The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

Sex-dependent placental methylation quantitative trait loci provide insight into the prenatal origins of childhood onset traits and conditions.

Molecular quantitative trait loci (QTLs) allow us to understand the biology captured in genome-wide association studies (GWASs). The placenta regulates fetal development and shows sex differences in DNA methylation. We therefore hypothesized that placental methylation QTL (mQTL) explain variation in genetic risk for childhood onset traits, and that effects differ by sex. We analyzed 411 term placentas from two studies and found 49,252 methylation (CpG) sites with mQTL and 2,489 CpG sites with sex-dependent mQTL. All mQTL were enriched in regions that typically affect gene expression in prenatal tissues. All mQTL were also enriched in GWAS results for growth- and immune-related traits, but male- and female-specific mQTL were more enriched than cross-sex mQTL. mQTL colocalized with trait loci at 777 CpG sites, with 216 (28%) specific to males or females. Overall, mQTL specific to male and female placenta capture otherwise overlooked variation in childhood traits.

Cytolytic circumsporozoite-specific memory CD4+ T cell clones are expanded during Plasmodium falciparum infection.

Clinical immunity against Plasmodium falciparum infection develops in residents of malaria endemic regions, manifesting in reduced clinical symptoms during infection and in protection against severe disease but the mechanisms are not fully understood. Here, we compare the cellular and humoral immune response of clinically immune (0-1 episode over 18 months) and susceptible (at least 3 episodes) during a mild episode of Pf malaria infection in a malaria endemic region of Malawi, by analysing peripheral blood samples using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses. In the clinically immune, we find increased proportions of circulating follicular helper T cells and classical monocytes, while the humoral immune response shows characteristic age-related differences in the protected. Presence of memory CD4+ T cell clones with a strong cytolytic ZEB2+ T helper 1 effector signature, sharing identical T cell receptor clonotypes and recognizing the Pf-derived circumsporozoite protein (CSP) antigen are found in the blood of the Pf-infected participants gaining protection. Moreover, in clinically protected participants, ZEB2+ memory CD4+ T cells express lower level of inhibitory and chemotactic receptors. We thus propose that clonally expanded ZEB2+ CSP-specific cytolytic memory CD4+ Th1 cells may contribute to clinical immunity against the sporozoite and liver-stage Pf malaria.

Multi-Omics Data Integration Reveals Sex-Dependent Hippocampal Programming by Maternal High-Fat Diet during Lactation in Adult Mouse Offspring.

Early-life exposure to high-fat diets (HF) can program metabolic and cognitive alterations in adult offspring. Although the hippocampus plays a crucial role in memory and metabolic homeostasis, few studies have reported the impact of maternal HF on this structure. We assessed the effects of maternal HF during lactation on physiological, metabolic, and cognitive parameters in young adult offspring mice. To identify early-programming mechanisms in the hippocampus, we developed a multi-omics strategy in male and female offspring. Maternal HF induced a transient increased body weight at weaning, and a mild glucose intolerance only in 3-month-old male mice with no change in plasma metabolic parameters in adult male and female offspring. Behavioral alterations revealed by a Barnes maze test were observed both in 6-month-old male and female mice. The multi-omics strategy unveiled sex-specific transcriptomic and proteomic modifications in the hippocampus of adult offspring. These studies that were confirmed by regulon analysis show that, although genes whose expression was modified by maternal HF were different between sexes, the main pathways affected were similar with mitochondria and synapses as main hippocampal targets of maternal HF. The effects of maternal HF reported here may help to better characterize sex-dependent molecular pathways involved in cognitive disorders and neurodegenerative diseases.

Amyloid-Beta Peptides Trigger Premature Functional and Gene Expression Alterations in Human-Induced Neurons.

Alzheimer's disease (AD) is the most prevalent cause of dementia in the elderly, characterized by the presence of amyloid-beta (Aß) plaques, neurofibrillary tangles, neuroinflammation, synapse loss and neurodegeneration in the brain. The amyloid cascade hypothesis postulates that deposition of Aß peptides is the causative agent of AD pathology, but we still lack comprehensive understanding of the molecular mechanisms connecting Aß peptides to neuronal dysfunctions in AD. In this work, we investigate the early effects of Aß peptide accumulation on the functional properties and gene expression profiles of human-induced neurons (hiNs). We show that hiNs acutely exposed to low concentrations of both cell-secreted Aß peptides or synthetic Aß1-42 exhibit alterations in the frequency of calcium transients suggestive of increased neuronal excitability. Using single-cell RNA sequencing, we also show that cell-secreted Aß up-regulates the expression of several synapse-related genes and down-regulates the expression of genes associated with metabolic stress mainly in glutamatergic neurons and, to a lesser degree, in GABAergic neurons and astrocytes. These neuronal alterations correlate with activation of the SEMA5, EPHA and NECTIN signaling pathways, which are important regulators of synaptic plasticity. Altogether, our findings indicate that slight elevations in Aß concentrations are sufficient to elicit transcriptional changes in human neurons, which can contribute to early alterations in neural network activity.

Epigenetic and Transcriptomic Programming of HSC Quiescence Signaling in Large for Gestational Age Neonates.

Excessive fetal growth is associated with DNA methylation alterations in human hematopoietic stem and progenitor cells (HSPC), but their functional impact remains elusive. We implemented an integrative analysis combining single-cell epigenomics, single-cell transcriptomics, and in vitro analyses to functionally link DNA methylation changes to putative alterations of HSPC functions. We showed in hematopoietic stem cells (HSC) from large for gestational age neonates that both DNA hypermethylation and chromatin rearrangements target a specific network of transcription factors known to sustain stem cell quiescence. In parallel, we found a decreased expression of key genes regulating HSC differentiation including EGR1, KLF2, SOCS3, and JUNB. Our functional analyses showed that this epigenetic programming was associated with a decreased ability for HSCs to remain quiescent. Taken together, our multimodal approach using single-cell (epi)genomics showed that human fetal overgrowth affects hematopoietic stem cells' quiescence signaling via epigenetic programming.

T cell receptor and IL-2 signaling strength control memory CD8+ T cell functional fitness via chromatin remodeling.

Cognate antigen signal controls CD8+ T cell priming, expansion size and effector versus memory cell fates, but it is not known if and how it modulates the functional features of memory CD8+ T cells. Here we show that the strength of T cell receptor (TCR) signaling controls the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that competitively re-expand upon secondary antigen encounter. Combining strong TCR and intact IL-2 signaling during priming synergistically induces genome-wide chromatin accessibility in regions targeting a wide breadth of biological processes, consistent with greater T cell functional fitness. Chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins correlates with faster intracellular calcium accumulation, initiation of cell cycle and more robust expansion. High-dimensional flow-cytometry analysis of these T cells also highlights higher diversity of T cell subsets and phenotypes with T cells primed with stronger TCR and IL-2 stimulation than those primed with weaker strengths of TCR and/or IL-2 signals. These results formally show that epitope selection in vaccine design impacts memory CD8+ T cell epigenetic programming and function.

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes.

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 × 10-8) with GDM, mapping to/near MTNR1B (P = 4.3 × 10-54), TCF7L2 (P = 4.0 × 10-16), CDKAL1 (P = 1.6 × 10-14), CDKN2A-CDKN2B (P = 4.1 × 10-9) and HKDC1 (P = 2.9 × 10-8). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Memory CD8+ T cells mediate early pathogen-specific protection via localized delivery of chemokines and IFNγ to clusters of monocytes.

While cognate antigen drives clonal expansion of memory CD8+ T (CD8+ TM) cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early protection before clonal expansion occurs. Here, using distinct models of immunization, we establish that cognate antigen recognition by CD8+ TM cells on dendritic cells initiates their rapid and coordinated production of a burst of CCL3, CCL4, and XCL1 chemokines under the transcriptional control of interferon (IFN) regulatory factor 4. Using intravital microscopy imaging, we reveal that CD8+ TM cells undergo antigen-dependent arrest in splenic red pulp clusters of CCR2+Ly6C+ monocytes to which they deliver IFNγ and chemokines. IFNγ enables chemokine-induced microbicidal activities in monocytes for protection. Thus, rapid and effective CD8+ TM cell responses require spatially and temporally coordinated events that quickly restrict microbial pathogen growth through the local delivery of activating chemokines to CCR2+Ly6C+ monocytes.

Epigenome-Wide Association Study Reveals Methylation Loci Associated With Offspring Gestational Diabetes Mellitus Exposure and Maternal Methylome.

OBJECTIVE: Gestational diabetes mellitus (GDM) is associated with an increased risk of obesity and insulin resistance in offspring later in life, which might be explained by epigenetic changes in response to maternal hyperglycemic exposure. RESEARCH DESIGN AND METHODS: We explored the association between GDM exposure and maternal blood and newborn cord blood methylation in 536 mother-offspring pairs from the prospective FinnGeDi cohort using Illumina MethylationEPIC 850K BeadChip arrays. We assessed two hypotheses. First, we tested for shared maternal and offspring epigenetic effects resulting from GDM exposure. Second, we tested whether GDM exposure and maternal methylation had an epigenetic effect on the offspring. RESULTS: We did not find any epigenetic marks (differentially methylated CpG probes) with shared and consistent effects between mothers and offspring. After including maternal methylation in the model, we identified a single significant (false discovery rate 1.38 × 10-2) CpG at the cg22790973 probe (TFCP2) associated with GDM. We identified seven additional FDR-significant interactions of maternal methylation and GDM status, with the strongest association at the same cg22790973 probe (TFCP2), as well as cg03456133, cg24440941 (H3C6), cg20002843 (LOC127841), cg19107264, and cg11493553 located within the UBE3C gene and cg17065901 in FAM13A, both susceptibility genes for type 2 diabetes and BMI, and cg23355087 within the DLGAP2 gene, known to be involved in insulin resistance during pregnancy. CONCLUSIONS: Our study reveals the potential complexity of the epigenetic transmission between mothers with GDM and their offspring, likely determined by not only GDM exposure but also other factors indicated by maternal epigenetic status, such as maternal metabolic history.

Prenatal Hyperglycemia Exposure and Cellular Stress, a Sugar-Coated View of Early Programming of Metabolic Diseases.

Worldwide, the number of people with diabetes has quadrupled since 1980 reaching 422 million in 2014 (World Health Organization). This distressing rise in diabetes also affects pregnant women and thus, in regard to early programming of adult diseases, creates a vicious cycle of metabolic dysfunction passed from one generation to another. Metabolic diseases are complex and caused by the interplay between genetic and environmental factors. High-glucose exposure during in utero development, as observed with gestational diabetes mellitus (GDM), is an established risk factor for metabolic diseases. Despite intense efforts to better understand this phenomenon of early memory little is known about the molecular mechanisms associating early exposure to long-term diseases risk. However, evidence promotes glucose associated oxidative stress as one of the molecular mechanisms able to influence susceptibility to metabolic diseases. Thus, we decided here to further explore the relationship between early glucose exposure and cellular stress in the context of early development, and focus on the concept of glycemic memory, its consequences, and sexual dimorphic and epigenetic aspects.

DNA methylation loci in placenta associated with birthweight and expression of genes relevant for early development and adult diseases.

BACKGROUND: Birthweight marks an important milestone of health across the lifespan, including cardiometabolic disease risk in later life. The placenta, a transient organ at the maternal-fetal interface, regulates fetal growth. Identifying genetic loci where DNA methylation in placenta is associated with birthweight can unravel genomic pathways that are dysregulated in aberrant fetal growth and cardiometabolic diseases in later life. RESULTS: We performed placental epigenome-wide association study (EWAS) of birthweight in an ethnic diverse cohort of pregnant women (n = 301). Methylation at 15 cytosine-(phosphate)-guanine sites (CpGs) was associated with birthweight (false discovery rate (FDR) < 0.05). Methylation at four (26.7%) CpG sites was associated with placental transcript levels of 15 genes (FDR < 0.05), including genes known to be associated with adult lipid traits, inflammation and oxidative stress. Increased methylation at cg06155341 was associated with higher birthweight and lower FOSL1 expression, and lower FOSL1 expression was correlated with higher birthweight. Given the role of the FOSL1 transcription factor in regulating developmental processes at the maternal-fetal interface, epigenetic mechanisms at this locus may regulate fetal development. We demonstrated trans-tissue portability of methylation at four genes (MLLT1, PDE9A, ASAP2, and SLC20A2) implicated in birthweight by a previous study in cord blood. We also found that methylation changes known to be related to maternal underweight, preeclampsia and adult type 2 diabetes were associated with lower birthweight in placenta. CONCLUSION: We identified novel placental DNA methylation changes associated with birthweight. Placental epigenetic mechanisms may underlie dysregulated fetal development and early origins of adult cardiometabolic diseases. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT00912132.

Retargeting of macroH2A following mitosis to cytogenetic-scale heterochromatic domains.

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here, we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly incorporated macroH2A retargets the same large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of a new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell.

Correction: Genetic variants influence on the placenta regulatory landscape.

[This corrects the article DOI: 10.1371/journal.pgen.1007785.].

Effect of diet in females (F1) from prenatally undernourished mothers on metabolism and liver function in the F2 progeny is sex-specific.

PURPOSE: Poor maternal nutrition sensitises to the development of metabolic diseases and obesity in adulthood over several generations. The prevalence increases when offspring is fed with a high-fat (HF) diet after weaning. This study aims to determine whether such metabolic profiles can be transmitted to the second generation and even aggravated when the mothers were exposed to overnutrition, with attention to potential sex differences. METHODS: Pregnant Wistar rats were subjected to ad libitum (control) or 70% food-restricted diet (FR) during gestation (F0). At weaning, F1 females were allocated to three food protocols: (1) standard diet prior to and throughout gestation and lactation, (2) HF diet prior to and standard diet throughout gestation and lactation, and (3) HF diet prior to and throughout gestation and lactation. F2 offspring was studied between 16 and 32 weeks of age. RESULTS: FR-F2 offspring on standard diet showed normal adiposity and had no significant metabolic alterations in adulthood. Maternal HF diet resulted in sex-specific effects with metabolic disturbances more apparent in control offspring exposed to HF diet during gestation and lactation. Control offspring displayed glucose intolerance associated with insulin resistance in females. Female livers overexpressed lipogenesis genes and those of males the genes involved in lipid oxidation. Gene expression was significantly attenuated in the FR livers. Increased physical activity associated with elevated corticosterone levels was observed in FR females on standard diet and in all females from overnourished mothers. CONCLUSIONS: Maternal undernutrition during gestation (F0) improves the metabolic health of second-generation offspring with more beneficial effects in females.

Genetic variants influence on the placenta regulatory landscape.

From genomic association studies, quantitative trait loci analysis, and epigenomic mapping, it is evident that significant efforts are necessary to define genetic-epigenetic interactions and understand their role in disease susceptibility and progression. For this reason, an analysis of the effects of genetic variation on gene expression and DNA methylation in human placentas at high resolution and whole-genome coverage will have multiple mechanistic and practical implications. By producing and analyzing DNA sequence variation (n = 303), DNA methylation (n = 303) and mRNA expression data (n = 80) from placentas from healthy women, we investigate the regulatory landscape of the human placenta and offer analytical approaches to integrate different types of genomic data and address some potential limitations of current platforms. We distinguish two profiles of interaction between expression and DNA methylation, revealing linear or bimodal effects, reflecting differences in genomic context, transcription factor recruitment, and possibly cell subpopulations. These findings help to clarify the interactions of genetic, epigenetic, and transcriptional regulatory mechanisms in normal human placentas. They also provide strong evidence for genotype-driven modifications of transcription and DNA methylation in normal placentas. In addition to these mechanistic implications, the data and analytical methods presented here will improve the interpretability of genome-wide and epigenome-wide association studies for human traits and diseases that involve placental functions.

Late-life targeting of the IGF-1 receptor improves healthspan and lifespan in female mice.

Diminished growth factor signaling improves longevity in laboratory models, while a reduction in the somatotropic axis is favorably linked to human aging and longevity. Given the conserved role of this pathway on lifespan, therapeutic strategies, such as insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibodies (mAb), represent a promising translational tool to target human aging. To this end, we performed a preclinical study in 18-mo-old male and female mice treated with vehicle or an IGF-1R mAb (L2-Cmu, Amgen Inc), and determined effects on aging outcomes. Here we show that L2-Cmu preferentially improves female healthspan and increases median lifespan by 9% (P = 0.03) in females, along with a reduction in neoplasms and inflammation (P ≤ 0.05). Thus, consistent with other models, targeting IGF-1R signaling appears to be most beneficial to females. Importantly, these effects could be achieved at advanced ages, suggesting that IGF-1R mAbs could represent a promising therapeutic candidate to delay aging.

Intrauterine Hyperglycemia Is Associated with an Impaired Postnatal Response to Oxidative Damage.

Hyperglycemia and other adverse exposures early in life that reprogram stem cells may lead to long-lasting phenotypic influences over the lifetime of an individual. Hyperglycemia and oxidative stress cause DNA damage when they exceed the protective capabilities of the cell, in turn affecting cellular function. DNA damage in response to hyperglycemia and oxidative stress was studied in human umbilical cord mesenchymal stem cells (hUC-MSCs) from large-for-gestational-age (LGA) infants of mothers with gestational diabetes mellitus (LGA-GDM) and control subjects. We tested the response of these cells to hyperglycemia and oxidative stress, measuring reactive oxygen species (ROS) levels and antioxidant enzyme activities. We find that hUC-MSCs from LGA-GDM infants have increased DNA damage when exposed to oxidative stress. With the addition of hyperglycemic conditions, these cells have an increase in ROS and a decrease in antioxidant glutathione peroxidase (GPx) activity, indicating a mechanism for the increased ROS and DNA damage. This study demonstrates that a memory of in utero hyperglycemia, mediated through downregulation of GPx activity, leads to an increased susceptibility to oxidative stress. The alteration of GPx function in self-renewing stem cells, can mediate the effect of intrauterine hyperglycemia to be propagated into adulthood and contribute to disease susceptibility.

Apc inactivation, but not obesity, synergizes with Pten deficiency to drive intestinal stem cell-derived tumorigenesis.

Obesity is a major risk factor for colorectal cancer and can accelerate Lgr5+ intestinal stem cell (ISC)-derived tumorigenesis after the inactivation of Apc However, whether non-canonical pathways involving PI3K-Akt signaling in ISCs can lead to tumor formation, and if this can be further exacerbated by obesity is unknown. Despite the synergy between Pten and Apc inactivation in epithelial cells on intestinal tumor formation, their combined role in Lgr5+-ISCs, which are the most rapidly dividing ISC population in the intestine, is unknown. Lgr5+-GFP mice were provided low-fat diet (LFD) or high-fat diet (HFD) for 8 months, and the transcriptome was evaluated in Lgr5+-ISCs. For tumor studies, Lgr5+-GFP and Lgr5+-GFP-Ptenflox/flox mice were tamoxifen treated to inactivate Pten in ISCs and provided LFD or HFD until 14-15 months of age. Finally, various combinations of Lgr5+-ISC-specific, Apc- and Pten-deleted mice were generated and evaluated for histopathology and survival. HFD did not overtly alter Akt signaling in ISCs, but did increase other metabolic pathways. Pten deficiency, but not HFD, increased BrdU-positive cells in the small intestine (P < 0.05). However, combining Pten and Apc deficiency synergistically increased proliferative markers, tumor pathology and mortality, in a dose-dependent fashion (P < 0.05). In summary, we show that HFD alone fails to drive Akt signaling in ISCs and that Pten deficiency is dispensable as a tumor suppressor in Lgr5+-ISCs. However, combining Pten and Apc deficiency in ISCs synergistically increases proliferation, tumor formation and mortality. Thus, aberrant Wnt/ß-catenin, rather than PI3K-Akt signaling, is requisite for obesity to drive Lgr5+ ISC-derived tumorigenesis.