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Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex.

Significance The PIK3CAH1047R mutation is a common cancer “driver” and also causes an array of benign but highly disfiguring overgrowth disorders. Human induced pluripotent stem cells engineered to express two copies of PIK3CAH1047R undergo cancer-like transcriptional remodeling and lose their ability to exit the stem cell state. A single mutant copy of PIK3CAH1047R, as observed in noncancerous overgrowth, had minimal effect on the stem cells and was fully compatible with normal differentiation. Combined with the finding of multiple PIK3CA mutant copies in human cancers, this suggests that a signaling threshold determines the disease consequences of PIK3CAH1047R, one of the commonest human oncogenic mutations., 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.

Activating PIK3CA mutations are known ‘drivers’ of human cancer and developmental overgrowth syndromes. We recently demonstrated that the ‘hotspot’ PIK3CAH1047R variant exerts unexpected allele dose-dependent effects on stemness in human pluripotent stem cells (hPSCs). In this study, we combine high-depth transcriptomics, total proteomics and reverse-phase protein arrays to reveal potentially disease-related alterations in heterozygous cells, and to assess the contribution of activated TGFβ signalling to the stemness phenotype of homozygous PIK3CAH1047R cells. We demonstrate signalling rewiring as a function of oncogenic PI3K signalling strength, and provide experimental evidence that self-sustained stemness is causally related to enhanced autocrine NODAL/TGFβ signalling. A significant transcriptomic signature of TGFβ pathway activation in heterozygous PIK3CAH1047R was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in homozygous PIK3CAH1047R hPSCs was reversed by pharmacological inhibition of NODAL/TGFβ signalling, but not by pharmacological PI3Kα pathway inhibition. Altogether, this provides the first in-depth analysis of PI3K signalling in hPSCs and directly links strong PI3K activation to developmental NODAL/TGFβ signalling. This work illustrates the importance of allele dosage and expression when artificial systems are used to model human genetic disease caused by activating PIK3CA mutations. This article has an associated First Person interview with the first author of the paper., Summary: Transcriptomic and proteomic analyses of iPSCs with allele-dose dependent expression of the PIK3CAH1047R oncogene confirm network rewiring in homozygous mutants, with self-sustained stemness likely driven by constitutive TGFβ/NODAL pathway activation.

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Activating PIK3CA mutations are known “drivers” of human cancer and developmental overgrowth syndromes. We recently demonstrated that the “hotspot” PIK3CAH1047R variant exerts unexpected allele dose-dependent effects on stemness in human pluripotent stem cells (hPSCs). In the present study, we combine high-depth transcriptomics, total proteomics and reverse-phase protein arrays to reveal potentially disease-related alterations in heterozygous cells, and to assess the contribution of activated TGFβ signalling to the stemness phenotype of PIK3CAH1047R homozygous cells. We demonstrate signalling rewiring as a function of oncogenic PI3K signalling dose, and provide experimental evidence that self-sustained stemness is causally related to enhanced autocrine NODAL/TGFβ signalling. A significant transcriptomic signature of TGFβ pathway activation in PIK3CAH1047R heterozygous was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in PIK3CAH1047R homozygous iPSCs was reversed by pharmacological inhibition of TGFβ signalling, but not by pharmacological PI3Kα pathway inhibition. Altogether, this provides the first in-depth analysis of PI3K signalling in human pluripotent stem cells and directly links dose-dependent PI3K activation to developmental NODAL/TGFβ signalling.

Isolated macrodactyly (OMIM 155500) belongs to a heterogeneous group of overgrowth syndromes. It is a congenital anomaly resulting in enlargement of all tissues localized to the terminal portions of a limb and caused by somatic mutations in the phosphatidylinositol 3-kinase catalytic alpha (PIK3CA, OMIM 171834) gene. Here we report a Hungarian girl with macrodactyly and syndactyly. Genetic screening at hotspots in the PIK3CA gene identified a mosaic mutation (c.1624G > A, p.Glu542Lys) in the affected tissue, but not in the peripheral blood. To date, this somatic mutation has been reported in eight patients affected by different forms of segmental overgrowth syndromes. Detailed analysis of the Hungarian child and previously reported cases suggests high phenotypic diversity associated with the p.Glu542Lys somatic mutation. The identification of the mutation provides a novel therapeutic modality for the affected patients: those who carry somatic mutations in the PIK3CA gene are potential recipients of a novel "repurposing" approach of rapamycin treatment.

Objective Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. Methods The human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. Results Like humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. Conclusions Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted., Highlights • SHORT syndrome features insulin resistance and reduced adiposity without dyslipidaemia and fatty liver. • A mouse model with a pathogenic human PI 3-Kinase mutation recapitulates this uncoupling. • Surprisingly, no adipose injury nor increased liver de novo lipogenesis is seen. • Energy expenditure is increased, causing resistance to diet-induced obesity. • This increases evidence for some beneficial metabolic effects of PI 3-Kinase inhibition.

Summary The organization of Rhodopsin-family G protein-coupled receptors (GPCRs) at the cell surface is controversial. Support both for and against the existence of dimers has been obtained in studies of mostly individual receptors. Here, we use a large-scale comparative study to examine the stoichiometric signatures of 60 receptors expressed by a single human cell line. Using bioluminescence resonance energy transfer- and single-molecule microscopy-based assays, we found that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appear to be monomeric. Overall, the analysis predicted that fewer than 20% of ∼700 Rhodopsin-family receptors form dimers. The clustered distribution of the dimers in our sample and a striking correlation between receptor organization and GPCR family size that we also uncover each suggest that receptor stoichiometry might have profoundly influenced GPCR expansion and diversification., Graphical Abstract, Highlights • Systematic analysis of 60 Rhodopsin-family GPCRs reveals that most are monomers • Rhodopsin-family root ancestor GPCRs are also monomers • Across families, rates of receptor diversification correlate with stoichiometry • Skewed family structure suggests dimerization increases receptor “fitness density”, The quaternary organization of Rhodopsin-family GPCRs is controversial. Felce et al. show that 60 receptors are mostly monomeric. They propose a simple explanation for the remarkable asymmetry in GPCR family structure, i.e., that it is underpinned by the lineage expansion of monomers rather than dimers.

SummaryHeterodimeric class IA phosphatidylinositol-3-kinases (PI3K) transduce signals from many receptor tyrosine kinases including the insulin receptor. PI3K recruitment to phosphotyrosines is mediated by Pik3r1 gene products including the most intensely studied PI3K regulatory subunit, p85α, which also binds and regulates the PIP3 phosphatase Pten, and the lipogenic transcription factor Xbp1. Mutations in human PIK3R1 cause SHORT syndrome, featuring lipodystrophy and severe insulin resistance which, uniquely, are uncoupled from fatty liver and dyslipidemia. We describe a novel mouse model of SHORT syndrome made by knock in of the Pik3r1 Y657X mutation. Homozygous embryos die at E11.5, while heterozygous mice exhibit pre-and postnatal growth impairment with diminished placental vascularity. Adipose tissue accretion on high fat feeding was reduced, however adipocyte size was unchanged and preadipocyte differentiation ex vivo unimpaired. Despite severe insulin resistance, heterozygous mice were hypolipidemic, and plasma adiponectin, liver weight, cholesterol, glycogen and triglyceride content were unchanged. Mild downregulation of lipogenic Srebp1, Srebp2 and Chrebp transcriptional activity but no suppression of Xbp1 target genes was seen after fasting. These findings give new insights into the developmental role of Pik3r1, and establish a model of lipodystrophic insulin resistance dissociated from dyslipidemia as seen in SHORT syndrome.

Background Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited and multi-disciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). Project To investigate the pathogenesis of 160 sporadic VMs in which known genetic causes had been excluded, sequencing of affected tissue DNA was undertaken using deep next generation sequencing with analysis optimised for detection of low mutant allele frequency. Results Mosaic activating variants in KRAS, NRAS, BRAF and MAP2K1 were identified, most commonly in AVM, both intracranial and extracranial. Transgenic zebrafish expressing BRAFV600E only in the vasculature recapitulated the human phenotype. Treatment of zebrafish with the BRAF inihibitor, vemurafinib, restored blood flow in AVM. Conclusions These findings reveal an important unifying cause of sporadic vascular malformations of different clinical types, and offer the potential of personalised medical treatment for affected individuals by repurposing of existing licensed cancer therapies.

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies. DOI: http://dx.doi.org/10.7554/eLife.23813.001, eLife digest Obesity and the diseases associated with it are among the biggest healthcare problems in developed countries. The word obesity means, simply, the accumulation of too much fat tissue in the body, but this ignores growing evidence that fat tissue is highly complex. Fat tissue is important for “mopping up” and storing excess calories safely, but also sends messages to the brain and other organs to report how full the body’s energy stores are. Understanding how fat tissues perform these roles will aid the development of strategies to treat or prevent obesity. A hormone called leptin acts as a signal of the status of the body’s fat stores. High levels of leptin in the blood tell the brain that the body has plenty of fat stored. On the other hand, if the levels of leptin in the blood become very low it tells the brain to prioritize finding food and shut down any nonessential processes. This helps to prevent the body from starving. It is not clear how the production of leptin is controlled, in part because fat tissues in different parts of the body behave very differently. Individuals who have a particular rare genetic mutation accumulate large amounts of fat tissue in their upper bodies and gradually lose fat tissue in their arms and legs. Despite accumulating a lot of fat tissue in the upper body, these individuals have extremely low levels of leptin in their blood. To investigate this genetic condition, Rocha et al. studied two children with the mutation and their healthy parents. The experiments show that this mutation alters a protein called mitofusin 2, which is found in cell compartments called mitochondria. Mitofusin 2 helps the mitochondria to bind to each other and to other parts of the cell, which is important for the mitochondria to generate the energy needed for vital cell processes. The mitochondria in the fat cells of the children are less closely linked to each other and have an unusual appearance compared to the mitochondria in the parents’ fat cells. Further experiments showed that some genes, including the one that produces leptin, are less active in the children compared to their parents – while other genes that are involved in starvation or stress responses are more active. This work suggests that mitochondria play an important role in regulating the production of leptin. Furthermore, it suggests that leptin or drugs that switch off stress-related genes may have the potential to be used to treat individuals with this particular mutation. DOI: http://dx.doi.org/10.7554/eLife.23813.002

WhetherRhodopsin-family G protein-coupled receptors (GPCRs) form dimers is highly controversial, with much data both for and against emerging from studies of mostly individual receptors. The types of large-scale comparative studies from which a consensus could eventually emerge have not previously been attempted. Here, we sought to determine the stoichiometric “signatures” of 60 GPCRs expressed by a single human cell-line using orthogonal bioluminescence resonance energy transfer-based and single-molecule microscopy assays. We observed that a relatively small fraction ofRhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appeared to be monomeric. Mapped onto the entire family the analysis predicted that fewer than 20% of the ~700Rhodopsin- family receptors form dimers. The clustered distribution ofRhodopsin-familydimers, and a striking correlation between receptor stoichiometry and GPCR family-size that we also identified, suggested that evolution has tended to favor the lineage expansion of monomers rather than dimers.One Sentence SummaryAnalysis of 71 GPCRs from a single cell reveals the strong tendency ofRhodopsin-family receptors to exist as monomers rather than form dimers.

Wilms tumor and nephroblastomatosis are associated with syndromic conditions including hemihyperplasia. Hemihyperplasia is genetically heterogeneous and may be the result of genomic abnormalities seen in Beckwith-Wiedemann syndrome, mosaic chromosome or genomic abnormalities, or somatic point mutations. Somatic missense mutations affecting the PI3K-AKT-MTOR pathway result in segmental overgrowth and are present in numerous benign and malignant tumors. Here, we report a fourth patient with asymmetric overgrowth due to a somatic PIK3CA mutation who had nephroblastomatosis or Wilms tumor. Similar to two of three reported patients with a somatic PIK3CA mutation and renal tumors, he shared a PIK3CA mutation affecting codon 1047, presented at birth with asymmetric overgrowth, and had fibroadipose overgrowth. Codon 1047 is most commonly affected by somatic mutations in PIK3CA-related overgrowth spectrum (PROS). While the fibroadipose overgrowth phenotype appears to be common in individuals with PIK3CA mutations at codon 1047, individuals with a clinical diagnosis of Klippel-Trenaunay syndrome or isolated lymphatic malformation also had mutations affecting this amino acid. Screening for Wilms tumor in individuals with PROS-related hemihyperplasia may be considered and, until the natural history is fully elucidated in larger cohort studies, may follow guidelines for Beckwith-Wiedemann syndrome, or isolated hemihyperplasia. It is not known if the specific PIK3CA mutation, the mosaic distribution, or the clinical presentation affect the Wilms tumor or nephroblastomatosis risk in individuals with PROS. © 2016 Wiley Periodicals, Inc.

We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.

Introduction L’hypomelanose d’Ito (HI) est definie par une hypopigmentation suivant les lignes de Blaschko, isolee ou associee a des manifestations principalement neurologiques. Nous rapportons ici une anomalie genetique en mosaique commune chez des patients atteints d’HI avec megalencephalie. Materiel et methodes Cinq enfants (3 filles, 2 garcons), sans antecedents familiaux, ayant des troubles pigmentaires et une megalencephalie ont ete etudies. Quatre avaient une hypopigmentation blaschko-lineaire, et un avait une meche blanche avec hypochromie scapulaire en quadrant et heterochromie irienne. Tous avaient une macrocrânie precoce (PC > 3 DS) et un retard des acquisitions. La patiente princeps (P1) et 3 autres patients avaient une epilepsie severe. L’IRM cerebrale montrait une megalencephalie diffuse ou unilaterale, sans anomalie de la gyration ni dysplasie corticale. Chez P1, un sequencage d’exome a haut debit a la recherche de variants de novo a ete effectue sur ADN de biopsie cutanee, et compare a l’ADN sanguin des parents. Chez les 4 autres patients, un sequencage cible sur ADN cutane a directement ete effectue. Resultats Le sequencage d’exome a identifie chez P1 une mutation activatrice du gene mTOR sur la peau, absente du sang et de l’ADN parental, representant 29 % des alleles. Chacun des 4 autres patients etait porteur d’une mutation mTOR differente sur ADN cutane, indetectable ou a un taux tres faible dans le sang, confirmant leur nature post-zygotique. L’etude de la voie mTOR sur fibroblastes cutanes de P1 apres depletion en acides amines a montre qu’elle etait constitutivement activee avec phosphorylation de la S6-kinase et Akt. De ce fait un traitement par everolimus, inhibiteur specifique de mTOR, a ete instaure mais interrompu en l’absence d’amelioration de l’epilepsie ou des fonctions cognitives. Discussion Meme si des anomalies chromosomiques en mosaique avaient ete rapportees dans l’HI, aucune anomalie genetique recurrente n’avait jusqu’ici ete identifiee. Les mutations de mTOR etaient ici en mosaique, ne touchant qu’une fraction des cellules. Leur role causal direct dans l’hypopigmentation est probable, l’activation de mTOR entrainant une diminution de la melanogenese par repression de MITF et des proteines qu’il active. Ce mecanisme rapproche l’HI de l’hypochromie de la sclerose tubereuse, due a une inactivation de TSC1 et TSC2, proteines inhibitrices de mTOR. Les troubles neurologiques qui resultent probablement aussi de l’activation de mTOR sont theoriquement accessibles aux analogues de la rapamycine malgre l’echec de l’everolimus chez notre patiente. Conclusion L’hypomelanose d’Ito avec megalencephalie constitue une nouvelle affection en mosaique touchant la voie de signalisation PI3K-Akt-mTOR. Nos resultats illustrent l’interet de l’individualisation de phenotypes homogenes d’affections en mosaique et du sequencage a haut debit.

Background The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting. Results Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an “average” clone and ~40% that of the “best” clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein. Conclusion Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where

Background Somatic activating mutations in PIK3CA , which encodes the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K) are frequently found in cancers and have been identified in a spectrum of mosaic overgrowth disorders ranging from isolated digit enlargement to more extensive overgrowth of the body, brain, or vasculature. We aimed to study affected dermal fibroblasts with a view to inform therapeutic studies, and to observe cancer-associated mutations in isolation. Methods We measured PIP 3 concentrations in dermal fibroblasts with endogenous PIK3CA mutations and in wild type fibroblasts using mass spectrometry, and we measured downstream signalling events with ELISA and immunoblotting. Cellular proliferation was evaluated with 5-bromo-2'-deoxyuridine incorporation, and cell size assessed by fluorescence-activated cell sorting (FACS). Glycolysis and mitochondrial tests were performed with an extracellular flux analyser (Seahorse Bioscience, Billerica, MA, USA), and mitochondrial potential was measured by FACS-based JC1 staining. Experiments were repeated after exposure to 5 nmol everolimus for 72 h. Findings Mutant fibroblasts had two times higher basal PIP 3 concentrations than wild-type fibroblasts (p=0·0017), with concomitant AKT and p70S6 activation downstream. The rate of cellular proliferation was higher in mutant cells under low serum conditions, but median cell size was not statistically different. Glycolytic capacity was similar between mutant and wild type fibroblasts, but subtle differences in mitochondrial function were detected with blunted responses to uncoupling agents and reduced membrane potentials. Treatment with everolimus reversed aberrant AKT ser473 and p70S6 signalling, slowed cellular proliferation, and reversed mitochondrial abnormalities, but was associated, paradoxically, with increases in PIP 3 concentrations. Interpretation These experiments demonstrate activation of the PI3K-AKT pathway in affected fibroblasts with increased proliferation, but no hypertrophy. Moreover, we identified changes in mitochondrial function in keeping with the known propensity of AKT to modulate elements of the Warburg effect. These results suggest that inhibitors of the mammalian target of rapamycin (mTOR) might be beneficial, but these inhibitors will require formal evaluation in clinical trials. More targeted therapy with p110α inhibitors is an enticing future option. Funding Wellcome Trust, Sackler Fund, National Instititute for Health Research.