,encoding the PI3K regulatory subunits p85a, p55a, and p50a, havedefects in normal lymphangiogenesis and develop lymphatic malfor-mations in the intestines and skin (30).

Thus, to assess whether our VM model might exhibit a substantiallymphatic component, we stained for the lymphatic-specific markerslymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (31)and prospero homeobox 1 (PROX-1) (32). Our IHC staining with theselymphatic markers did not detect any relevant reactivity in the areascomprising the malformation, indicating that these lesions are entirelyVM (fig. S1, E and F).

We hypothesized that the Sprr2f-Cre strain drives the expressionof the Cre recombinase in mature or precursor ECs, in addition to thereported endometrial epithelial cells. Thus, we crossed the LSL-LacZ re-porter strain with the Sprr2f-Cre mouse and performed b-galactosidasestaining in spinal sections. In LacZSprr2f-Cre sections, we detected discretepositive cells resembling ECs that were sparsely distributed within thewhite and gray matter of the spinal cord (fig. S1G). Because of technicalconstraints, we were not able to obtain double staining for LacZ andCD31. However, double immunofluorescence staining against Cre andCD31 on VM from the Sprr2f-Cre mice confirmed the presence of Crerecombinase in the CD31-positive lesions that explain the vascular phe-notype observed in these mice (fig. S1H).

PIK3CA activating mutation affects normal ECsAnalogous to recent studies (11), we transduced primary human skinECs with retrovirus encoding the PIK3CAwild type or H1047R variantsto study the cellular mechanisms by which PIK3CA mutations mightalter EC function. PIK3CAH1047R mutant cells exhibited amplified down-stream PI3K/AKT/mTOR signaling with increased phosphorylation ofAKT at S473 and T308, and the mTOR downstream targets S6 kinase atT389 and ribosomal S6 protein at S235/6 and S240/4 (Fig. 2A).We undertooktube formation assays to assess the ability of these cells to form a normalcapillary network in a three-dimensional matrix, an approach widelyused to assess the normal function of EC (33). PIK3CAH1047R mutantcells formed aberrant EC clusters, as opposed to their wild-type counter-parts, which generated normal vascular tubes in vitro (Fig. 2B). Wefurther confirmed these results using HUVECs infected with PIK3CAwild type and the H1047R mutant, which recapitulated the increasedPI3K/AKT signaling (fig. S2A) and aberrant tube formation in vitro(fig. S2B). Because PI3K regulates cell proliferation (18), we tested theproliferation ratio of our primary cells in vitro using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays and found that the mutantcells exhibited a slightly higher, but reproducible, proliferation rate ascompared with wild-type cells and empty vector controls, which wasreversed upon treatment with a PI3Ka inhibitor (Fig. 2C) in a dose-dependent manner (fig. S2, C and D).

ECs, which are key players in the development of vascular malfor-mations, create a pathological niche that involves the mural cell com-partment, probably in part as a result of aberrant cytokine secretion(12, 34). To test the impact of the PIK3CA activating mutation H1047Ron the secretion of angiogenic factors, we performed antibody arraysin our primary ECs carrying wild-type PIK3CA or the H1047R mu-tation. We found that angiopoietin-2 (ANG2) protein expression was

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decreased in PIK3CAH1047R but not in the PIK3CAWT or control cells(fig. S2E).

Because ANG2 is a cytokine that is regulated by forkhead O (FOXO)transcription factors downstream of the PI3K/AKT pathway, inhibitsblood vessel leakage (35), and plays a role in the pathogenesis of lym-phatic malformations and VM (12, 20, 36), we sought to confirm whetherour primary ECs displayed decreased expression of ANG2. Consistently,PIK3CAH1047R mutant ECs had lower expression of ANGPT2 mRNAand secreted ANG2 protein compared to PIK3CAWT cells and emptyvector controls (fig. S2F). Next, we treated PIK3CAH1047R ECs with thedifferent inhibitors of the PI3K/AKT/mTOR pathway, namely, BYL719(PI3Ka), MK2206 (AKT), and everolimus (mTOR). We observed thatboth PI3Ka and AKT inhibitors were able to rescue the mRNA andprotein expression of ANG2, but the mTOR inhibitor everolimus wasnot (fig. S2F). These results are in agreement with the previous evidencedescribing this secretory phenotype in VM, where PDGFB and ANG2are down-regulated in TEK mutant ECs (12).

Human VM harbor PIK3CA mutationsNext, to ascertain whether the same genetic alterations triggering thephenotype in our mouse and cellular models were also present in thehuman condition, we examined clinical specimens from 32 patients,mainly adults (median age, 36 years), diagnosed with VM (table S1).Patients diagnosed with VM at our institution, a cancer center, mainlypresented with deep-seated and infiltrative masses in the skeletal muscle(53% of the cases were intramuscular, 34% involved skin, and 13% werein other locations) (table S1). Histologically, these lesions displayed amixed pattern of vascular proliferation, including thick-walled mal-formed vessels, cavernous spaces filled with erythrocytes, and capillaryareas (Fig. 2D), and these were radiologically detected by routinemagnetic resonance imaging (MRI) scans (Fig. 2E). We analyzed theseVM by targeted exome sequencing of 341 cancer-related genes usingthe MSK-IMPACT (Memorial Sloan Kettering–Integrated MutationProfiling of Actionable Cancer Targets) assay (37) developed at our in-stitution, yielding a median coverage of 588X. This assay wascomplemented with the next-generation deep sequencing targeted tothe TEK locus (table S2). Deep sequencing detected PIK3CAmutationsin 25% (8 of 32) of cases in previously described (38) hotspots encod-ing for the gain-of-function mutations H1047R (3 of 32) and E542K(3 of 32) with an allele frequency ranging from 3 to 15% (Fig. 2F andfig. S2G). We also identified two other mutations in PIK3CA, coding forC420R and I143V. In addition, we found gain-of-function mutationsin other genes related to the PI3K pathway, such as AKT2, AKT3, andIRS2, resulting in an overall frequency of about 30% of mutations inthe PI3K/AKT pathway (Fig. 2G and table S1). Because it was im-possible to asses copy number variations in our cohort as a result oflow cellularity and considering that amplification of PIK3CA and lossof PTEN are two common alterations of the PI3K pathway in hu-man cancer, we performed fluorescence in situ hybridization anal-ysis in all the VM patient samples sequenced. We did not find anyamplification of PIK3CA or deletion of PTEN in any of the samplesanalyzed (fig. S2H). Furthermore, we found isolated mutations in genesinvolved in the MAPK pathway (GNAQ, NF1,MAP2K1, andMAP3K1)in 13% of the cases (Fig. 2G).

Previously described mutations in the tyrosine kinase receptor TEK(6, 7) were found in 35% of the patients of our cohort, with allele fre-quencies ranging from 4 to 15%. These mutations were mutually ex-clusive with the mutations in the PI3K pathway, with the exception of

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one case (Fig. 2H). Considering that in ECs, the TIE2 receptor, en-coded by TEK, is immediately upstream from PI3K and signals viaPI3K itself (9, 13, 39), VM may be defined as a disease state character-ized by the presence of somatic activating mutations in the TIE2-PI3K-AKT axis.

We were not able to detect any mutation in 5 of the 32 samples ana-lyzed (table S1). This could be a result of low mutation allele frequencythat is below the detection limit of our assay, the presence of a muta-tion that is not represented in our MSK-IMPACT assay, or a non-genetic etiology.

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Ubiquitous expression of PIK3CAH1047R spontaneouslyinduces VM in miceWe hypothesized that the cell of origin giving rise to VM might beparticularly sensitive to oncogenic PIK3CA transformation. Thus, wegenerated PIK3CACAG-CreER mice, in which the PIK3CAH1047R allele isubiquitously expressed upon tamoxifen administration (fig. S3A) (40).Six- to eight-week-old mice fed with tamoxifen rapidly developed cuta-neous VM with 100% penetrance compared to their PIK3CAWT litter-mates (Fig. 3A). Histologic assessment confirmed a combined capillaryand cavernous phenotype exhibiting dilated blood channels filled with

Fig. 2. PIK3CAmutations cause sporadicVM inhumans. (A) Western blot of humanskin ECs infected with empty vector (EV),PIK3CA WT, and H1047R mutation probedwith the indicated antibodies. Cells wereserum-starved overnight before lysis. S6K,S6 kinase; pS6K, phosphorylated S6K; pS6,phosphorylated S6; pAKT, phospshorylatedAKT; HFSEC, human female skin endothe-lial cell. (B) Representative images from thetube formation assays of primary ECs in-fected with EV, PIK3CAWT, and H1047R mu-tation and serum-starved overnight beforeseeding. Pictures were taken 6 hours afterseeding. Note the reticular network formedin the EV and PIK3CA WT cells that fails toform in the PIK3CA H1047R mutant cells.Scale bars, 200 mm. (C) EdU incorporationassay quantification of ECs infected with EV,PIK3CA WT, and H1047R mutation, serum-starved overnight, and labeled with EdUfor 4 hours. Graph shows mean fold change± SEM. n = 3 biological replicates. P valueswere calculated using Student’s t test. (D) H&Estaining highlighting the representativemorphology of one of the human VM pa-tients sequenced in this study. Blood poolsand thick mural cell layer are evident inthe histological sections of these patients.(E) Characteristic MRI scan from an intra-muscular sporadic VM patient sequencedin this study. T1 axial and coronal sectionsare shown. Dashed line delimits the radio-logical extension of the malformation. [R]and [L] indicate right and left, respectively.(F) PI3Ka domains and specific sites foundto be mutated in this study. The p85-bindingdomain is represented in green, the Ras-binding domain in red, the C2 domain inblue, the helical domain in yellow, and thekinase domain in purple. aa, amino acid.(G) Schematic pathway depicting TIE2,PI3K, and MAPK pathway gene componentsfound to be mutated in sporadic VM byMSK-IMPACT in this study. Activating muta-tions are indicated in red and inactivatingmutations in blue. Dark red designates mu-tations found in more than 20% of the pa-

tients. Unknownmutations are shown in gray. (H) Mutual exclusivity of the gene mutations present in the TEK and PIK3CA pathways. The activating mutationsin TEK are indicated in blue, and the activating mutations in PIK3CA are in red. The alterations affecting genes involved in the PI3K or MAPK pathway arerepresented in green.

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erythrocytes (Fig. 3B) and immunoreactivity for CD31 (Fig. 3C) andphosphorylated AKT (S473), a surrogate marker of PI3K activation(Fig. 3D).

Similar to human VM, murine vascular lesions from thePIK3CACAG-CreER mice were negative for GLUT-1, WT-1, LYVE-1, andPROX-1 (fig. S3, B to E) and contained high amounts of hemosiderindeposition (Fig. 3E). Although the skin phenotype was readily evident,additional lesions were observed at necropsy at multiple sites, including

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mesentery, genitourinary tract, kidney, and retina (Fig. 3F), with noapparent difference in the incidence by anatomic site. Histological analy-ses of these lesions revealed large spaces filled with blood and lined byflattened ECs, with a similar immunophenotype, positive for CD31and Prussian blue staining (Fig. 3, G and H).

We confirmed our findings using the UBC-CreER strain, anothertransgenic strain in which the ubiquitin C promoter drives the expressionof a tamoxifen-inducible Cre recombinase in all the cells of the organism

Fig. 3. Ubiquitous expression of PIK3CAH1047R induces VM. (A) Disease-free survival plot of PIK3CACAG-CreER (n= 20) and PIK3CAWT (n= 25) littermates

mouse skin VM lesions. Arrow indicates a normal blood vessel. (F) Histologicalrepresentation of mesenteric vasculature and VM harvested during necropsy

on tamoxifen diet assessed by the appearance of visible cutaneous VM.Dotted line represents the time when tamoxifen diet was administered(day 21). P value was calculated using log-rank test. (B) H&E staining of arepresentative normal blood vessel and a VM lesion developed in the skinof PIK3CACAG-CreER mice. Arrow indicates normal blood vessel. (C) CD31 IHCstaining showingpositivity for the ECs of a normal blood vessel andVM.Notethat erythrocytes exhibit nonspecific staining. Arrow indicates normal bloodvessel. (D) Phosphorylated AKT (S473) IHC. The arrow in the bottom panelindicates lining ECs that show positivity for the staining. Note the negativityfor phosphorylatedAKT in thenormal bloodvessel (top, indicatedbyanarrow).(E) Prussian blue staining of normal blood vessels and the PIK3CACAG-CreER

and detailed view to highlight the blood pools observed in the preparations.Arrows indicate normal blood vessels. (G andH) CD31 (G) andPrussianblue (H)positivity for theVMdescribed in (F). (I) BrdU incorporation (red) inPIK3CACAG-CreER

VMcompared to normal blood vessels. CD31 (green) andDAPI (4′,6-diamidino-2-phenylindole) (blue) show ECs and nuclei, respectively. Note the encasedBrdU-positive nuclei in the CD31-positive lining EC layer. (J) Quantification ofBrdU-positive nuclei in normal blood vessels and VM. P value was calculatedusingStudent’s t test. Graph showsmeans±SD.n=45 fields from fivebiologicalreplicates. (K) Morphological quantification of the maximal blood vessel diam-eter of normal vessels and VM. P value was calculated using Student’s t test.Graph shows means ± SD. n = 45 fields from five biological replicates.

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(fig. S4) (41). Consistently, our resultsindicated that upon ubiquitous expres-sion of the oncogenic PIK3CA trans-gene, the cell of origin for VM mightbe more sensitive to transformationthan other cell types, resulting in thegenesis of VM.

To test whether the formation of VMin our mouse model results, at least inpart, from increased proliferation causedby hyperactive PI3K signaling (42, 43),we measured 5-bromo-2′-deoxyuridine(BrdU) incorporation and Ki-67 stainingin both PIK3CAWT and PIK3CACAG-CreER

littermates. Whereas normal blood ves-sels were negative for BrdU incorpora-tion as a consequence of EC quiescence(44), VM displayed a marked increasein proliferative cells (Fig. 3, I and J). Inagreement with the BrdU data, Ki-67positivity was found in our VM (fig.S5, A and B). We cannot rule out wheth-er the proliferative enhancement ob-served is caused by the direct effect ofthe PIK3CA mutant allele or a result ofautocrine and/or paracrine signalingfrom proliferating ECs, which plays animportant role in the complex develop-ment of human VM (12, 45). At the mor-phological level, quantification of thelumen diameter from normal blood ves-sels and VM revealed a 10-fold increasein the size of these structures in VMsamples (Fig. 3K).

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PI3K inhibitors are effective in the treatment of PIK3CA-induced VMThe presence of oncogenic PIK3CAmutations in human specimens ofVM, together with the observed phenotypes in mice, prompted us toevaluate the full growth potential of these lesions, despite the fact thatthey are considered to be vascular malformations. To this end, we in-jected PIK3CACAG-CreER VM cells into recipient immunocompromisednude mice. These cells formed highly vascularized and proliferativemasses a few weeks after injection, with a histology and appearancehighly resembling that of the original lesions (Fig. 4A). AlthoughVM do not have metastatic potential in patients, our finding that theymay be successfully transplanted and grown in animals suggests thatthese lesions display some tumorigenic behaviors and highlight thefine line between malignant and benign tumors in some cases. Allo-transplanted VM formed new cystic structures that contained bloodand exhibited intravascular coagulopathy, as measured by the increasedconcentration of D-dimers in plasma (Fig. 4B), a useful tool for the dif-ferential diagnosis of VM in human patients (46).

Of clinical relevance, the presence of activating PIK3CA mutationsin VM opens the door for the treatment of this condition with PI3Kainhibitors, currently under clinical development for several cancer indica-tions (47). Treatment of VM with the PI3Ka selective inhibitor BYL719resulted in a marked response as measured by a decrease in VM volume

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(Fig. 4C), reduced proliferation, and increased apoptosis (Fig. 4D and fig.S5C). On the contrary, treatment with the b-adrenergic antagonist pro-pranolol, an active agent against IH (28), did not yield any effect (Fig.4, E and F, and fig. S5D). In support of the role of the aberrant activa-tion of the PI3K/mTOR pathway in VM, treatment with the mTORinhibitor everolimus (48, 49) partially decreased VM size and prolifera-tion in a similar fashion as PI3K inhibition, although it did not increaseapoptosis (Fig. 4, E and F, and fig. S5D).

As mentioned above, VMmay be defined as a disease characterizedby the presence of somatic activating mutations in the TIE2-PI3K-AKT axis (Fig. 2, G and H). Because the TIE2 receptor, encoded byTEK, is immediately upstream from PI3K and signals via PI3K itself,we postulated that treatment with PI3K inhibitors might also be effica-cious in VM harboring TEKmutations. Indeed, treatment of HUVECsstably expressing the TEK mutation L914F with the PI3Ka inhibitorBYL719 decreased the amount of phosphorylated AKT (T308 and S473)(fig. S6A), suggesting that PI3K inhibitors may be efficacious for VMwith either PIK3CA or TEK mutations.

Given that a large number of VM are detected in the skin or su-perficial tissues, together with the substantial toxicity of systemic ad-ministration of PI3K inhibitors in patients (50), we wondered whetherthe topical application of PI3K inhibitors might be of therapeutic in-terest in this context. To this end, we formulated two different cream

Fig. 4. PI3K inhibitors are effective for the treatment of VM. (A) Schematic representation and imagesfrom the allotransplantation assays. (B) Quantification of plasma D-dimers measured in animals with or

without VM. P value was calculated using Student’s t test. (C) VM volume measured in PIK3CACAG-CreER

VM-derived allografts treated with vehicle or PI3Ka inhibitor for 1 week [BYL719, 50 mg kg−1, daily, per os(po)]. P value was calculated using Student’s t test. (D) Quantification of BrdU incorporation and cleavedcaspase-3 (clC3) in CD31-positive cells from (B). P value was calculated using Student’s t test. n = 10. (E) VMvolume measured in PIK3CACAG-CreER VM-derived allografts treated with vehicle, everolimus (10 mg kg−1,daily, po), or propranolol (40 mg kg−1, daily, po) for 1 week. P value was calculated using Student’s t test.(F) Quantification of BrdU incorporation and cleaved caspase-3 in CD31-positive cells from (E). P value wascalculated using Student’s t test. n = 8. (G) VM volume in PIK3CACAG-CreER VM-derived allografts treatedtopically with BYL719 at 1% (w/w) using two different formulations (free and soluble BYL719) for 3 weeks.The pretreatment time point indicates when the treatment was started. All treatments in weeks 1, 2, and 3have a P < 0.001 as compared to the vehicle control VM. P values were calculated using Student’s t test.

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preparations containing the PI3Ka inhibitor BYL719 at 1% (w/w):one preparation with the inhibitor dispersed directly into the creambase and another one with the inhibitor presolubilized in dimethylsulfoxide. Topical administration of the PI3Ka inhibitor using thesetwo different cream formulas achieved a rapid and sustained regres-sion of skin lesions (Fig. 4G and fig. S6B). Together, our results indi-cate that VM have tumorigenic growth potential as evidenced by theirability to engraft in nude mice and that the treatment with PI3K in-hibitors either systemically or locally is a suitable pharmacological ap-proach to control the disease.

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Expression of mutant PIK3CA impairs normal vasculogenesisVM may occur as a result of defects during angiogenesis, a process inwhich PI3Ka is actively involved (17, 18, 51). To explore the biologicalrelevance of PI3K hyperactivation specifically in blood vessels, wecrossed PIK3CAH1047R mice with the Tie2-Cre strain (52), which drivesthe expression of the transgene in ECs (fig. S7A). PIK3CATie2-Cre micewere not viable because of early embryonic lethality [embryonic day10 (E10)] resulting from vascular defects (Fig. 5A). CD31 staining ofcoronal sections revealed dilated blood vessels and vascular anomalies(Fig. 5B, upper panel) present in meningeal vessels, cardinal vein, anddorsal aorta. Moreover, small intersomitic vessels failed to form (Fig. 5B,lower panel), suggesting that deregulated PI3K activity results in lethalimpairment of small vessel formation (17). These malformations werealso evident in whole-mount embryo CD31 staining, with aberrant for-mation of the cephalic and intersomitic vessels (Fig. 5, C and D, upperpanels). Morphology, proliferation, and apoptosis were not altered inPIK3CATie2-Cre embryos’ hearts (fig. S7, B to D), suggesting that theobserved phenotype is caused by a defect specifically affecting bloodvessel formation. Aiming to validate the implication of excessive PI3Ksignaling in aberrant vasculogenesis, as well as to evaluate whether phar-macological inhibition could overcome this effect, we attempted to re-vert the phenotype by treating pregnant mice with the PI3Ka inhibitorBYL719. PIK3CATie2-Cre E9.5 embryos treated with the PI3Ka inhibitorshowed an overall body size comparable to PIK3CAWT littermates, sug-gesting improved vascular function (fig. S7E). CD31 whole-mount stain-ing revealed restored cephalic and intersomitic small blood vesselformation (Fig. 5, C and D, lower panels). Phosphorylated AKT stainingshowed a strong reduction in both PIK3CATie2-Cre and PIK3CAWT

embryos after PI3Ka inhibitor treatment, in contrast with untreatedcontrol embryos (fig. S7F). At the histologic level, treatment reestab-lished meningeal, cardinal vein, and dorsal aorta blood vessel morphol-ogy (Fig. 5E), indicating that aberrant PI3K pathway hyperactivationimpairs normal embryonic angiogenesis in mice.

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DISCUSSION

VM are the most common vascular malformation in humans (5) andare a cause of pain, functional limitations of the affected areas, aesthet-ic disfigurements, and coagulopathies. In severe cases, sclerotherapy orsurgical resection may be considered; however, these procedures ofteninvolve complications such as cutaneous necrosis or extended inflam-matory reactions (53), and depending on the anatomic location and ex-tension may have limited applicability. Moreover, VM are prone torecur and recanalize (54), raising the need for developing more effec-tive therapies.

GEMMs represent reliable tools for investigating the etiology, bio-logy, and progression of human diseases, as well as for exploring newtherapeutic approaches (55, 56). The first somatic molecular altera-tions linked to the development of sporadic VM were the acquisitionof gain-of-function mutations in the gene encoding the EC-specifictyrosine kinase receptor TIE2 (TEK) (6, 8, 57, 58). Ligand-independentreceptor activation drives constitutive activation of the PI3K/AKT andMAPK pathways, resulting in increased proliferation and survival ofEC that could account for increased EC accumulation in VM and ab-normal recruitment of smooth muscle cells. However, only a subgroupof VM harbor defects in TEK, suggesting that other genomic or mo-lecular alterations may be at play in this disease. In this line, it would

Fig. 5. PIK3CAH1047R impairs embryonic angiogenesis. (A) Embryonicphenotype observed in PIK3CAWT (left) and PIK3CATie2-Cre (right) littermates at

E10. For morphological studies, a minimum of four dissections for each geno-type were performed yielding n ≥ 15 embryos. (B) CD31 staining of coronalsections from PIK3CAWT and PIK3CATie2-Cre embryos at E9.5. Arrowheads indi-cate blood vessel enlargement defects in the meningeal vessels (upperpanel), and arrows indicate the defects in the intersomitic vessels (lowerpanel). For CD31 histologic studies, a minimum of four embryos for eachphenotype obtained from two different dissections were used. Scale bars,100 mm. (C) Whole-embryo CD31 staining of PIK3CAWT and PIK3CATie2-Cre em-bryos from mice treated with vehicle or PI3Ka inhibitor (BYL719, 50 mg kg−1,daily, po; 48, 24, and 2 hours before embryos are harvested). For CD31 his-tologic studies, a minimum of four embryos for each condition were used.(D) Detailed view of the cephalic and intersomitic blood vessels from (C).Arrow indicates defects in the meningeal (upper panel) and the intersomiticvessels (lower panel). (E) CD31 coronal sections from embryos in (D). CV,cardinal vein; DA, dorsal aorta. Scale bars, 100 mm.

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be interesting to develop a GEMM expressing activating TEK muta-tions in the endothelial compartment to characterize in detail theirrole in the histopathology and the mechanism of pathogenesis of thisdisease.

Recent studies performing xenograft experiments with HUVECstransduced with the most frequent TEK mutation, L914F, have dem-onstrated its functional relevance in inducing VM (11). Treatment ofmurine xenografts with rapamycin proved the efficacy of inhibitingmTOR activity, which also showed clinical activity in VM patientsin a pilot trial. Intriguingly, three of five patients that responded tomTOR inhibition in this study did not harbor any genetic defect inTEK (11). It is thus tempting to speculate that additional molecular al-terations enhancing the activity of the PI3K/AKT/mTOR pathwaycould be driving the formation of VM in these patients.

We report the generation of a GEMM for VM by inducing theexpression of the gain-of-function PIK3CAH1047R mutant allele in mice.The histopathologic resemblance of the lesions arising in mice to thoseaffecting humans prompted us to evaluate the existence of similar al-terations in clinical specimens. Through targeted exome sequencing,we found that 25% of the evaluated samples bear activating mutationsin PIK3CA or additional genetic defects predicted to stimulate con-stitutive downstream signaling. To reconcile our findings with thosepreviously reported, we confirmed that 35% of the patients harboredmutations in TEK, yet these were mutually exclusive with the presenceof activating PI3K mutations, consistent with a functional redundancy.These results are in agreement with the high prevalence of TEKmuta-tions reported by others, mainly in pediatric patients (6, 7).

TIE2 activated as a result of TEKmutations mainly signals throughPI3K (9, 39, 59), consistent with the unifying hypothesis that aberrantactivation of PI3K signaling by mutations in the upstream receptor orin the pathway itself causes the development of VM. Somatic mutationof PIK3CA is frequently detected in several cancer types, and geneticalterations driving hyperactivation of the PI3K/AKT pathway have alsobeen reported in nonhereditary postzygotic tissue overgrowth syn-dromes that often exhibit mixed capillary, lymphatic, and venousanomalies. Because of the clinical overlap of these overgrowth syn-dromes with PIK3CAmutations, the term PIK3CA-related overgrowthsyndrome has been proposed (19). Patients suffering from congenitallipomatous overgrowth, vascular malformations, epidermal nevi, andskeletal/spinal abnormalities (CLOVES) syndrome harbor somatic mo-saicism for activating PIK3CA mutations resulting in hyperactive PI3K/AKT signaling (60). The presence of somatic mutations in PIK3CA wasalso detected in patients affected by Klippel-Trenaunay-Weber syndrome,an overgrowth condition with features overlapping those of CLOVESsyndrome: isolated lymphatic malformations, fibroadipose hyperpla-sia, and fibroadipose vascular anomalies (61, 62). Additional geneticalterations in PTEN, GNAQ, AKT isoforms, or the regulatory subunitof PI3K PIK3R1, which enhance PI3K/AKT/mTOR andMAPK pathwayactivation, have also been reported in other malformation syndromesincluding Proteus (63), megalencephaly-capillary malformation (MCAP)(64), Sturge-Weber (65), and Bannayan-Riley-Ruvalcaba (66) syn-dromes, underscoring the involvement of aberrant PI3K/AKT/mTORsignaling in developmental disorders. The MCAP syndrome exhibits apredominant brain overgrowth phenotype in which PIK3CA mutationsare also involved. A recent report has described the first mouse modelfor brain overgrowth using GEMM of PIK3CA mutants E545K andH1047R, validating the importance of these mouse models in the studyof PIK3CA-driven syndromes (67). Nevertheless, sporadic and solitary

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VM lesions are a different and much more prevalent entity that is notnecessarily associated with overgrowth.

Despite the fact that most lymphatic malformations carry PIK3CAmutations, our mouse model does not present detectable lymphaticanomalies. This is a limitation of our model that could be explainedby a number of factors including the moment and time of recombina-tion, or possibly different sets of precursor cells giving rise to each mal-formation. Our observations are further supported by those made byCastillo and colleagues, who found that mosaic somatic mutations in-duced in a PIK3CAH1047R mouse model cause VM that are associatedwith neither tissue overgrowth nor lymphatic malformations (45), sug-gesting that the cell of origin giving rise to VM may be more suscep-tible to hyperactive PI3K signaling than other cell lineages and thatadditional genetic or environmental cues are required to reproducethe complex phenotypes observed in overgrowth syndromes. In the fu-ture, it would also be interesting to identify the cell of origin of VM bymeans of lineage-tracing experiments.

Given the ability of our mouse model to recapitulate the patho-genesis of human VM, we determined whether it could be used asa platform for testing pharmacological inhibition using PI3K inhibitorscurrently under clinical development. To put it into context, we alsoevaluated the efficacy of other agents that have been proposed to in-hibit the growth of VM, including rapamycin analogs and propranolol(11, 68). The greatest growth inhibition was achieved when treatingallograft transplants with a PI3Ka inhibitor or the rapamycin analogeverolimus, but no effect was observed with propranolol. Rapamycin im-proves quality of life in patients with VM (11), lymphatic malformations(69), and other vascular syndromes (48), probably because long-termtreatment with rapamycin has the ability to inhibit AKT in ECs; thisobservation is also seen in adipocytes, but not in all epithelial cells (70).

In contrast to the antiproliferative effect of rapamycin analogs, we pro-pose that the proapoptotic effect achieved upon PI3K inhibition is likely toyield improved therapeutic efficacy by diminishing the recurrence of VM.Topical administration of PI3Ka inhibitor further demonstrated the ef-ficacy of this treatment with an approach that would be devoid of thesubstantial side effects associated with systemic drug administration(hyperglycemia, nausea, gastrointestinal effects, and fatigue) (50). Theimpaired vasculogenesis observed in mouse embryos as a consequenceof endothelial-restricted expression of the PIK3CAH1047R allele was alsorescued when pregnant mice were treated with the PI3Ka inhibitor, furthersupporting a functional requirement for controlled PI3K signaling innormal embryonic vasculogenesis as has been demonstrated by others (51).

In summary, our study provides a GEMM recapitulating human VMcaused by hyperactivation of the PI3K/AKT pathway, reveals the impactof PIK3CA somatic mutations in the pathogenesis of VM, and providesa potential therapeutic approach to treat advanced or recurrent lesions inthese patients.

MATERIALS AND METHODS

Materials and methods can be found in the Supplementary Materials.

SUPPLEMENTARY MATERIALS

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Fig. S1. Histologic characterization of PIK3CASprr2f-Cre mice.Fig. S2. PIK3CA mutation in ECs.Fig. S3. Histologic characterization of PIK3CACAG-CreER mice.Fig. S4. Histologic characterization of PIK3CAUBC-CreER mice.Fig. S5. Cell proliferation in mouse VM with or without in vivo treatments.Fig. S6. Treatment of VM with PI3K inhibitors.Fig. S7. Histological assessment of PIK3CATie2-Cre embryos.Table S1. Clinical features and genomic findings in VM patients.Table S2. Bait sequences used for TEK targeted sequencing.

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Acknowledgments: We thank P. Zanzonico and V. Longo from the Animal Imaging Core; M. Turkekul,V. Boyko, and D. Yarilin from the Molecular Cytology Core; and G. Nanjangud from the MolecularCytogenetics Core. We also thank M. Lepherd from the Laboratory of Comparative Pathology. Wethank the MSKCC (Memorial Sloan Kettering Cancer Center) Center for Molecular Oncology, theIntegrated Genomics Operation, K. Huberman, and N. Socci for the assistance with DNA sequencingand data analysis. We thank J. Bischoff and M. Valiente for providing cell lines and G. Minuesa for helpwith flow cytometry. Funding: Supported by Geoffrey Beene Cancer Center. The Molecular CytologyCore is supported by grant P30 CA008748J. The Integrated Genomics Operation is supported by grantP30 CA008748. J.G.-B. is supported by the Secretary for Universities and Research of the Governmentof Catalonia and the COFUND program of the Marie Curie Actions of the 7th R&D Framework Pro-gramme of the European Union. F.J.C. holds a fellowship from the Terri Brodeur Breast Cancer Foun-dation. K.V.A. was funded by NIH R01 NS 044385. Author contributions: P.C. and J.B. conceived theproject. P.C., M.S., K.V.A., E.B., and J.B. supervised the research. P.C., F.J.C., and J.G.-B. performed thein vitro and in vivo experiments. C.R.A. reviewed the human and mice cases. C.R.A., S.B., and E.B.provided patients’ samples. M.F.B. and A.V. supervised and analyzed the sequencing. P.C. preparedthe figures. P.C., F.J.C., E.B., and J.B. wrote the manuscript. Competing interests: J.B. is on the Board ofDirectors for Aura Biosciences and Infinity Pharmaceuticals. He has performed consulting and/or ad-visory work for PMV Pharma, ApoGen, Juno, Seragon, Aragon, Roche, Lilly, Novartis, AstraZeneca,Verastem, and Northern Biologics. He has stock or other ownership interests in PMV Pharma, Juno,Aura, Infinity, and ApoGen. He has previously received honoraria or travel expenses from Roche,Novartis, and Lilly. M.S. has consulted for Menarini Ricerche and for Vincen Tech. P.C., E.B., and J.B.have applied for a patent “Use of phosphoinositide 3-kinase inhibitors for treatment of vascular mal-formations” (filing no. US20180117055A1). Data and materials availability: MSK-IMPACT raw se-quencing data are available upon request. For the reagents, please contact the corresponding author.

Submitted 29 October 2015Accepted 2 March 2016Published 30 March 201610.1126/scitranslmed.aaf1164

Citation: P. Castel, F. J. Carmona, J. Grego-Bessa, M. F. Berger, A. Viale, K. V. Anderson, S. Bague,M. Scaltriti, C. R. Antonescu, E. Baselga, J. Baselga, Somatic PIK3CA mutations as a driver ofsporadic venous malformations. Sci. Transl. Med. 8, 332ra42 (2016).

eTranslationalMedicine.org 30 March 2016 Vol 8 Issue 332 332ra42 10

mutations as a driver of sporadic venous malformationsPIK3CASomatic

Bague, Maurizio Scaltriti, Cristina R. Antonescu, Eulàlia Baselga and José BaselgaPau Castel, F. Javier Carmona, Joaquim Grego-Bessa, Michael F. Berger, Agnès Viale, Kathryn V. Anderson, Silvia

DOI: 10.1126/scitranslmed.aaf1164, 332ra42332ra42.8Sci Transl Med

PI3K activity, paving the way for future clinical trials.studied these in numerous mouse models, and demonstrated that they can be effectively treated by inhibitingidentified mutations in the phosphatidylinositol 3-kinase (PI3K) pathway as a cause of venous malformations,

. have bothet al. and Castillo et althese malformations are invasive and not particularly effective. Now, Castel forwhich can cause a variety of complications such as pain, disfigurement, and bleeding. The available treatments

Venous malformations are a type of congenital vascular anomalies composed of dilated blood vessels,PI3K-ing the best treatment

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