PTEN hamartoma tumour syndrome (PHTS), a rare disease caused by germline heterozygous PTEN variants, is associated with multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer risk. Phenotypic variability in PHTS is partly due to diverse PTEN variants and the protein's multifaceted functions. PTEN is primarily a phosphatidylinositol(3,4,5)trisphosphate (PIP3) phosphatase regulating PI3K/AKT signalling but also maintains chromosomal stability through nuclear functions such as double-stranded (ds)DNA damage repair. Here, we show that PTEN-R173C, a pathogenic variant frequently found in PHTS and somatic cancer, has elevated PIP3 phosphatase activity that effectively regulates canonical PI3K/AKT signalling. However, PTEN-R173C is unstable and excluded from the nucleus. We generated Pten+/R173C mice which developed few tumours during their lifetime, aligning with normal PI3K/AKT signalling. However, they exhibited lymphoid hyperplasia, macrocephaly and brain abnormalities, associated with impaired nuclear functions of PTEN-R173C, demonstrated by reduced dsDNA damage repair. We integrated PHTS patient data with our mouse model results, and propose that defective nuclear functions of PTEN variants can predict the onset of PHTS phenotypes and that late-onset cancer in these individuals may arise from secondary genetic alterations, facilitated by compromised dsDNA repair.

Abstract PI3Kδ is a predominantly leukocyte-enriched class I PI3K consisting of the p110δ catalytic subunit (encoded by PIK3CD) and most commonly the p85α regulatory subunit (encoded by PIK3R1). PI3Kδ is an important regulator of B-cell function and has been the target of extensive drug development efforts for B-cell malignancies, with multiple inhibitors approved for this indication. Based on our pre-clinical studies, PI3Kδ inhibition is now also emerging as an immunotherapy approach for solid tumours. Regulatory T-cells (Treg) are exquisitely sensitive to PI3Kδ inhibition, therefore pharmacological PI3Kδ inhibitors preferentially target the Tregs, rebalancing the immune system in favour of an effector T cell-mediated anti-tumour immune response. This concept is currently under investigation in clinical trials in uveal melanoma and non-small cell lung cancer. Notably, we and others have also observed multiple solid tumour types including glioblastoma, melanoma, breast cancer, prostate cancer, neuroblastoma and hepatocellular carcinoma also express wild-type PIK3CD, often at much higher levels than their non-transformed cell types of origin. Some studies suggest PI3Kδ promotes proliferation in these tumour cells, but this remains unclear with the potential that inhibitors were used at non-PI3Kδ-specific concentrations, and these findings are not consistently confirmed with genetic approaches. This raises the questions of what function PI3Kδ plays in solid tumours and how the use of PI3Kδ inhibitors for immunotherapy will affect the tumour cells themselves via inhibition of cell-intrinsic PI3Kδ. Using highly-selective PI3Kδ inhibitors and CRISPR-Cas9-mediated PIK3CD deletion in solid tumour cell lines, we demonstrate that PI3Kδ inhibition does not affect cell proliferation in these models. However, through both in vitro and xenograft studies, we have identified cancer-relevant roles for PI3Kδ in solid tumours, the implications of which will be presented. Our data suggest the solid tumour cell-intrinsic PI3Kδ expression should be considered to inform PI3Kδ-targeting immunotherapy studies. Citation Format: Sarah E. Conduit, Elena Lopez-Guadamillas, Daniele Morelli, Harriet Howard, Wayne Pearce, Cheryl Scudamore, Bart Vanhaesebroeck. Solid tumor cell-intrinsic function of PI3Kδ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5750.

Abstract Cancer cells maintain oncogenic signalling within a survival “fitness zone”. While conventional targetαed therapies suppress this signalling below a critical threshold, excessive activation of the same pathways can also be lethal to cell, an observation currently being explored as a novel therapeutic approach in cancer. In this study, we report that hyperactivation of PI3Kα, one of the most frequently oncogenically activated PI3K isoforms, using the small-molecule PI3Kα activator UCL-TRO-1938 (further referred to as 1938) induces cytotoxicity in cancer cell lines while sparing non-transformed cells. This PI3K activation-induced cell death (PI3K-AICD) depends on AKT/mTORC1 activity, only occurs under serum starvation and is enhanced by low O2 levels. This hyperactivation lethality is mechanistically linked to an irreconcilable metabolic conflict by simultaneously activating anabolic PI3K/mTORC1 signalling in a catabolic (hypoxic) state, resulting in an unresolvable energy crisis and ultimately cell death. In serum-deprived lung cancer cell lines, 1938 induces a magnified endoplasmic reticulum stress response which, along with PI3K-AICD, can be mitigated by supplementation with unsaturated fatty acids, suggesting a critical metabolic dependency on lipid metabolism for driving this cell death response. Consequently, co-treatment with 1938 and inhibitors of stearoyl-CoA desaturase-1 (SCD1), an O2-dependent enzyme essential for fatty acid desaturation, amplifies the PI3K-AICD response. In summary, these findings demonstrate that enhancing an oncogenic pathway central to metabolic control can selectively kill cancer cells. Citation Format: Benoit Bilanges, Ralitsa Madsen, Daniele Morelli, Thomas Jones, Wayne Pearce, Etienne Leveille, Mustafa Kocak, Eden Bramson, Mark Bekala, Roger Williams, Nicholas McGranahan, Markus Muschen, William R. Sellers, Henning Walczak, Bart Vanhaesebroeck. Pharmacological activation of PI3Kα triggers cancer cell death under metabolic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4738.

The placenta is essential for embryonic development, in part by mediating nutrient transfer from mother to embryo. Placental insufficiency is the most common cause of intrauterine growth restriction which has long-term health consequences lasting into adulthood. p110β is a class IA phosphoinositide 3-kinase (PI3K) catalytic subunit, a family of lipid kinases which are critical regulators of adult metabolism, immunity and embryonic and placental development. However, unlike the other class IA PI3K isoforms, the in vivo functions of p110β remain unclear. While homozygous p110β kinase-dead mice are mostly embryonically lethal, some survive into adulthood with no apparent phenotypes, other than reduced fertility. The mechanism(s) underlying this embryonic lethality remain unclear. Therefore, we performed an in-depth characterisation of p110β kinase-dead embryos, revealing a previously unrecognised role for p110β in controlling the expression of system A amino acid transporters. We show that homozygous p110β kinase-dead embryos are phenotypically normal, but growth-restricted and exhibit placental insufficiency. The placenta is small with a reduced nutrient storing junctional zone and downregulation of the system A amino acid transporters, required for maternal-to-embryo amino acid transfer. These data suggest defective amino acid transfer drives embryonic growth restriction and partial lethality of p110β kinase-dead embryos. This predominantly embryonic p110β phenotype is consistent with the notion that system A amino acid transporters are more critical during development than in adult physiology. The greater significance of p110β in development than in adult homeostasis may also help explain why p110β inhibitors, compared to inhibitors of other PI3K isoforms, are well-tolerated in adults.

The organismal roles of the class II PI3K isoform PI3K-C2α remain poorly understood. Recent studies have found PI3K-C2α to promote arterial thrombosis and breast cancer metastasis, generating interest in this kinase as a drug target, with small molecule PI3K-C2α inhibitors now available. However, the consequences of systemic PI3K-C2α inactivation in the nondiseased, postnatal state are largely unknown. Here, we show that induction of genetic PI3K-C2α inactivation in adult mice is well tolerated, without adverse effects on normal physiology. Surprisingly, however, mice with inactive PI3K-C2α display strong sensitization to challenge with bacterial lipopolysaccharide (LPS), a model of endotoxic shock. This sensitization is recapitulated by vascular endothelial-specific deletion of PI3K-C2α. Furthermore, sensitization to LPS can be fully rescued by disabling extrinsic induction of cell death by combined caspase-8- and RIPK3 deficiency. These observations validate the tolerability of systemic PI3K-C2α inhibition in principle but reveal an unexpected role for PI3K-C2α in the regulation of extrinsic cell death pathways.

PTEN Hamartoma Tumour Syndrome (PHTS) is a rare disorder characterized by germline heterozygous mutations in the PTEN tumour suppressor gene, leading to multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer risk. PHTS individuals display heterogeneity in phenotypes, which has been linked in part to the diverse genetic alterations in the PTEN gene and the multifaceted functions of this protein. Indeed, while PTEN primarily functions as a PIP3 lipid phosphatase in the cytosol, regulating PI3K/AKT signalling, a pathway commonly deregulated in cancer, it also plays crucial roles in maintaining chromosomal stability through nuclear activities such as double strand (ds) DNA damage repair. Recent studies have identified a subset of missense PHTS variants that cause nuclear exclusion of PTEN, impairing its nuclear functions. Here, we present our findings from one such pathogenic variant, PTEN-R173C , frequently found in PHTS and somatic cancers. Using cell biological and mouse modelling approaches, we show that PTEN-R173C has higher PIP3 phosphatase activity than wild-type PTEN, resulting in effective regulation of canonical PI3K/AKT signalling. However, PTEN-R173C is unstable and excluded from the nucleus. Aligning with their near normal PI3K/AKT signalling, Pten+/R173C mice display a low incidence of solid tumours compared to Pten+/- mice. Pten+/R173C mice also exhibit lymphoid hyperplasia and macrocephaly which correlates with compromised nuclear functions of PTEN-R173C. That nuclear functions are compromised is demonstrated by reduced dsDNA damage repair in Pten+/R173C mice. Integrating PHTS patient data with findings from our mouse model, our study indicates that nuclear dysfunction of pathogenic PTEN variants is a key factor in predicting the onset of the different PHTS-associated phenotypes. We speculate that late-onset cancer in individuals with nuclear-excluded PTEN results from genetic alterations unrelated to PTEN itself, facilitated by impaired PTEN-mediated dsDNA damage repair. ### Competing Interest Statement BV is a consultant for Pharming (Leiden, The Netherlands) and iOnctura (Geneva, Switzerland) and a shareholder of Open Orphan (Dublin, Ireland) PTEN Research Foundation, https://ror.org/04b98dz04, UCL-16-001, UCL-20-001, UOC-17-001 Cancer Research UK, https://ror.org/054225q67, C416/A29287, CANTAC721\100022, C23338/A25722, C416/A25145 European Commission, https://ror.org/00k4n6c32, H2020-MSCA-IF-2018 GA: 838559 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/W007460/1, BB/N009061/1 Jean Shanks Foundation, https://ror.org/00wf6bs47 Fondation ARC pour la Recherche sur le Cancer, https://ror.org/0489qz649, ARCPJA2022060005118 La Ligue Contre le Cancer, https://ror.org/00rkrv905 Wellcome Trust, https://ror.org/029chgv08, 108726/Z/15/Z NIHR Cambridge Biomedical Research Centre, NIHR203312 Medical Research Council, https://ror.org/03x94j517, MC-A024-5PF91

Abstract Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA-related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kβ produce the PIP3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered ‘Disorders with Ciliary Contributions’, a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction.

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer‐predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3‐kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53 . However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short‐term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro‐intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long‐term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68 Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.

PTEN negatively regulates the Class I PI 3-kinases by metabolizing phosphatidylinositol(3,4,5)trisphosphate (PIP3) and acts as a tumor suppressor. Heterozygous germline PTEN mutations in humans manifest into a complex multiorgan disorder known as PTEN hamartoma tumor syndrome (PHTS). Patients present with hamartomatous skin lesions, benign GI polyps, have an increased risk of developing certain types of cancer (breast, colon, endometrium, thyroid, GI tract and kidney) and have macrocephaly and autism spectrum disorders (ASD). There is huge variability in disease symptoms in PHTS patients, the cause of which is unclear. It has been proposed that this correlates with the nature of the aberration in PTEN and its impact on PTEN expression and function, allowing to discriminate the following cancer-risk groups in PHTS patients: 1. High cancer risk: Patients expressing a catalytically inactive but stable PTEN protein (mis-sense PTEN mutations such as the C124S, G129R, R130Q etc.) have a severe phenotype, with benign tumors and aggressive malignant cancers at a young age of one of more tissues. 2. Medium risk: Patients with complete loss of PTEN expression due to insertions, deletions and truncations in the PTEN gene present with an intermediate severity with benign and malignant tumors. 3. Low risk: Patients with PTEN protein characteristics that do not fall into Group 1 or 2 but with some detectable suppression of AKT signaling. This covers a range of PTEN mutants, with often a mix of characteristics such as being unstable but active or even overactive, stable but partially inactive or no apparent activity defect. These patients have a mild phenotype, mainly related to a broad range of ASD symptoms such as macrocephaly, developmental delay and mental retardation. Some patients are severely autistic. Patients may have benign tumors and skin hamartomas, but malignant cancer is rare. Here we expand our analysis by characterizing a range of additional PHTS PTEN mutations using biochemical methods. Of the several mutations characterized, the R173C mutation leads to a PTEN enzyme that retains its activity but becomes partially unstable, putting it in Group 3. This mutation was of particular interest because the R173 site in PTEN is also the third most commonly somatically mutated PTEN site in cancer (COSMIC database; accessed September 2018). Mouse models such as the heterozygous PTEN heterozygous mice (PTEN +/- mice) and PTEN +/C124S represent Groups 1 and 2, and their characteristics coincide with the predicted phenotypes. However, no clinically relevant mouse models for the low-risk PHTS group have been reported. We have generated and characterized a PTEN +/R173C PHTS mouse model. We show that the PTEN +/R173C mice have macrocephaly, fewer tumors and a significantly longer tumor-free survival compared to the PTEN +/-, thus providing further evidence that the above-proposed PTEN genotype-phenotype correlation holds true in PHTS. Acknowledgment: This work has been supported by PTEN Research. Citation Format: Wayne Pearce, Nicoletta Kessaris, Nicholas R. Leslie, Bart Vanhaesebroeck, Priyanka Tibarewal, Gala Classen, Virginia A Garcia, Victoria Rathbone, Nisha Kriplani, Georgia Constantinou. Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo approaches [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B22.

PTEN negatively regulates the Class I PI 3-kinases by metabolizing phosphatidylinositol(3,4,5)trisphosphate (PIP3) and acts as a tumor suppressor. Heterozygous germline PTEN mutations in humans manifest into a complex multiorgan disorder known as PTEN hamartoma tumor syndrome (PHTS). Patients present with hamartomatous skin lesions, benign GI polyps, have an increased risk of developing certain types of cancer (breast, colon, endometrium, thyroid, GI tract and kidney) and have macrocephaly and autism spectrum disorders (ASD). There is huge variability in disease symptoms in PHTS patients, the cause of which is unclear. It has been proposed that this correlates with the nature of the aberration in PTEN and its impact on PTEN expression and function, allowing to discriminate the following cancer-risk groups in PHTS patients: 1. High cancer risk: Patients expressing a catalytically inactive but stable PTEN protein (mis-sense PTEN mutations such as the C124S, G129R, R130Q etc.) have a severe phenotype, with benign tumors and aggressive malignant cancers at a young age of one of more tissues. 2. Medium risk: Patients with complete loss of PTEN expression due to insertions, deletions and truncations in the PTEN gene present with an intermediate severity with benign and malignant tumors. 3. Low risk: Patients with PTEN protein characteristics that do not fall into Group 1 or 2 but with some detectable suppression of AKT signaling. This covers a range of PTEN mutants, with often a mix of characteristics such as being unstable but active or even overactive, stable but partially inactive or no apparent activity defect. These patients have a mild phenotype, mainly related to a broad range of ASD symptoms such as macrocephaly, developmental delay and mental retardation. Some patients are severely autistic. Patients may have benign tumors and skin hamartomas, but malignant cancer is rare. Here we expand our analysis by characterizing a range of additional PHTS PTEN mutations using biochemical methods. Of the several mutations characterized, the R173C mutation leads to a PTEN enzyme that retains its activity but becomes partially unstable, putting it in Group 3. This mutation was of particular interest because the R173 site in PTEN is also the third most commonly somatically mutated PTEN site in cancer (COSMIC database; accessed September 2018). Mouse models such as the heterozygous PTEN heterozygous mice (PTEN +/- mice) and PTEN +/C124S represent Groups 1 and 2, and their characteristics coincide with the predicted phenotypes. However, no clinically relevant mouse models for the low-risk PHTS group have been reported. We have generated and characterized a PTEN +/R173C PHTS mouse model. We show that the PTEN +/R173C mice have macrocephaly, fewer tumors and a significantly longer tumor-free survival compared to the PTEN +/-, thus providing further evidence that the above-proposed PTEN genotype-phenotype correlation holds true in PHTS. Acknowledgment: This work has been supported by PTEN Research. Citation Format: Wayne Pearce, Nicoletta Kessaris, Nicholas R. Leslie, Bart Vanhaesebroeck, Priyanka Tibarewal, Gala Classen, Virginia A Garcia, Victoria Rathbone, Nisha Kriplani, Georgia Constantinou. Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo approaches [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B22.

The PIK3CA gene, which encodes the p110α catalytic subunit of PI3 kinase (PI3K), is mutationally activated in cancer and in overgrowth disorders known as PIK3CA-related overgrowth spectrum (PROS). To determine the consequences of genetic PIK3CA activation in a developmental context of relevance to both PROS and cancer, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous knockin of PIK3CAH1047R While heterozygous iPSCs remained largely similar to wild-type cells, homozygosity for PIK3CAH1047R caused widespread, cancer-like transcriptional remodeling, partial loss of epithelial morphology, up-regulation of stemness markers, and impaired differentiation to all three germ layers in vitro and in vivo. Genetic analysis of PIK3CA-associated cancers revealed that 64% had multiple oncogenic PIK3CA copies (39%) or additional PI3K signaling pathway-activating "hits" (25%). This contrasts with the prevailing view that PIK3CA mutations occur heterozygously in cancer. Our findings suggest that a PI3K activity threshold determines pathological consequences of oncogenic PIK3CA activation and provide insight into the specific role of this pathway in human pluripotent stem cells.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca(H1047R) hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA(H1047R) mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.

Nature 510, 407–411 (2014); doi:10.1038/nature13444 Queen Mary University London notified Nature and University College London that there is reason to question the provenance of the data for Fig. 5b, d, e of this Letter (Fig. 5a, c data are unaffected). Ongoing studies are investigating the reportedeffect of p110δ inhibition in the pancreatic cancer mouse model.

Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.

AIMS/HYPOTHESIS:While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS:We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS:While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION:Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS:All reagents are available upon request.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact.