CRISPR-Cas13d screens identify KILR, a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

BACKGROUND: Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. METHODS: We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. RESULTS: We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. CONCLUSIONS: Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis.

BACKGROUND: Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. METHODS: We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. FINDINGS: The S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. INTERPRETATION: These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. FUNDING: Australian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179.

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.

Redefining normal breast cell populations using long noncoding RNAs.

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

CRISPR screens identify gene targets at breast cancer risk loci.

BACKGROUND: Genome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in non-coding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS. RESULTS: Here, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants. CONCLUSIONS: We demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase.

Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

CRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni.

Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

CRISPR/Cas9: A new tool for the study and control of helminth parasites.

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9-targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9-mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.

eQTL Colocalization Analyses Identify NTN4 as a Candidate Breast Cancer Risk Gene.

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

Chromatin interactome mapping at 139 independent breast cancer risk signals.

BACKGROUND: Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. RESULTS: We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region. CONCLUSIONS: Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

Non-coding RNAs underlie genetic predisposition to breast cancer.

BACKGROUND: Genetic variants identified through genome-wide association studies (GWAS) are predominantly non-coding and typically attributed to altered regulatory elements such as enhancers and promoters. However, the contribution of non-coding RNAs to complex traits is not clear. RESULTS: Using targeted RNA sequencing, we systematically annotated multi-exonic non-coding RNA (mencRNA) genes transcribed from 1.5-Mb intervals surrounding 139 breast cancer GWAS signals and assessed their contribution to breast cancer risk. We identify more than 4000 mencRNA genes and show their expression distinguishes normal breast tissue from tumors and different breast cancer subtypes. Importantly, breast cancer risk variants, identified through genetic fine-mapping, are significantly enriched in mencRNA exons, but not the promoters or introns. eQTL analyses identify mencRNAs whose expression is associated with risk variants. Furthermore, chromatin interaction data identify hundreds of mencRNA promoters that loop to regions that contain breast cancer risk variants. CONCLUSIONS: We have compiled the largest catalog of breast cancer-associated mencRNAs to date and provide evidence that modulation of mencRNAs by GWAS variants may provide an alternative mechanism underlying complex traits.

MiR-29b-1-5p is altered in BRCA1 mutant tumours and is a biomarker in basal-like breast cancer.

Depletion of BRCA1 protein in mouse mammary glands results in defects in lactational development and increased susceptibility to mammary cancer. Extensive work has focussed on the role of BRCA1 in the normal breast and in the development of breast cancer, the cell of origin for BRCA1 tumours and the protein-coding genes altered in BRCA1 deficient cells. However, the role of non-coding RNAs in BRCA1-deficient cells is poorly understood. To evaluate miRNA expression in BRCA1 deficient mammary cells, RNA sequencing was performed on the mammary glands of Brca1 knockout mice. We identified 140 differentially expressed miRNAs, 9 of which were also differentially expressed in human BRCA1 breast tumours or familial non-BRCA1 patients and during normal gland development. We show that BRCA1 binds to putative cis-elements in promoter regions of the miRNAs with the potential to regulate their expression, and that four miRNAs (miR-29b-1-5p, miR-664, miR-16-2 and miR-744) significantly stratified the overall survival of basal-like tumours. Importantly the prognostic value of miR-29b-1-5p was higher in significance than several commonly used clinical biomarkers. These results emphasise the role of Brca1 in modulating expression of miRNAs and highlights the potential for BRCA1 regulated miRNAs to be informative biomarkers associated with BRCA1 loss and survival in breast cancer.

Association analysis identifies 65 new breast cancer risk loci.

Breast cancer risk is influenced by rare coding variants in susceptibility genes, such as BRCA1, and many common, mostly non-coding variants. However, much of the genetic contribution to breast cancer risk remains unknown. Here we report the results of a genome-wide association study of breast cancer in 122,977 cases and 105,974 controls of European ancestry and 14,068 cases and 13,104 controls of East Asian ancestry. We identified 65 new loci that are associated with overall breast cancer risk at P < 5 × 10-8. The majority of credible risk single-nucleotide polymorphisms in these loci fall in distal regulatory elements, and by integrating in silico data to predict target genes in breast cells at each locus, we demonstrate a strong overlap between candidate target genes and somatic driver genes in breast tumours. We also find that heritability of breast cancer due to all single-nucleotide polymorphisms in regulatory features was 2-5-fold enriched relative to the genome-wide average, with strong enrichment for particular transcription factor binding sites. These results provide further insight into genetic susceptibility to breast cancer and will improve the use of genetic risk scores for individualized screening and prevention.

Functional dissection of breast cancer risk-associated TERT promoter variants.

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

Evidence that the 5p12 Variant rs10941679 Confers Susceptibility to Estrogen-Receptor-Positive Breast Cancer through FGF10 and MRPS30 Regulation.

Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region (positions 44,394,495-45,364,167; NCBI build 37), we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen-receptor-positive (ER+) breast cancer (per-g allele OR ER+ = 1.15; 95% CI 1.13-1.18; p = 8.35 × 10-30). After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER-) breast cancer (lead SNP rs6864776: per-a allele OR ER- = 1.10; 95% CI 1.05-1.14; p conditional = 1.44 × 10-12), and a single signal 3 SNP (rs200229088: per-t allele OR ER+ = 1.12; 95% CI 1.09-1.15; p conditional = 1.12 × 10-05). Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis.

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

A Common Variant at the 14q32 Endometrial Cancer Risk Locus Activates AKT1 through YY1 Binding.

A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.