Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Mitotic bookmarking by SWI/SNF subunits.

For cells to initiate and sustain a differentiated state, it is necessary that a 'memory' of this state is transmitted through mitosis to the daughter cells1-3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4-7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.

cBAF complex components and MYC cooperate early in CD8+ T cell fate.

The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

Partitioning of Chemotherapeutics into Nuclear Condensates-Opening the Door to New Approaches for Drug Development.

Klein et al. (2020) demonstrate for the first time that small-molecule cancer therapeutics are selectively partitioned and concentrated within phase-separated nuclear condensates, providing new insights to drug efficacy and creating the opportunity for enhanced control of therapeutic targeting.

Current and future perspectives of liquid biopsies in genomics-driven oncology.

Precision oncology seeks to leverage molecular information about cancer to improve patient outcomes. Tissue biopsy samples are widely used to characterize tumours but are limited by constraints on sampling frequency and their incomplete representation of the entire tumour bulk. Now, attention is turning to minimally invasive liquid biopsies, which enable analysis of tumour components (including circulating tumour cells and circulating tumour DNA) in bodily fluids such as blood. The potential of liquid biopsies is highlighted by studies that show they can track the evolutionary dynamics and heterogeneity of tumours and can detect very early emergence of therapy resistance, residual disease and recurrence. However, the analytical validity and clinical utility of liquid biopsies must be rigorously demonstrated before this potential can be realized.

AP-1 Transcription Factors and the BAF Complex Mediate Signal-Dependent Enhancer Selection.

Enhancer elements are genomic regulatory sequences that direct the selective expression of genes so that genetically identical cells can differentiate and acquire the highly specialized forms and functions required to build a functioning animal. To differentiate, cells must select from among the ∼106 enhancers encoded in the genome the thousands of enhancers that drive the gene programs that impart their distinct features. We used a genetic approach to identify transcription factors (TFs) required for enhancer selection in fibroblasts. This revealed that the broadly expressed, growth-factor-inducible TFs FOS/JUN (AP-1) play a central role in enhancer selection. FOS/JUN selects enhancers together with cell-type-specific TFs by collaboratively binding to nucleosomal enhancers and recruiting the SWI/SNF (BAF) chromatin remodeling complex to establish accessible chromatin. These experiments demonstrate how environmental signals acting via FOS/JUN and BAF coordinate with cell-type-specific TFs to select enhancer repertoires that enable differentiation during development.

Atomic Layer Deposition of Cu Electrocatalysts on Gas Diffusion Electrodes for CO2 Reduction.

Electrochemical reduction of CO2 using Cu catalysts enables the synthesis of C2+ products including C2H4 and C2H5OH. In this study, Cu catalysts were fabricated using plasma-enhanced atomic layer deposition (PEALD), achieving conformal deposition of catalysts throughout 3-D gas diffusion electrode (GDE) substrates while maintaining tunable control of Cu nanoparticle size and areal loading. The electrochemical CO2 reduction at the Cu surface yielded a total Faradaic efficiency (FE) > 75% for C2+ products. Parasitic hydrogen evolution was minimized to a FE of ∼10%, and a selectivity of 42.2% FE for C2H4 was demonstrated. Compared to a line-of-sight physical vapor deposition method, PEALD Cu catalysts show significant suppression of C1 products compared to C2+, which is associated with improved control of catalyst morphology and conformality within the porous GDE substrate. Finally, PEALD Cu catalysts demonstrated a stable performance for 15 h with minimal reduction in the C2H4 production rate.

Utilizing graph machine learning within drug discovery and development.

Graph machine learning (GML) is receiving growing interest within the pharmaceutical and biotechnology industries for its ability to model biomolecular structures, the functional relationships between them, and integrate multi-omic datasets - amongst other data types. Herein, we present a multidisciplinary academic-industrial review of the topic within the context of drug discovery and development. After introducing key terms and modelling approaches, we move chronologically through the drug development pipeline to identify and summarize work incorporating: target identification, design of small molecules and biologics, and drug repurposing. Whilst the field is still emerging, key milestones including repurposed drugs entering in vivo studies, suggest GML will become a modelling framework of choice within biomedical machine learning.

Synthetic vulnerabilities of mesenchymal subpopulations in pancreatic cancer.

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Intense and Mild First Epidemic Wave of Coronavirus Disease, The Gambia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is evolving differently in Africa than in other regions. Africa has lower SARS-CoV-2 transmission rates and milder clinical manifestations. Detailed SARS-CoV-2 epidemiologic data are needed in Africa. We used publicly available data to calculate SARS-CoV-2 infections per 1,000 persons in The Gambia. We evaluated transmission rates among 1,366 employees of the Medical Research Council Unit The Gambia (MRCG), where systematic surveillance of symptomatic cases and contact tracing were implemented. By September 30, 2020, The Gambia had identified 3,579 SARS-CoV-2 cases, including 115 deaths; 67% of cases were identified in August. Among infections, MRCG staff accounted for 191 cases; all were asymptomatic or mild. The cumulative incidence rate among nonclinical MRCG staff was 124 infections/1,000 persons, which is >80-fold higher than estimates of diagnosed cases among the population. Systematic surveillance and seroepidemiologic surveys are needed to clarify the extent of SARS-CoV-2 transmission in Africa.

Individual and combined effects of 5-year exposure to hyperandrogenemia and Western-style diet on metabolism and reproduction in female rhesus macaques.

STUDY QUESTION: What is the impact of prolonged exposure to hyperandrogenemia (T), Western-style diet (WSD) and the combination on metabolic and reproductive function in female rhesus macaques, particularly in the post-partum period? SUMMARY ANSWER: Combined T + WSD worsened measures of insulin sensitivity and parameters of cyclicity following prolonged (5 years) exposure, but there was no effect on post-partum metabolic function. WHAT IS KNOWN ALREADY: Women with hyperandrogenemia due to polycystic ovary syndrome are at higher risk for gestational diabetes and Type 2 diabetes post-partum, but it is unknown if this is related to hyperandrogenemia. Hyperandrogenemia in the presence of a WSD worsens metabolic function in female nonhuman primates. STUDY DESIGN, SIZE, DURATION: Female rhesus macaques began treatment near menarche (roughly 2.5 years of age) consisting of either cholesterol (control; C) or testosterone (T) implants (average serum levels 1.4 ng/ml) and exposure to standard monkey chow or a WSD (15 vs 36% of calories from fat, respectively). The four groups were maintained on treatment for 3 years, underwent a fertility trial in Year 4 and continued with treatments through Year 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolic measurements (glucose tolerance tests and double X-ray absorptiometry scans) were performed yearly, and results from 5 years of treatment are reported for all animals. Animals were bled daily for 30 days at 5 years to capture changes in ovarian cycle hormones, and ultrasound measurements were performed during the early follicular and luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: After 5 years of treatment, WSD exposure moderately increased body weight and body fat, although control animals also had a high body mass index due to ad libitum feeding. Animals in the T + WSD group had increased fasting insulin and insulin secretion during an intravenous glucose tolerance test. WSD exposure also altered ovarian cycles, delaying the time to the E2 surge, decreasing progesterone and anti-Müllerian hormone levels and increasing the number of antral follicles present by ultrasound. Longitudinal assessment of metabolic function for only those animals that became pregnant in Year 4 of treatment revealed no differences in post-partum metabolism between groups, although WSD resulted in overall elevated weights, body fat and measures of insulin resistance. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The small sample size and heterogeneity in metabolic effects observed in the T + WSD group are limitations of the current study, with only a subset of animals in this group showing impaired insulin resistance relative to controls. In addition, obesity in the C group prevented comparisons to lean animals. WIDER IMPLICATIONS OF THE FINDINGS: Hyperandrogenemia combined with WSD had a greater impact on insulin sensitivity and ovarian function than either treatment alone. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH grant P50 HD071836 to C.T.R., J.H. and C.T. and P51 OD011092 for support of the Oregon National Primate Research Center. All authors declare no competing interests.

Improving rigor and reproducibility in nonhuman primate research.

Nonhuman primates (NHPs) are a critical component of translational/preclinical biomedical research due to the strong similarities between NHP and human physiology and disease pathology. In some cases, NHPs represent the most appropriate, or even the only, animal model for complex metabolic, neurological, and infectious diseases. The increased demand for and limited availability of these valuable research subjects requires that rigor and reproducibility be a prime consideration to ensure the maximal utility of this scarce resource. Here, we discuss a number of approaches that collectively can contribute to enhanced rigor and reproducibility in NHP research.

Effects of pre- and postnatal protein restriction on maternal and offspring metabolism in the nonhuman primate.

Women in low- and middle-income countries frequently consume a protein-deficient diet during pregnancy and breastfeeding. The effects of gestational malnutrition on fetal and early postnatal development can have lasting adverse effects on offspring metabolism. Expanding on previous studies in rodent models, we utilized a nonhuman primate model of gestational and early-life protein restriction (PR) to evaluate effects on the organ development and glucose metabolism of juvenile offspring. Offspring were born to dams that had consumed a control diet containing 26% protein or a PR diet containing 13% protein. Offspring were maintained on the PR diet and studied [body and serum measurements, intravenous glucose tolerance tests (ivGTTs), and dual-energy X-ray absorptiometry scans] up to 7 mo of age, at which time tissues were collected for analysis. PR offspring had age-appropriate body weight and were euglycemic but exhibited elevated fasting insulin and reduced initial, but increased total, insulin secretion during an ivGTT at 6 mo of age. No changes were detected in pancreatic islets of PR juveniles; however, PR did induce changes, including reduced kidney size, and changes in liver, adipose tissue, and muscle gene expression in other peripheral organs. Serum osteocalcin was elevated and bone mineral content and density were reduced in PR juveniles, indicating a significant impact of PR on early postnatal bone development.

Unveil early-stage nanocytotoxicity by a label-free single cell pH nanoprobe.

Single-cell analysis is an emerging research area that aims to reveal delicate cellular status and underlying mechanisms by conquering the intercellular heterogeneity. Current single-cell research methods, however, are highly dependent on cell-destructive protocols and cannot sequentially display the progress of cellular events. A recently developed pH nanoprobe in our lab conceptually showed its ability to detect intracellular pH (pHi) without cell labeling or disruption. In the present study, we took the cytotoxicity of nanoparticles (NPs) as a typical example of cell heterogeneity, to testify the practicality of the pH nanoprobe in interpreting cell status. Three types of NPs (CeO2, TiO2, and SiO2) were employed to generate varied toxic effects. Results showed that the traditional assays - including cell viability, intracellular ROS generation, and mitochondrial inner membrane depolarization - not only failed to report the nanotoxicity accurately and timely, but also drew confusing or misleading conclusions. The pH nanoprobe revealed explicit pHi changes induced by the NPs, which corresponded well with the cell damages found by the transmission electron microscopic (TEM) imaging. Besides, our results unveiled an unexpectedly devastating effect of SiO2 NPs on cells during the early stage NP-cell interaction. The developed novel pH nanoprobe demonstrated a rapid sensing capability at single-cell resolution with minimum invasiveness. Therefore, it may become a promising alternative for a wide range of applications in areas such as single-cell research and precision medicine.

Maternal serum glycosylated fibronectin as a short-term predictor of preeclampsia: a prospective cohort study.

BACKGROUND: Preeclampsia is a major pregnancy complication that results in significant maternal and infant mortality, most of which occurs in low and middle-income countries. The accurate and timely diagnosis of preeclampsia is critical in management of affected pregnancies to reduce maternal and fetal/neonatal morbidity and mortality, yet difficulties remain in establishing the rigorous diagnosis of preeclampsia based on clinical parameters alone. Biomarkers that detect biochemical disease have been proposed as complements or alternatives to clinical criteria to improve diagnostic accuracy. This cohort study assessed the performance of several biomarkers, including glycosylated fibronectin (GlyFn), to rule-in or rule-out preeclampsia within 4 weeks in a cohort of women at increased risk for preeclampsia. METHODS: 151 women with risk factors for or clinical signs and symptoms of preeclampsia were selected from a prospective cohort. Maternal serum samples were collected between 20 and 37 weeks of gestation. Clinical suspicion of preeclampsia was defined as presence of new-onset proteinuria, or clinical symptoms of preeclampsia. Subjects with a clinical diagnosis of preeclampsia at the time of enrollment were excluded. GlyFn, pregnancy-associated plasma protein-A2 (PAPPA2), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1) were measured by immunoassay. GlyFn was also determined using a rapid point-of care (POC) test format. Receiver-operating characteristic (ROC) curves derived from logistic regression analysis were used to determine the classification performance for each analyte. RESULTS: 32 of 151 (21%) women developed a clinical diagnosis of preeclampsia within 4 weeks. All biomarkers exhibited good classification performance [GlyFn (area under the curve (AUROC) = 0.94, 91% sensitivity, 86% specificity); PAPPA2 AUC = 0.92, 87% sensitivity, 77% specificity; PlGF AUC = 0.90, 81% sensitivity, 83% specificity; sFlt-1 AUC = 0.92, 84% sensitivity, 91% specificity. The GlyFn immunoassay and the rapid POC test showed a correlation of r = 0.966. CONCLUSIONS: In this prospective cohort, serum biomarkers of biochemical disease were effective in short-term prediction of preeclampsia, and the performance of GlyFn in particular as a POC test may meet the needs of rapid and accurate triage and intervention.

High Frequency of Ovarian Cyst Development in Vhl2B/+;Snf5+/- Mice.

The new paradigm of mutations in chromatin-modifying genes as driver events in the development of cancers has proved challenging to resolve the complex influences over disease phenotypes. In particular, impaired activities of members of the SWI/SNF chromatin remodeling complex have appeared in an increasing variety of tumors. Mutations in SNF5, a member of this ubiquitously expressed complex, arise in almost all cases of malignant rhabdoid tumor in the absence of additional genetic alterations. Therefore, we studied how activation of additional oncogenic pathways might shift the phenotype of disease driven by SNF5 loss. With the use of a genetically engineered mouse model, we examined the effects of a hypomorphic Vhl2B allele on disease phenotype, with a modest up-regulation of the hypoxia response pathway. Snf5+/-;Vhl2B/+ mice did not demonstrate a substantial difference in overall survival or a change in malignant rhabdoid tumor development. However, a high percentage of female mice showed complex hemorrhagic ovarian cysts, a phenotype rarely found in either parental mouse strain. These lesions also showed mosaic expression of SNF5 by immunohistochemistry. Therefore, our studies implicate that modest changes in angiogenic regulation interact with perturbations of SWI/SNF complex activity to modulate disease phenotypes.

Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity.

Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27(me3)) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122(+) NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

Combined androgen excess and Western-style diet accelerates adipose tissue dysfunction in young adult, female nonhuman primates.

STUDY QUESTION: What are the separate and combined effects of mild hyperandrogenemia and consumption of a high-fat Western-style diet (WSD) on white adipose tissue (WAT) morphology and function in young adult female nonhuman primates? SUMMARY ANSWER: Combined exposure to mild hyperandrogenemia and WSD induces visceral omental (OM-WAT) but not subcutaneous (SC-WAT) adipocyte hypertrophy that is associated with increased uptake and reduced mobilization of free fatty acids. WHAT IS KNOWN ALREADY: Mild hyperandrogenemia in females, principally in the context of polycystic ovary syndrome, is often associated with adipocyte hypertrophy, but the mechanisms of associated WAT dysfunction and depot specificity remain poorly understood. STUDY DESIGN, SIZE AND DURATION: Female rhesus macaques were randomly assigned at 2.5 years of age (near menarche) to receive either cholesterol (C; n = 20) or testosterone (T; n = 20)-containing silastic implants to elevate T levels 5-fold above baseline. Half of each of these groups was then fed either a low-fat monkey chow diet or WSD, resulting in four treatment groups (C, control diet; T alone; WSD alone; T + WSD; n = 10/group) that were maintained until the current analyses were performed at 5.5 years of age (3 years of treatment, young adults). PARTICIPANTS/MATERIALS, SETTING AND METHODS: OM and SC-WAT biopsies were collected and analyzed longitudinally for in vivo changes in adipocyte area and blood vessel density, and ex vivo basal and insulin-stimulated fatty acid uptake and basal and isoproterenol-stimulated lipolysis. MAIN RESULTS AND THE ROLE OF CHANCE: In years 2 and 3 of treatment, the T + WSD group exhibited a significantly greater increase in OM adipocyte size compared to all other groups (P < 0.05), while the size of SC adipocytes measured at the end of the study was not significantly different between groups. In year 3, both WAT depots from the WSD and T + WSD groups displayed a significant reduction in local capillary length and vessel junction density (P < 0.05). In year 3, insulin-stimulated fatty acid uptake in OM-WAT was increased in the T + WSD group compared to year 2 (P < 0.05). In year 3, basal lipolysis was blunted in the T and T + WSD groups in both WAT depots (P < 0.01), while isoproterenol-stimulated lipolysis was significantly blunted in the T and T + WSD groups only in SC-WAT (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: At this stage of the study, subjects were still relatively young adults, so that the effects of mild hyperandrogenemia and WSD may become more apparent with increasing age. WIDER IMPLICATIONS OF THE FINDINGS: The combination of mild hyperandrogenemia and WSD accelerates the development of WAT dysfunction through T-specific (suppression of lipolytic response by T), WSD-dependent (reduced capillary density) and combined T + WSD (increased fatty acid uptake) mechanisms. These data support the idea that combined hyperandrogenemia and WSD increases the risk of developing obesity in females. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number P50 HD071836 to C.T.R. and award number OD 011092 from the Office of the Director, National Institutes of Health, for operation of the Oregon National Primate Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.