17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Article The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib Piotr Kobialka1,† , Helena Sabata1,† , Odena Vilalta1, Leonor Gouveia1,2 , Ana Angulo-Urarte1, Laia Muixı1, Jasmina Zanoncello1, Oscar Mun ~oz-Aznar3, Nagore G Olaciregui3, Lucia Fanlo4, Anna Esteve-Codina5,6, Cinzia Lavarino3, Biola M Javierre4, Veronica Celis7, Carlota Rovira8, Susana Lopez-Fern andez9, Eul alia Baselga10 , Jaume Mora3,7 , Sandra D Castillo1,*,‡ & Mariona Graupera1,11,**,‡ Abstract DOI 10.15252/emmm.202115619 | Received 4 January 2022 | Revised 19 May 2022 | Accepted 20 May 2022 | Published online 13 June 2022 Low-flow vascular malformations are congenital overgrowths EMBO Mol Med (2022) 14: e15619 composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result Introduction in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation Vascular malformations are a congenital group of diseases composed into clinical trials of specific molecular therapies for these of abnormal vascular channels that can occur anywhere in the body diseases. Here, we demonstrate that the Pik3caH1047R activating and often have a major impact on the quality of life of patients. They mutation in endothelial cells triggers a transcriptome rewiring tend to be painful and disfiguring and many leading to bleeding, that leads to enhanced cell proliferation. We describe a new repro- recurrent infections, thrombosis, organ dysfunction and even death ducible preclinical in vivo model of PI3K-driven vascular malforma- (Van Damme et al, 2020). Vascular malformations can be classified tions using the postnatal mouse retina. We show that active as low-flow (venous, lymphatic and capillary) and fast-flow (arteri- angiogenesis is required for the pathogenesis of vascular malfor- ovenous) lesions, the former being the most frequent subtype. These mations caused by activating Pik3ca mutations. Using this model, vascular lesions appear during embryonic development, when we demonstrate that the AKT inhibitor miransertib both prevents vascular growth factors are produced at high levels, and expand and induces the regression of PI3K-driven vascular malformations. proportionally with the physiological growth of the patient (Pang We confirmed the efficacy of miransertib in isolated human et al, 2020). Of note, vascular malformations may occur in isolation endothelial cells with genotypes spanning most of human low- or as part of a syndrome (Canaud et al, 2021). At present, there is no flow vascular malformations. molecularly targeted therapy in the current management for these diseases. Instead, standard of care includes a broad spectra of mostly Keywords AKT; angiogenesis; endothelial cell; PI3K; vascular malformations inefficient and invasive techniques including bandage compression, Subject Categories Cardiovascular System; Vascular Biology & Angiogenesis surgical excision and sclerosing approaches (Castillo et al, 2019). 1 Endothelial Pathobiology and Microenvironment, Josep Carreras Leukaemia Research Institute, Barcelona, Spain 2 Department of Immunology, Genetics, and Pathology, Uppsala University, Uppsala, Sweden 3 Developmental Tumor Biology Laboratory, Institut de Recerca Sant Joan de Deu, Barcelona, Spain 4 3D Chromatin Organization, Josep Carreras Leukaemia Research Institute, Barcelona, Spain 5 CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain 6 Universitat Pompeu Fabra (UPF), Barcelona, Spain 7 Pediatric Cancer Center Barcelona, Hospital Sant Joan de Deu Barcelona, Barcelona, Spain 8 Department of Pathology, Hospital Sant Joan de Deu Barcelona, Barcelona, Spain 9 Department of Plastic Surgery, Hospital de la Santa Creu i de Sant Pau, Barcelona, Spain 10 Department of Dermatology, Hospital Sant Joan de Deu, Barcelona, Spain 11 CIBERONC, Instituto de Salud Carlos III, Madrid, Spain *Corresponding author. Tel: +34 93 557 28 00 (ext. 4441); E-mail: scastillo@carrerasresearch.org **Corresponding author. Tel: +34 93 557 28 00 (ext. 4440); E-mail: mgraupera@carrerasresearch.org † These authors contributed equally to this work as first authors ‡ These authors contributed equally to this work as last authors ª 2022 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine 14: e15619 | 2022 1 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al Overactivation of phosphoinositide 3-kinase (PI3K) signalling is factors; thus, active angiogenesis is required for the formation of a hallmark of most low-flow vascular malformations (Castillo et al, Pik3ca-driven vascular malformations. We report a unique in vivo 2016b, 2019; Canaud et al, 2021; M€akinen et al, 2021). Sporadic model of PI3K-driven vascular malformations that allows a more venous malformations, the most common type of vascular malfor- accurate understanding of the dynamic pathogenesis of these mations, are caused by gain-of-function mutations either in the diseases and thus a more efficient assessment of therapeutic strate- endothelial tyrosine kinase receptor TEK/TIE2 or in the PI3K cata- gies. In addition, we show how analysing patient-derived ECs from lytic subunit alpha PIK3CA (Limaye et al, 2015; Castel et al, 2016; vascular malformations allows for personalized medicine testing in Castillo et al, 2016a); with TEK and PIK3CA mutations largely being these diseases. Using a spectra of preclinical models, we demon- mutually exclusive. Also, about 85% of lymphatic malformations strate the efficacy of the AKT inhibitor miransertib for PI3K-driven are caused by activating PIK3CA mutations (Boscolo et al, 2015; vascular malformations both for prevention and treatment Luks et al, 2015; M€akinen et al, 2021). In addition, venous, strategies. lymphatic and/or capillary malformations are frequently present in overgrowth syndromes caused by PIK3CA mutations, the so-called PROS (PIK3CA-related overgrowth spectrum) (Keppler-Noreuil et al, Results 2015). PIK3CA mutations in vascular malformations are similar to those found in epithelial cancer, being the missense mutations in PI3K overactivation in ECs leads to a molecular signature for the helical (PIK3CAE542K and PIK3CAE545K) and the kinase (PIK3CAH1047R) enhanced cell cycle progression domains the most prevalent (Samuels et al, 2004). PIK3CA encodes the p110a lipid kinase protein, which is a major To understand the pathogenesis of PI3K-driven blood vascular signalling component downstream of growth factor receptor tyro- malformations, we analysed early changes in the transcriptome of sine kinases (RTKs) (Bilanges et al, 2019; Kobialka & Graupera, ECs upon Pik3caH1047R expression. For this, we isolated lung ECs 2019). Specifically in endothelial cells (ECs), p110a is activated by from mice, which express the Pik3caH1047R mutation under the the vascular endothelial growth factor receptors (VEGF-R) and TIE endogenous promoter (Kinross et al, 2012) specifically in ECs tyrosine kinase receptors (Graupera & Potente, 2013). Hence, it is (Pdgfb-iCreER) upon 4-hydroxytamoxifen (4-OHT) treatment (Clax- not surprising that p110a is the sole class I PI3K isoform required ton et al, 2008). First, we aim to define a time point to assess for an for blood and lymphatic vascular development (Graupera et al, early impact on the transcriptome driven by Pik3caH1047R. To this 2008; Stanczuk et al, 2015). p110a catalyses the phosphorylation of end, we first analysed the expression of Ang2 and Pdgfb, two bona- the lipid second messenger phosphatidylinositol-4,5-triphosphate fide transcriptomic readouts of PI3K activation in ECs (Potente et al, (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell 2005; Castel et al, 2016; Castillo et al, 2016a) at 6, 24, 48, and 72 h membrane (Bilanges et al, 2019). PIP3, in turn, contributes to the after induction of Pik3caH1047R expression (Appendix Fig S1A). We recruitment and activation of a wide range of downstream targets, found that analysis at 24 h post-4-OHT was sufficient to induce the serine-threonine protein kinase AKT (also known as protein PI3K-related transcriptional changes (Appendix Fig S1A). We con- kinase B, PKB) being critical in this cascade. The PI3K-AKT signal- firmed that PI3K signalling was overactivated at 24 h after 4-OHT ling pathway regulates many cellular processes that are key for EC by showing enhanced phospho (p)-Akt compared with non-induced biology, including cell proliferation, survival and motility (Manning cells (Fig 1A). Next, we performed transcriptomic analysis by RNA & Toker, 2017; Bilanges et al, 2019). There are three isoforms of sequencing of ECs at 24 h after 4-OHT and their vehicle-treated AKT (AKT1, 2 and 3), showing high homology but being not redun- counterparts. In total, 14,226 genes were analysed and 418 genes dant. AKT1 and AKT2 are broadly expressed, with AKT1 being the resulted differentially expressed (Padj < 0.05) (Fig 1B). Pathway predominant isoform in ECs (Ackah et al, 2005; Chen et al, 2005). enrichment analysis determined that the most affected biological The discovery that most low-flow lesions are caused by overacti- processes upon Pik3caH1047R expression in ECs included cell divi- vation of PI3K signalling has catalysed the repurpose of PI3K path- sion, mitosis and cell cycle progression (Fig 1C). In line with this, way inhibitors for the treatment of these diseases. Given that gene set enrichment analysis (GSEA) confirmed that the top pathological mutant ECs primarily depend on AKT signalling, inhibi- enriched hallmarks upon Pik3caH1047R expression include E2F and tion of AKT is a promising strategy for low-flow vascular malforma- MYC targets and G2 M checkpoints (Appendix Table S1, Appendix tions. Among AKT inhibitors, miransertib (ARQ 092, MK-7075) is a Fig S1B). potent and selective allosteric AKT inhibitor showing higher speci- To assess the biological relevance of the proliferative molecular ficity for the AKT1 isoform. Miransertib suppresses AKT activity by programme triggered by Pik3caH1047R expression, we next studied inhibiting membrane-bound active form of AKT and preventing acti- the impact of PI3K overactivation in endothelial cell cycle upon vation of the inactive form of AKT (Yu et al, 2015). This inhibitor both serum starvation and stimulation. Wild-type ECs showed PI3K/ has shown efficacy in preclinical studies for PI3K-driven tumours AKT signalling activation 30 min after serum stimulation, while (Yu et al, 2015, 2017) and Proteus syndrome (Lindhurst et al, Pik3caH1047R ECs showed stronger activation in both starved and 2015), which is caused by a somatic AKT gain-of-function mutation. serum-stimulated culture conditions (Fig 1D). Cell cycle analysis Also, compassionate use of this inhibitor has shown therapeutic effi- revealed that, even though PI3K/AKT signalling was similarly over- cacy in patients with Proteus and PROS (Leoni et al, 2019; Biesecker activated in both starved and serum-stimulated conditions of et al, 2020; Forde et al, 2021). Pik3caH1047R ECs, solely the presence of growth factors resulted in a Here, we show that Pik3caH1047R expression in ECs triggers a significant overrepresentation of ECs in the S and G2/M phases transcriptional programme favouring cell cycle progression. Cell (Fig 1E and F). The enhanced proliferative state upon growth factors cycle progression in Pik3caH1047R ECs is responsive to growth correlated with pS6 levels (Fig 1D), thereby indicating that the 2 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine A B C D E F Figure 1. PI3K pathway activation in ECs leads to enhanced cell cycle progression. A Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24 h after induction of Pik3caH1047R expression. B Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (Padj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24 h (grey, unchanged; orange, downregulated; purple, upregulated). n = 4 biological replicates per genotype. C Pathway enrichment analysis showing most represented biological functions altered upon Pik3caH1047R expression in ECs. D Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2 h starvation () and 30 min stimulation with 20% foetal bovine serum (FBS) (+). E, F (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum () or 20% serum (+) for 24 h. (F) Percentage of proliferative ECs (in S and G2 M phases) from Fig 1E. Error bars are SEM. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann–Whitney test. **P < 0.01 and ****P < 0.0001 were considered statistically significant. Source data are available online for this figure. AKT/mTOR axis is relevant for the pathological proliferation of different dependency on mitogenic signals (Ehling et al, 2013) to Pik3caH1047R ECs. induce the endogenous expression of the Pik3caH1047R mutation in ECs in vivo. 4-OHT was administered at postnatal day (P)1, P7 or Active angiogenesis is required for the formation of PI3K-driven P15, and retinas were isolated 1 week later (Fig 2A). By analysing vascular malformations the extent of vascular overgrowth, we found that both early and intermediate stages showed full penetrance, with all retinas anal- Our data suggest that Pik3caH1047R-driven EC proliferation requires ysed developing vascular malformations (Fig 2B and C). However, the presence of growth factors to induce a pathological response. the degree of enhanced vascularity was more prominent and gener- Given that vascular malformations appear during embryonic devel- alized in the early developmental stage than in the intermediate opment when endothelial mitogenic signals are produced at high period (Fig 2B–D). In contrast, only one-third of the retinas showed concentration, we hypothesized that the onset of these lesions malformed vascular areas when Pik3caH1047R mutation was induced depends on angiogenesis. To test this idea, we took advantage of at P15 (Fig 2B–D). By taking advantage of ROSAmTmG; Pdgfb-iCreER the mouse retinal vasculature, which allows the study of the dif- (later referred to as EC-mTmG) reporter mice (Muzumdar et al, ferent phases of vascular development. We chose three develop- 2007), which expresses cell membrane-localized EGFP following mental stages (early, intermediate and late) in which ECs exhibit Cre recombination, we demonstrated that the Pdgfb-iCreER line is ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 3 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al A B C D E F Figure 2. Pathogenesis of Pik3ca-driven vascular malformations depends on active angiogenesis. A Experimental setup scheme showing analysed angiogenic stages in the retina models. B Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 lm. C Pie charts showing the incidence of vascular lesions at different angiogenic stages. Lesions were categorized according to their expanse (generalized or focal). Quantification was performed per retina petal. Numbers below show the presence of any type of lesions per retina petal. D Quantification of IB4-positive areas per retina. Error bars are SEM. n ≥ 3 retinas per genotype. E Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 lm. F Quantification of GFP/IB4 ratio per retina. Error bars are SEM. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann–Whitney test. *P < 0.05 was considered statistically significant. Source data are available online for this figure. 4 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine similarly active at all time points tested (Fig 2E and F). Thus, pene- AKT inhibitor miransertib prevents the formation of PI3K-driven trance and severity of vascular malformations in our model were vascular malformations independent of the number of ECs recombined at the different stages. These results indicated that the expression of mutant Pik3ca Targeting AKT, the main player of PI3K-driven signalling in ECs has in ECs is not sufficient for the acquisition of a malformed vascular not yet been assessed in vivo in vascular malformations. First, we phenotype and that active angiogenesis is required for PI3K-driven assessed PI3K/AKT signalling in vitro in Pik3caH1047R ECs upon AKT vascular malformations to occur. inhibition with miransertib. P-Akt and p-Pras40 levels were reduced in a dose-response manner (Appendix Fig S2A). In line with A new preclinical model of PI3K-driven vascular malformations this, 24 h after miransertib treatment (2 µM), expression levels of Ang2 and specific cell cycle regulators found deregulated upon Our data showed that expression of Pik3caH1047R in ECs at an early Pik3caH1047R expression were normalized (Appendix Fig S2B–D). stage of postnatal angiogenesis leads to generalized vascular malfor- Next, we took advantage of our unique in vivo model to examine mations. However, sporadic vascular malformations appear as a the impact of miransertib in the dynamic pathophysiology of PI3K- mosaic disease, where malformed vascular lesions tend to be driven vascular malformations. Previous preclinical studies of isolated and focal. Hence, to better reproduce the aetiology of miransertib using tumour xenografts showed that the minimum human disease, we studied the impact of a decreasing range dose that has an impact on tumour volume is 75 mg/kg (Yu et al, of 4-OHT doses during the early developmental stage in EC- 2017); thus, we first evaluated this dose to assess miransertib for Pik3caWT/H1047R mouse retinas (Fig 3A). To validate mosaicism and preventing the formation of Pik3ca-driven vascular malformations. identify targeted ECs in our dosing strategy, we treated For this, we treated P1 EC-Pik3caH1047R mice with 4-OHT and we EC-mTmG P1 mice in parallel with the same doses (Fig 3B and C). dosed these mice with either 75 mg/kg of miransertib or vehicle at This approach allowed us to identify the lowest 4-OHT dose P1 and P2, followed by the analysis of P6 retinas (Fig 5A). Miran- (0.125 mg/kg) that led to distinguishable vascular malformations sertib prevented the formation of vascular malformations, assessed with a total vascular density significantly increased compared with by the vascular area and total EC number (Fig 5B, D and E) by wild-type counterparts (Fig 3A and D). We noticed that low dose of inhibiting Pik3caH1047R-driven EC hyperproliferation assessed by 4-OHT targeted all vessel subtypes shown by mTmG-positive cells in EdU incorporation (Fig 5B and F). Also, the treatment partially arteries, veins and capillaries (Fig 3B). Instead, expression of prevented the loss of NG2-positive mural cell coverage (Fig EV3). Pik3caH1047R upon low dose of 4-OHT resulted in the formation of By pS6 immunostaining, we confirmed that miransertib treatment vascular malformations only in veins and capillaries (Fig 4A). inhibited PI3K signalling in the vasculature (Fig 5C and G). Of note, Pik3caH1047R-vascular malformations in postnatal retinas showed wild-type control retinas treated with miransertib showed a slight enriched phospho (p)-S6 (Ser235/236) levels, a read-out for PI3K/ impact on vasculature density and EC proliferation (Fig 5B, D and F) AKT/mTORC1 signalling, compared with the surrounding normal confirming a key role of AKT in angiogenesis and vascular home- vasculature and wild-type retinas (Fig 4B and D). These lesions also ostasis (Ackah et al, 2005; Chen et al, 2005; Kerr et al, 2016). exhibited loss of pericyte coverage, a hallmark of vascular malfor- Molecularly targeted treatment of vascular malformations may mations, in contrast to non-malformed vasculature in the same require long-term, even chronic, therapeutic approaches in paedi- retina and the control (Fig EV1). A 2-h snapshot of the number atric patients. This points to the importance of identifying the mini- of ECs in S-phase unveiled that Pik3caH1047R mutant retinas exhib- mal effective dose of any candidate treatment. Thus, we next asked ited higher number of proliferative ECs (Fig 4C and E). Also, whether reducing the dose of miransertib would provide similar effi- Pik3caH1047R mutant retinas also showed that the malformed vascu- cacy and reduce knock-on effects on normal vasculature in our lature was composed of more ECs, which may result in no dif- preclinical model. To test this, we reduced miransertib dose by half ference in the mitotic index between genotypes at this time point (35 mg/kg) and evaluated its efficacy on preventing Pik3ca-driven (Fig 4C and F). To further understand whether Pik3ca-related vascu- vascular malformations (Fig 6A). We observed that the therapeutic lar malformations emerge as a consequence of enhanced EC prolifer- efficacy of AKT inhibition by miransertib was maintained at the ation, we postulated that the analysis of ECs in S-phase had to be lower dose, with decreased EC proliferation leading to reduced done before the accumulation of too many ECs. To test this, we first number of total ECs and vascular density in EC-Pik3caH1047R induced the formation of vascular malformations by treating EC- miransertib-treated retinas compared with vehicle-treated counter- Pik3caH1047R mice with 4-OHT at P1 and retinas were analysed at P4 parts (Fig 6B and D–F). This dose of miransertib efficiently inacti- (Fig EV2A). At this time point, Pik3caH1047R retinas showed vascular vated PI3K signalling as shown by reduced pS6 levels (Fig 6C and G). malformations (Fig EV2B and F), a modest increase in the number Importantly, this dose had no impact on non-mutant vasculature of ECs (Fig EV2C and G), as well as a substantial increase in EdU- (Fig 6B–G). Altogether, our data demonstrate that miransertib effi- positive ECs (Fig EV2C and H). This allowed to ascertain that during ciently inhibits the formation of PI3K-driven vascular malformations the early onset of vascular malformations, Pik3caH1047R leads to an and that lower doses than reported for oncological purposes of increased mitotic index in ECs (Fig EV2I). In addition, P4 mutant miransertib appear equally effective for preventive strategies. retinas exhibited other hallmarks of PI3K-driven vascular malforma- tions such as enhanced pS6 levels (Fig EV2D and J) and impaired Treatment with miransertib induces the regression of pericyte coverage (Fig EV2E and K). Collectively, these data PI3K-driven vascular malformations show that EC-specific mosaic induction of endogenous Pik3caH1047R expression during active vascular growth faithfully recapitulates Since vascular malformations are congenital diseases, therapeutic human low-flow vascular malformations. interventions should aim for inducing the regression of mass ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 5 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al A B C D Figure 3. Modelling PI3K-vascular malformations in murine retinas by mosaic expression of Pik3caH1047R in ECs. A, B Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 lm. C Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are SEM. n ≥ 3 retinas per genotype. D Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are SEM. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann–Whitney test. *P < 0.05 was considered statistically significant. Source data are available online for this figure. 6 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine 7 of 19 EMBO Molecular Medicine 14: e15619 | 2022 G F E Piotr Kobialka et al ª 2022 The Authors Figure 4. A B C D 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al ◀ Figure 4. Pik3ca-vascular malformations in murine retinas reproduce hallmarks of human disease. A Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 lm (left panel) and 30 lm (right, high magnification panels). B Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/ 236) and blood vessels (IB4). Scale bars: 150 lm (left panels) and 30 lm (right panels, high magnification). C Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 lm (left panels) and 30 lm (right panels, high magnification). D–G Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg- positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are SEM. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann–Whitney test. **P < 0.01 was considered statistically significant. Source data are available online for this figure. lesions. Indeed, we assessed the therapeutic effect of miransertib on was similar in both PIK3CA and TEK-mutant ECs (Fig 8E and F). We PI3K-driven vascular malformations. For this, we first induced the then studied the functional impact of miransertib-mediated AKT formation of vascular malformations by treating EC-Pik3caWT/H1047R signalling inhibition by determining the dose-response effect on cell mice with 4-OHT at P1 and then treated these mice with 35 mg/kg viability in ECs with either PIK3CA- or TEK-mutant genotype and (low dose) of miransertib at P4 and P5 (Fig 7A). As shown above, wild-type HUVECs. Miransertib robustly decreased EC viability in in our in vivo preclinical model, vascular malformations are all genotypes showing low IC50 with overlapping (not significantly already present at P4, showing all hallmarks of PI3K-driven vascu- different) confident intervals (Fig 8G). Interestingly, cell viability of lar malformations (Fig EV2). In contrast to vehicle-treated, wild-type ECs was slightly less sensitive to miransertib treatment. miransertib-treated EC-Pik3caH1047R retinas effectively regressed To assess whether this in vitro effect is reproduced by other AKT vascular malformations, confirmed by the normalization of vascular inhibitor, we assessed cell viability after treating wild-type and density, EC number and proliferation rate (Fig 7B and D–F). Impor- PIK3CA and TEK-mutant ECs with the AKT inhibitor MK2206 tantly, elevated PI3K signalling induced by Pik3caH1047R was blunted (Appendix Fig S3E). The impact of MK2206 on EC viability is signif- by miransertib (Fig 7C and G). These data demonstrate that miran- icantly reduced in both PIK3CA and TEK-mutant genotypes sertib is effective in inducing regression of PI3K-driven vascular compared with miransertib treatment. Moreover, wild-type ECs are malformations, thereby setting the grounds for a promising clinical slightly more sensitive to MK2206 treatment than mutant ECs. strategy for these diseases. These data show that miransertib impacts the viability of patient- derived ECs at low concentrations and that it might constitute a Miransertib inhibits PI3K/AKT signalling and reduces cell viability promising therapeutic strategy for both PIK3CA and TEK-mutant in patient-derived PIK3CA- and TEK-mutant ECs vascular malformations. Next, we assessed the therapeutic potential of miransertib in a preclinical human setting. For this, we isolated and cultured ECs Discussion derived from human vascular malformations carrying mutations in PIK3CA and TEK/TIE2 (Appendix Table S2), together spanning the Activation of the PI3K/AKT signalling pathway by genetic mutations genetic causes of more than 80% of low-flow lesions in patients in the endothelium is the primary etiological cause of most, if not all, (Limaye et al, 2009, 2015; Castel et al, 2016; Castillo et al, 2016a). low-flow vascular malformations (Boscolo et al, 2015; Limaye et al, To isolate and culture these cells, fresh surgical resections of low- 2015; Luks et al, 2015; Castel et al, 2016; Castillo et al, 2016a). flow vascular malformations were subjected to tissue digestion and Despite of this, there is no molecular targeted therapy approved for EC-positive selection (Fig 8A). Sanger sequencing and droplet digital their clinical management today. This is partially due to poor biologi- PCR revealed that causing mutations were present in the EC culture cal understanding of PI3K overactivation in ECs. Here, we provide with variant allelic frequencies (VAFs) of around 50% (Fig 8B; the first evidence that endothelial expression of a hotspot Pik3ca Appendix Fig S3A and B), demonstrating that these cell cultures mutation in these diseases triggers the activation of early molecular were enriched in mutant cells. Characterization of these cells vali- programmes involved in cell cycle progression and cell proliferation; dated their specific EC properties—cobblestone morphology and thus, shedding light into how these lesions form. The precise mecha- expression of the EC-specific markers VE-cadherin and ERG (Fig 8C; nism by which Pik3caH1047R leads to enhanced cell cycle progression Appendix Fig S3C). Next, we analysed the impact of these muta- still remains unclear. Nevertheless, our results show that pathologi- tions in PI3K signalling by assessing AKT and S6 phosphorylation cal expansion of Pik3caH1047R ECs primary occurs in the presence of levels. Indeed, PIK3CA- and TIE2-mutant ECs exhibited constitutive growth factors. Given that pS6, a downstream effector of AKT/ activation of the pathway compared with wild-type HUVECs mTOR, is selectively activated in the presence of growth factors in (Fig 8D). To evaluate the therapeutic efficacy of miransertib in mutant cells, it suggests that the AKT/mTOR axis orchestrates the patient-derived PIK3CA and TEK-mutant ECs, we first assessed its onset of Pik3caH1047R-vascular malformations. impact on PI3K/AKT signalling. At very low doses, miransertib Using the postnatal mouse retina as a model of vascular malfor- strongly inhibited AKT signalling in wild-type and mutant ECs, mations in combination with a tamoxifen-induced strategy, which shown by reduced levels of AKT and S6 phosphorylation in a dose- allows to mosaically express Pik3caH1047R at different developmen- dependent manner (Fig 8E and F; Appendix Fig S3D). This effect tal times, we demonstrate that active angiogenesis is required for 8 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine A B C D E F G Figure 5. Miransertib prevents the formation of Pik3ca-vascular malformations in mice. A 4-OHT and miransertib dosing scheme used for a prevention therapeutic experimental setup. B, CRepresentative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 lm (upper panel) and 30 lm (lower panels). D–G Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean  SEM. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonpara- metric Mann–Whitney test. *P < 0.05 and **P < 0.01 were considered statistically significant. Source data are available online for this figure. Pik3ca mutants to generate vascular malformations. In line with arteriovenous malformations rely on growth factor signals to be this, cultured patient-derived TEK or PIK3CA-mutant ECs lead to induced and expand (Garrido-Martin et al, 2014; Martinez-Corral pathological proliferative response only upon stimulation of et al, 2020; M€akinen et al, 2021; Ren et al, 2021). The growth- growth signals (Cai et al, 2019; Le Cras et al, 2020). Also, in vivo, push concept fits with clinical observations where low-flow vascu- other types of PI3K-related vascular malformations such as lar malformations appear during embryonic development, expand Pik3ca-driven lymphatic malformations and cerebral cavernous proportionally with the physiological growth of the patient and are malformations, and hereditary haemorrhagic telangiectasia-like largely quiescent during adulthood, when the production of growth ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 9 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al A B C D E F G Figure 6. Low dose of miransertib prevents the growth of Pik3ca-vascular malformations. A 4-OHT and miransertib dosing scheme used for a prevention experimental setup. B, CRepresentative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 lm (upper panels) and 30 lm (lower panels). D–G Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean  SEM. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonpara- metric Mann–Whitney test. *P < 0.05 and **P < 0.01 were considered statistically significant. Source data are available online for this figure. factors is residual (Pang et al, 2020). This also explains why low- Another important observation of our model is that Pik3ca- flow vascular malformations are histopathologically characterized related blood vascular malformations only occur in veins and in the by a low rate of EC proliferation. However, acute production of capillary bed, excluding arteries which remain unaltered. This extrinsic inputs such as hormonal changes, injury and wound resembles the human spectrum of vascular malformations where healing can reactivate proliferation in the malformed vasculature PIK3CA mutations have not been reported in arterial malformations (Pang et al, 2020); being a key aspect to mind for preventive (Peyre et al, 2021; Ren et al, 2021). Why arteries are unaffected by approaches. Pik3ca mutation remains unclear. This could be related to the 10 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine A B C D E F G Figure 7. Low-dose miransertib induces regression of Pik3ca-vascular malformations in vivo. A 4-OHT and miransertib dosing scheme used for a curative experimental setup. B, CRepresentative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 lm (upper panels) and 30 lm (lower panels). D–G Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean  SEM. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonpara- metric Mann–Whitney test. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant. Source data are available online for this figure. refractory behaviour that arterial cells exhibit upon reaching their 2021; Vanhaesebroeck et al, 2021). At least in part, the lack of definitive maturation state (Orsenigo et al, 2020; Luo et al, 2021), robust and rapid preclinical models for drug evaluation has and the cell cycle arrested state which fluid shear stress imposes on hampered the field. In this study, we provide evidence that the arterial ECs (Fang et al, 2017). While this would indicate that flow mouse retina model offers an opportunity for preclinical drug testing overrules high PI3K signalling, further experiments are required to of blood vascular disorders. The key aspects of this model are as address this. follows: (i) it develops in a short time frame (1 week); (ii) it allows Inhibition of the PI3K/AKT signalling pathway has been priori- for a dynamic preclinical testing, which covers prevention and cura- tized in targeted therapy strategies in medical oncology (Castel et al, tive strategies; (iii) it grants the quantification of disease hallmarks ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 11 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al including vascularity, EC proliferation and PI3K signalling, thus malformations microenvironment including the impact of blood providing a robust and non-biased setting to evaluate treatment flow, extracellular matrix, surrounding pericytes and smooth muscle efficacy; (iv) it recapitulates relevant features of vascular cells, which may influence drug response. Of relevance, this A B C D E F G Figure 8. 12 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine ◀ Figure 8. Miransertib impairs cell viability of PIK3CA- and TEK-mutant patient-derived ECs. A Illustration showing the strategy of EC isolation from patient-derived biopsies. B Representative sequencing chromatograms of PIK3CA and TEK-mutant VM-derived ECs. Arrows show the detected point mutations. C Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific tran- scription factor). Cell nuclei were visualized with DAPI. Scale bars: 30 lm. D Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control. E, F Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK- mutant patient-derived ECs. G Wild-type (HUVECs), PIK3CA and TEK-mutant EC viability upon the treatment with miransertib for 72 h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates. PIK3CA-mutant versus TEK-mutant P = 0.501; PIK3CA-mutant versus HUVECs P = 0.0815; TEK-mutant versus HUVECs: P = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test. Source data are available online for this figure. experimental system has been previously used for targeted therapy malformation, which should be considered proliferative vascular validation in other vascular disorders (Ola et al, 2016, 2018; Alsina- disorders. Sanchis et al, 2018). However, this is not a systemic model, and thus, it lacks some important aspects of the disease such as throm- bosis, haemorrhages, phlebectasia or infection (Pang et al, 2020). Materials and Methods We have previously modelled systemic vascular malformations by embryonic expression of Pik3caH1047R mutation in mice recapitulat- Reagents ing some of these knock-on effects (Castillo et al, 2016a). This preclinical model showed that therapeutic reduction of vascular All chemical reagents were purchased from Sigma-Aldrich, unless malformation may also eliminate and relieve some of these clinical stated otherwise. Cell culture media and buffers were purchased manifestations such as haemorrhages or phlebectasia of main inter- from Lonza and Gibco. Primers were obtained from Invitrogen. nal veins (Castillo et al, 2016a). Nevertheless, the high heterogene- Normal Primary Umbilical Vein Endothelial Cells (HUVECs) were ity of this model and the long-time frame needed to assess purchased from ATCC (PCS-100-010). therapeutic effectivity are important drawbacks for preclinical stud- ies of molecular therapies. Mice Blocking AKT signalling has been a promising therapy for cancer; however, rewiring PI3K signalling due to the complex The in vivo experiments were performed in agreement with the genetic landscape and instability of malignant cells have dampened guidelines and legislations of the Catalan Ministry of Agriculture, these expectations (Jansen et al, 2016). In contrast, vascular malfor- Livestock, Fisheries and Food (Catalonia, Spain), following proto- mations are considered largely monogenic diseases caused by acti- cols approved by the local Ethics Committees of IDIBELL-CEEA (ani- vating point mutations in an otherwise stable genetic context mal Use Protocol number 6809). Mice were kept in individually (Castel et al, 2020). Our data indicate that, similar to alpelisib, a ventilated cages under specific pathogen-free conditions (IDIBELL selective p110a inhibitor, low-dose miransertib may be sufficient to Animal Facility). All mice were crossed onto the C57BL/6J genetic prevent and induce regression of these diseases (Venot et al, 2018). background. Pik3caWT/H1047R mice carry a single, germline Cre- This is in line with previous studies reporting the effect of miran- inducible point mutation in Pik3ca allele (H1047R) (Kinross et al, sertib in PIK3CA-mutant ECs (Boscolo et al, 2019). Our results might 2012). This mice are crossed onto Pdgfb-iCreER mice (Claxton et al, indicate that this therapy is also effective for TEK-mutant vascular 2008) that express an inducible iCreER recombinase from the malformations; however, the lack of TEK-mutant mouse lines endogenous Pdgfb locus (EC-specific). Control mice were CreiER- precludes the assessment in vivo. In addition, comparison of different negative littermates injected with 4-hydroxytamoxifen. iCreER- PI3K signalling pathway inhibitors may contribute to the refinement mediated recombination in Pik3caWT/H1047R mice was induced by of therapeutic strategies for these diseases. Since these are early days intraperitoneal injection of 4-hydroxytamoxifen (doses indicated in for patient stratification for PI3K-related disorders, preclinical assess- the figure legends). ROSA-mTmG double fluorescent reporter mouse ment of new molecular drugs is critical to maximize therapeutic (Muzumdar et al, 2007) was crossed to Pdgfb-iCreER mice. The opportunities for these patients. Indeed, these patients may need ROSA-mTmG allele was kept heterozygous. 4-Hydroxytamoxifen long-term or even chronic treatment; thus alternation therapies was injected intraperitoneally in indicated doses (see the figure over time using effective low dose of inhibitor may be required to legends) and mouse retinas isolated at indicated time points. Cre- avoid side effects and the development of drug resistance. We antici- mediated recombination was assessed by the expression of pate that these results will have a direct impact on future clinical membrane-bound GFP. For the analysis of mouse postnatal retinas, strategies. male and female mice were analysed; the gender of mice was not Collectively, our data suggest that blocking EC growth should be assessed given the difficulty to determine this by anatomical dif- considered during potential reactivation scenarios. Furthermore, our ferences at postnatal age. For the RNAseq analysis, primary study calls for revisiting current classification of low-flow vascular endothelial cells were isolated from two female and two male mice. ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 13 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al For the rest of experiments, primary endothelial cells were isolated Rpl32 gene (coding for the 60S ribosomal protein L32) was used for from male mice. gene expression normalization. Mouse ECs isolation and culture RNA sequencing and analysis Mouse lung ECs were isolated from adult mice between 3 and The quantity and quality of the total RNA sample were determined 6 weeks old. Briefly, lungs were homogenized with a scalpel blade by Qubit RNA BR Assay kit (Thermo Fisher Scientific) and RNA and incubated in dispase II in Hank’s Balanced Salt Solution at 6000 Nano Bioanalyser 2100 Assay (Agilent). The RNASeq libraries 4 U/ml concentration for 1 h at 37°C. The digested tissue was were prepared following the TruSeq Stranded mRNA Sample Prep disintegrated by pipetting into a single-cell solution, following Kit protocol. Briefly, total RNA (500ng) was enriched for the polyA enzyme inactivation with DMEM supplemented with 10% FBS mRNA fraction and fragmented by divalent metal cations at high and 1% penicillin/streptomycin. Cells were resuspended in 100 µl temperature. In order to achieve the directionality, the second of PBS and incubated with rat anti-CD-144 (BD Biosciences, strand cDNA synthesis was performed in the presence of dUTP. The 555289) antibody-coated magnetic beads for 30 min at room blunt-ended double-stranded cDNA was 30 adenylated and Illumina temperature. CD-144-positive fraction was washed with PBS with Truseq adaptors with single indexes were ligated. The ligation 0.5% BSA and sorted using a magnet. Cells were resuspended and product was enriched with 15 PCR cycles, and the final library was cultured in 0.5% gelatine-coated culture well (12-well format) in validated on an Agilent 2100 Bioanalyser with the DNA 7500 assay F12/DMEM medium supplemented with 20% FBS, 1% penicillin/ (Agilent). For the analysis of RNAseq data, generated fastq files streptomycin and 4 ml EC growth factors, later referred to as F12 were first quality controlled using FASTQC software (version complete until they reached 80–90% confluency. Cells were 0.11.9). Then, these were aligned to the UCSC mm10 reference subjected for a second selection with the CD-144 antibody-coated genome using STAR (version 2.7.2b) (Dobin et al, 2013). Qualimap magnetic beads for 1 h at room temperature, then trypsinized, (version 2.2) (Okonechnikov et al, 2016) was used for mapping magnetic-sorted and resuspended in F12 complete medium and quality control and IGV (version 2.3.86) (Thorvaldsd ottir et al, further cultured (6-well format). Cells were cultured at 37°C 2013) for visualization of the aligned reads. Mapped counts were in 5% CO2 atmosphere until passage 5. Primary cells were not quantified with featureCounts (Liao et al, 2014) from the SubRead R tested for mycoplasma contamination. To induce Pik3caH1047R package (Liao et al, 2019) using the UCSC mm10 gene annotation, mutation, ECs were treated with 2 µM 4-hydroxytamoxifen for and normalization was performed with the DESeq2 R package (ver- 6 h. Ethanol-treated cells were used as a control. Recombined cells sion 3.10) (Love et al, 2014). Differential genes expression was also were re-seeded for an experiment and cultured at 37°C in 5% CO2 analysed using DESeq2, with 0.05 as the significance cut-off, and atmosphere. the obtained P-values were corrected for multiple testing using the Benjamini and Hochberg method. Gene set enrichment analysis Protein extraction and immunoblotting (GSEA, Subramanian et al, 2005) was performed on the log 2 fold change ranked list of genes in our dataset using the fgsea R package Cells were lysed in ice-cold lysis buffer (50 mM Tris–HCl pH 7.4, (preprint: Korotkevich et al, 2021) with Msigdb (Liberzon et al, 5 mM EDTA, 150 mM NaCl and 1% Triton X-100) containing 2011) and clusterProfiler (Yu et al, 2012) with BP Gene Ontology protease (Roche, 11836153001) and phosphatase inhibitors (Sigma- terms (Ashburner et al, 2000). Volcano plot in Fig 1B was created Aldrich, 4906837001). Total cell lysates were resolved on 10% poly- with VolcaNoseR (Goedhart & Luijsterburg, 2020). acrylamide gels, transferred onto nitrocellulose membranes and incubated with appropriate primary and secondary antibodies. The Cell cycle analysis by flow cytometry following primary antibodies were used: p-AKT S473 (CST, 4060, diluted 1:1,000), p-AKT T308 (CST, 4056S, diluted 1:500), AKT For cell cycle analysis, 106 cells per condition were harvested, (CST, 9272, diluted 1:2,000), p-PRAS40 T246 (CST, 2640, diluted washed in PBS and fixed by adding dropwise 0.9 ml ice-cold 70% 1:1,000), p-S6 S235/236 (CST, 4857, diluted 1:1,000), p-S6 S240/244 ethanol while vortexing and stored 24 h at 20°C. On the day of (CST, 2215S, diluted 1:1,000), S6 (CST, 2212, diluted 1:1,000), VE- analysis, cells were spun down at 300g 5 min and washed with cadherin (Santa Cruz Biotechnology, sc-6458, diluted 1:500) and PBS. Cell pellet was stained 30 min at RT using FxCycle PI/RNase vinculin (Abcam, ab49900, diluted 1:10,000). The following Staining solution (Life Technologies F10797) (0.5 ml per sample). secondary antibodies from DAKO were used (all diluted 1:5,000): Cell cycle was assessed using BD FACSCantoTM II flow cytometer, swine anti-rabbit (P0399), rabbit anti-goat (P0449), rabbit anti- and data were analysed using the FlowJoTM software v10.8.0 (BD mouse (P0260) and rabbit anti-sheep (P0163). Life Sciences). cDNA synthesis and quantitative polymerase chain reaction Pharmacological in vivo treatment RNA was extracted using RNeasy Qiagen kit following manufacturer Miransertib (ARQ 0922MSA salt) (ArQule, Inc., a wholly owned instructions. Reverse transcription was performed from 250 ng of subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA) was prepared RNA for cell lysate samples by using High-Capacity cDNA Reverse at a stock concentration of 10 mgA/ml in 20% Captisol (m/v) in Transcription Kit. For quantitative polymerase chain reaction, a 0.02 M citrate/saline buffer. 20% Captisol (m/v) in 0.02 M citrate/ LightCycler 480 System was used with LightCycler 480 SYBR Green saline buffer was used as a vehicle. Mice were injected intraperi- I Master kit and specific primers detailed in Appendix Table S3. toneally with either 75 mg/kg or 35 mg/kg dose of miransertib. 14 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine Mouse retina isolation and immunostaining region of interest (ROI). Then, the integrated density of p-S6 was measured in IB4-positive areas. The background measurements Mice were sacrificed by decapitation and eyes were isolated, (mean grey values) were taken from areas in close proximity to followed by an hour incubation on ice in 4% PFA in PBS. Isolated the vasculature, but negative for IB4. The corrected total fluores- retinas were fixed for additional hour, permeabilized overnight at cence (CTF) was calculated based on the following equation: 4°C in permabilization/blocking buffer (1% BSA, 0.3% Triton X-100 CTF = integrated density—(vascular area × mean grey background in PBS). Afterwards, the retinas were incubated overnight at 4°C value). Veins, arteries and capillaries in the mouse postnatal with specific primary antibodies, diluted in permabilization/block- retina are identified by morphology as previously described (Pit- ing buffer (ERG (Abcam, AB92513, diluted 1:400), NG2 (Milipore, ulescu et al, 2010). AB5320, diluted 1:200), p-S6 235/236 (Cell Signalling Technology, 4857, diluted 1:100). Samples were washed three times in PBS Isolation, culture and sequencing of ECs from patient-derived containing 1% Tween-20 (PBST), following incubation with PBlec vascular malformations buffer (1% Triton X- 100, 1 mM CaCl2, 1 mM MgCl2 and 1 mM MnCl2 in PBS, pH 6.8) for 30 min at RT. Secondary AlexaFluor- Patient tissue samples were obtained under therapeutic surgical conjugated antibodies, diluted in PBlec, were added to the retinas resection. Informed consent was obtained from all subjects with the and incubated for another 2 h (Invitrogen, A11001, A11011, A11008 approval of the Committees on Biomedical Investigation at Hospital and A31573). Blood vessels were visualized with AlexaFluor- Sant Joan de Deu, Hospital Santa Creu i Sant Pau and Hospital conjugated Isolectin GS-B4 (Molecular Probes, I21411, I21412). Universitari Bellvitge (Barcelona, Spain) under the codes PR264/16 Following three washes with PBST, the tissues were flat-mounted and PIC-96-16. Experiments conformed to the principles set out in on a microscope slide. the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Collected data were stored in a In vivo proliferation assay secure database maintained by Hospital Sant Joan de Deu. Human ECs were isolated from patient-derived biopsies of vascular malfor- 5-ethynyl-20 -deoxyuridine (EdU)-incorporation assay has been mations. Briefly, the biopsy was homogenized with a scalpel blade performed using a commercially available kit (Invitrogen, C10340). and digested in dispase II (4 U/ml) and collagenase A (0.9 mg/ml) Animals were injected intraperitoneally with 60 µl of EdU (0.5 mg/ in Hank’s Balanced Salt Solution for maximum 1.5 h at 37°C, ml in 50% DMSO and 50% PBS solution), and after 2 h, the animals vortexing the sample every 30 min. The digested tissue was disinte- were sacrificed and retinas isolated. EdU incorporation was grated by pipetting into a single-cell solution, following enzyme detected with Click-iT EdU Alexa Fluor-647 Imaging Kit, following inactivation with DMEM supplemented with 10% FBS and 1% peni- manufactures instructions. Afterwards, standard protocol for retina cillin/streptomycin. Cells were resuspended in 100 µl of 0.5% BSA immunostaining was applied. in PBS and incubated with mouse anti-human CD31 (Agilent Dako, M0823, clone JC70A) antibody-coated magnetic beads (Thermo- Confocal imaging and image quantification Fisher Scientific, 11041) for 1 h at room temperature. CD31-positive fraction was washed with 0.5% BSA in PBS and sorted with a Microscopy imaging was done with Leica TCS SP5 confocal magnet. Cells were resuspended and cultured in 0.5% gelatine- microscope. Volocity, Adobe Photoshop 2021 and ImageJ soft- coated culture well (12-well format) in EGM2 medium (PromoCell, wares were used for image editing and quantification, respec- C30140) supplemented with 10% FBS, 1% penicillin/streptomycin tively. Images were taken from at least 4 retina areas in each (later referred to as EGM2 complete) at 37°C and 5% CO2 until they genotype. At least three biological replicates per genotype were reach confluency. Cells were subjected for a second selection. performed. To quantify the vascular lesion area, an IB4-positive Primary cells were not tested for mycoplasma contamination. area was manually selected and the percentage of IB4 area per Genomic DNA was isolated according to the manufacturer’s proto- retina area was quantified. To determine the recombination effi- col (Thermo Fisher Scientific, K182001). The regions of interest in ciency of mTmG allele in ECs, the ratio of GFP-positive area to the genomic DNA were amplified by PCR using PlatinumTM Taq IB4-positive area was calculated and presented as percentage. DNA Polymerase High Fidelity (Thermo Fisher Scientific, Retina vascularity was measured using IB4 channel by adjusting 11304011). Exons 10 and 21 of PIK3CA, and exon 17 of TEK were the threshold to select the IB4-positive area, followed by quan- amplified. PCR products were purified according to the manufac- tification of the percentage of IB4-positive area in the total image turer’s protocol (GE Healthcare, 28-9034-70) followed by Sanger area (104 µm2). EC number was determined manually based on sequencing. Sequences of the primers used: PIK3CA exon 10: EC-specific nuclei staining (Erg) in 104 µm2 image area. Quan- (forward) 50 -TGGTTCTTTCCTGTCTCTGAAAA-30 and (reverse) tification of EC proliferation was done using EdU and Erg co- 50 -CCATTTTAGCACTTACCTGTGAC-30 . PIK3CA exon 21: (forward) immunostaining—both EdU- and Erg-positive ECs were quantified 50 -CATTTGCTCCAAACTGACCA-30 and (reverse) 50 -TGTGTGGAA in the 104 µm2 image area. The coverage of vessels by NG2- GATCCAATCCA-30 . TEK exon 17: (forward) 50 -TAGGCAATTT positive pericytes was quantified from both NG2 and IB4 chan- CCACAGCACA-30 and (reverse) 50 -GGCAAACCAGGCTAAGAGAG- nels by adjusting the threshold and selecting the positive NG2 30 . Droplet digital PCR was done on genomic DNA extracted from and IB4 areas, respectively. Then, the percentage of NG2 to IB4 cell cultures. PIK3CA genotyping assays from Bio-Rad were used to ratio was calculated. The vascular-specific p-S6 intensity was specifically detect the PIK3CAE542K mutation on DNA samples. The measured using both p-S6 and IB4 channels. First, a manual Bio-Rad QX200 ddPCR system was used, and allelic frequencies threshold was set to obtain the IB4-positive area and define the were calculated using Quantasoft Analysis Pro (BioRad) software. ª 2022 The Authors EMBO Molecular Medicine 14: e15619 | 2022 15 of 19 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EMBO Molecular Medicine Piotr Kobialka et al vehicle) were plotted against the logarithm of inhibitor concentra- The paper explained tion. IC50 and 95% CI values were calculated by non-linear regres- sion (variable slope) using GraphPad Prism software. Problem Low-flow vascular malformations are congenital diseases caused by a focal overgrowth of vessels. They may cause pain, bleeding, infections Statistics and obstruction of organs. While their genetic causes, leading to hyperactivation of PI3K signalling, have been known for a few years, Statistical analysis was performed using Prism 8 (GraphPad Soft- no targeted molecular therapy has been approved so far. ware Inc.). All figures are displayed with individual data points that indicate biological replicates and with the standard error of the Results mean (SEM) as error bars. At least three biological replicates were Pik3ca mutation in ECs activates transcriptomic changes, leading to used. P-values considered as statistically significant were as follows: enhanced cell cycle progression in the presence of growth factors. We have generated a robust and fast preclinical in vivo model that allows *P < 0.05; **P < 0.01 and ***P < 0.0001. for testing of targeted drugs. With this model, we demonstrated that PI3K-driven vascular malformations rely on active angiogenesis. Our preclinical studies show that AKT inhibition using miransertib Data availability prevents the disease and fully regresses established vascular malfor- mations. RNAseq data are deposited in the BioProject repository (NCBI) Impact under the accession ID PRJNA780473 (https://www.ncbi.nlm.nih. Our new in vivo model of PI3K-driven vascular malformations consti- gov/bioproject/PRJNA780473). tutes a reliable and fast preclinical setting to test new or repurposed targeted drugs. Our studies support that Pik3ca-mutant ECs cause Expanded View for this article is available online. vascular malformations upon growth stimuli, highlighting the impor- tance of preventive therapeutic approaches after invasive treatments. We provide proof of concept for the use of the AKT inhibitor miran- Acknowledgements sertib in PI3K-driven vascular malformations, which opens a new We thank members of the Endothelial Pathobiology and Microenvironment window for targeted therapeutic intervention for these diseases. We Group for helpful discussions. This work was partially founded by ArQule, Inc., further demonstrate in vitro that this targeted therapy is similarly a wholly owned subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. We thank effective in both PIK3CA and TEK-mutant vascular malformations. CERCA Programme/Generalitat de Catalunya and the Josep Carreras Founda- tion for institutional support. The research leading to these results has received funding by the Spanish Ministry of Science and Innovation MICINN Cell immunofluorescence (PID2020-116184RB-I00 /AEI/10.13038/501100011033). M.G. laboratory is supported by the research grants SAF2017-89116R-P (FEDER/EU) from MCIU Human VM-derived ECs were seeded on gelatine-coated coverslips (Spain) co-funded by European Regional Developmental Fund (ERDF), a Way in a way to reach confluency the next day and incubated overnight to Build Europe and PID2020-116184RB-I00 from MCEI; PTEN RESEARCH at 37°C in 5% CO2. Then, cells were washed with warm PBS with Foundation (IJC-21-001); la Caixa Banking Foundation (LCF/PR/PR16/51110035 Mg2+ and Ca2+ and then fixed with 4% PFA for 15 min at room n Espan and LCF/PR/HR19/52160023; also to E.B. and J.M.); by la Asociacio ~ola temperature, followed by triple wash with PBS with Mg2+ and Ca2+. contra el Cancer (AECC)-Grupos Traslacionales (GCTRA18006CARR); by la Cells were permeabilized with PBS containing 0.4% Triton X-100 Fundacion BBVA (Ayuda Fundacion BBVA a Equipos de Investigacio n Cientıfica for 5 min and blocked with 2% BSA in PBS for 1 h at RT. The 2019); World Cancer Research (21-0159). The computations and data handling following primary antibodies were used for 1 h at RT: VE-cadherin were enabled by resources in projects SNIC 2019/30-26 and SNIC 2019/8-129, F8 (Santa Cruz, SC-9989, 1:100), ERG (Abcam, ab92513, 1:400). provided by the Swedish National Infrastructure for Computing (SNIC) at Then, coverslips were washed three times with PBS for 5 min, UPPMAX, partially funded by the Swedish Research Council through grant followed by 45 min incubation at RT with appropriate secondary agreement no. 2018-05973. Personal support was from Marie-Curie ITN Actions antibody in PBS: goat anti-mouse Alexa Fluor-488 (Invitrogen (P.K. and J.Z.) grant agreement 675392. L.G. is funded by the Swedish Research A11001, diluted 1:300) and goat anti-rabbit Alexa Fluor-568 (Invitro- Council (2018-06591). S.D.C. is a recipient of a fellowship from the European gen A11011, diluted 1:300). Then, coverslips were washed with PBS Union’s Horizon 2020 Research and Innovation Programme under the Marie three times for 5 min, and in the last wash DAPI (Invitrogen, Sklodowska-Curie grant agreement No 749731. S.D.C. is currently funded by la D1306, diluted 1:10 000) was added to visualize cell nuclei. Cover- Caixa Banking Foundation Junior Leader project (LCF/BQ/PR20/11770002). E.B. slips were mounted on a microscope slide in a mounting medium n (Proyectos de investigaci is funded by the Agencia Estatal de Investigacio on (ThermoFisher Scientific, 9990402). en salud PI20/00102). The authors thank the Xarxa de Bancs de Tumors de Catalunya (XBTC; sponsored by Pla Director d’Oncologia de Catalunya). We are MTS viability assay and calculation of IC50 grateful to the Band of Parents at Hospital Sant Joan de Deu for supporting the overall research activities of the Developmental Tumor laboratory (Pedi- MTS assay was used in order to determine cell viability. Briefly, atric Cancer Center Barcelona). 2103 cells were seeded on gelatine-coated 96-well plates (5 technical replicates per condition) and incubated overnight at 37°C in 5% CO2 Author contributions atmosphere. The next day, cells were treated for 3 days with miran- Piotr Kobialka: Conceptualization; Data curation; Formal analysis; Investiga- sertib. MTS assay (Abcam, ab197010) was performed for 2.5 h, and tion; Methodology; Writing—original draft; Writing—review & editing. Helena the absorbance was measured at 490 nm. Data (the percentage of a Sabata: Conceptualization; Data curation; Formal analysis; Investigation; 16 of 19 EMBO Molecular Medicine 14: e15619 | 2022 ª 2022 The Authors 17574684, 2022, 7, Downloaded from https://www.embopress.org/doi/10.15252/emmm.202115619 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Piotr Kobialka et al EMBO Molecular Medicine Methodology; Writing—original draft; Writing—review & editing. Odena from patients with kaposiform lymphangiomatosis. Pediatr Blood Cancer Vilalta: Methodology. Leonor Gouveia: Data curation; Formal analysis. Ana 66: e27790 Angulo-Urarte: Methodology. Laia Muixi: Methodology. Jasmina Zanon- Cai Y, Schrenk S, Goines J, Davis GE, Boscolo E (2019) Constitutive active ~ oz-Aznar: Methodology. Nagore G Olacir- cello: Methodology. Oscar Mun mutant TIE2 induces enlarged vascular lumen formation with loss of egui: Methodology. Lucia Fanlo: Methodology. Anna Esteve-Codina: Data apico-basal polarity and pericyte recruitment. Sci Rep 9: 12352 curation; Formal analysis. Cinzia Lavarino: Resources; Supervision. Biola M Canaud G, Hammill AM, Adams D, Vikkula M, Keppler-Noreuil KM (2021) A Javierre: Supervision; Methodology. Veronica Celis: Resources; Supervision. review of mechanisms of disease across PIK3CA-related disorders with  pez-Ferna Carlota Rovira: Resources; Supervision. Susana Lo ndez: vascular manifestations. Orphanet J Rare Dis 16: 306 lia Baselga: Resources; Supervision. Jaume Mora: Resources; Supervision. Eula Castel P, Carmona FJ, Grego-Bessa J, Berger MF, Viale A, Anderson KV, Bague Resources; Supervision. Sandra D Castillo: Conceptualization; Data curation; S, Scaltriti M, Antonescu CR, Baselga E (2016) Somatic PIK3CA mutations Formal analysis; Supervision; Funding acquisition; Investigation; Methodology; as a driver of sporadic venous malformations. Sci Transl Med 8: 332ra42 Writing—original draft; Writing—review & editing. Mariona Graupera: Castel P, Rauen KA, McCormick F (2020) The duality of human oncoproteins: Conceptualization; Resources; Data curation; Formal analysis; Supervision; drivers of cancer and congenital disorders. Nat Rev Cancer 20: 383 – 397 Funding acquisition; Investigation; Writing—original draft; Writing—review & Castel P, Toska E, Engelman JA, Scaltriti M (2021) The present and future of editing. PI3K inhibitors for cancer therapy. Nat Cancer 2: 587 – 597 In addition to the CRediT author contributions listed above, the contribu- Castillo SD, Baselga E, Graupera M (2019) PIK3CA mutations in vascular tions in detail are: malformations. Curr Opin Hematol 26: 170 – 178 MG, SDC, PK and HS were the main contributors in the conception, design, Castillo SD, Tzouanacou E, Zaw-Thin M, Berenjeno IM, Parker VER, Chivite I, acquisition and interpretation of the data and in writing the article. PK HS, OV, Mila-Guasch M, Pearce W, Solomon I, Angulo-Urarte A et al (2016a) AA-U, LM, JZ, LF. NGO, CL, BMJ and SDC performed experiments and data anal- Somatic activating mutations in Pik3ca cause sporadic venous ysis with input from SDC and MG. RNAseq analysis was performed by LG and malformations in mice and humans. Sci Transl Med 8: 332ra43 AE-C. CR and OM-A interpreted histopathology. VC, SL-F, EB and JM liaised Castillo SD, Vanhaesebroeck B, Sebire NJ (2016b) Phosphoinositide 3-kinase: a with human subjects and provided access to human tissue samples and clini- new kid on the block in vascular anomalies. J Pathol 240: 387 – 396 cal input in the study. Chen J, Somanath PR, Razorenova O, Chen WS, Hay N, Bornstein P, Byzova TV (2005) Akt1 regulates pathological angiogenesis, vascular maturation Disclosure and competing interests statement and permeability in vivo. Nat Med 11: 1188 – 1196 M.G. has a research agreement with ArQule, Inc., a wholly owned subsidiary of Claxton S, Kostourou V, Jadeja S, Chambon P, Hodivala-Dilke K, Fruttiger M Merck & Co., Inc., Kenilworth, NJ, USA, and Venthera. E.B. is founder and CAB of (2008) Efficient, inducible cre-recombinase activation in vascular Venthera; PI and Advisor for Pierre Fabre; PI of the clinical trial NCT04589650 endothelium. Genesis 46: 74 – 80 (Novartis). Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15 – 21 References Ehling M, Adams S, Benedito R, Adams RH (2013) Notch controls retinal blood vessel maturation and quiescence. 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