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.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Technological Convergence: Highlighting the Power of CRISPR Single-Cell Perturbation Toolkit for Functional Interrogation of Enhancers.

Higher eukaryotic enhancers, as a major class of regulatory elements, play a crucial role in the regulation of gene expression. Over the last decade, the development of sequencing technologies has flooded researchers with transcriptome-phenotype data alongside emerging candidate regulatory elements. Since most methods can only provide hints about enhancer function, there have been attempts to develop experimental and computational approaches that can bridge the gap in the causal relationship between regulatory regions and phenotypes. The coupling of two state-of-the-art technologies, also referred to as crisprQTL, has emerged as a promising high-throughput toolkit for addressing this question. This review provides an overview of the importance of studying enhancers, the core molecular foundation of crisprQTL, and recent studies utilizing crisprQTL to interrogate enhancer-phenotype correlations. Additionally, we discuss computational methods currently employed for crisprQTL data analysis. We conclude by pointing out common challenges, making recommendations, and looking at future prospects, with the aim of providing researchers with an overview of crisprQTL as an important toolkit for studying enhancers.

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.

Heritable methylation marks associated with prostate cancer risk.

DNA methylation marks that are inherited from parents to offspring are known to play a role in cancer risk and could explain part of the familial risk for cancer. We therefore conducted a genome-wide search for heritable methylation marks associated with prostate cancer risk. Peripheral blood DNA methylation was measured for 133 of the 469 members of 25 multiple-case prostate cancer families, using the EPIC array. We used these families to systematically search the genome for methylation marks with Mendelian patterns of inheritance, then we tested the 1,000 most heritable marks for association with prostate cancer risk. After correcting for multiple testing, 41 heritable methylation marks were associated with prostate cancer risk. Separate analyses, based on 869 incident cases and 869 controls from a prospective cohort study, showed that 9 of these marks near the metastable epiallele VTRNA2-1 were also nominally associated with aggressive prostate cancer risk in the population.

Immortalised Cas9-expressing Cell lines for Gene interrogation.

The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.

Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies.

INTRODUCTION: Genes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes. RESULTS: Here, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome. CONCLUSIONS: Our work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

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.

Engineered Plant-Based Nanocellulose Hydrogel for Small Intestinal Organoid Growth.

Organoids are three-dimensional self-renewing and organizing clusters of cells that recapitulate the behavior and functionality of developed organs. Referred to as "organs in a dish," organoids are invaluable biological models for disease modeling or drug screening. Currently, organoid culture commonly relies on an expensive and undefined tumor-derived reconstituted basal membrane which hinders its application in high-throughput screening, regenerative medicine, and diagnostics. Here, we introduce a novel engineered plant-based nanocellulose hydrogel is introduced as a well-defined and low-cost matrix that supports organoid growth. Gels containing 0.1% nanocellulose fibers (99.9% water) are ionically crosslinked and present mechanical properties similar to the standard animal-based matrix. The regulation of the osmotic pressure is performed by a salt-free strategy, offering conditions for cell survival and proliferation. Cellulose nanofibers are functionalized with fibronectin-derived adhesive sites to provide the required microenvironment for small intestinal organoid growth and budding. Comparative transcriptomic profiling reveals a good correlation with transcriptome-wide gene expression pattern between organoids cultured in both materials, while differences are observed in stem cells-specific marker genes. These hydrogels are tunable and can be combined with laminin-1 and supplemented with insulin-like growth factor (IGF-1) to optimize the culture conditions. Nanocellulose hydrogel emerges as a promising matrix for the growth of organoids.

GeNets: a unified web platform for network-based genomic analyses.

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.

Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression.

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in ß-Catenin-Active Cancers.

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive ß-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of ß-catenin, define functional networks required for the survival of ß-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.

Characterizing genomic alterations in cancer by complementary functional associations.

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of ß-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.

RNF43 is frequently mutated in colorectal and endometrial cancers.

We report somatic mutations of RNF43 in over 18% of colorectal adenocarcinomas and endometrial carcinomas. RNF43 encodes an E3 ubiquitin ligase that negatively regulates Wnt signaling. Truncating mutations of RNF43 are more prevalent in microsatellite-unstable tumors and show mutual exclusivity with inactivating APC mutations in colorectal adenocarcinomas. These results indicate that RNF43 is one of the most commonly mutated genes in colorectal and endometrial cancers.

KRAS and YAP1 converge to regulate EMT and tumor survival.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

Genomic insights into WNT/ß-catenin signaling.

The canonical WNT pathway regulates the stability of the proto-oncogene ß-catenin and is aberrantly activated in many cancer types. Studies in a wide range of experimental models confirm that ß-catenin activity is required for tumor initiation in cancers where this pathway is deregulated. However, to date this pathway has proven to be challenging to target therapeutically. Moreover, several lines of evidence suggest that other components and regulators of ß-catenin exist. Here we will describe recent structural and functional studies describing genomic alterations and new regulators of ß-catenin that lead to aberrant activation of the WNT/ß-catenin pathway. These findings provide new insights into the biology of WNT/ß-catenin signaling and suggest potential therapeutic opportunities.

Systematic interrogation of 3q26 identifies TLOC1 and SKIL as cancer drivers.

UNLABELLED: 3q26 is frequently amplified in several cancer types with a common amplified region containing 20 genes. To identify cancer driver genes in this region, we interrogated the function of each of these genes by loss- and gain-of-function genetic screens. Specifically, we found that TLOC1 (SEC62) was selectively required for the proliferation of cell lines with 3q26 amplification. Increased TLOC1 expression induced anchorage-independent growth, and a second 3q26 gene, SKIL (SNON), facilitated cell invasion in immortalized human mammary epithelial cells. Expression of both TLOC1 and SKIL induced subcutaneous tumor growth. Proteomic studies showed that TLOC1 binds to DDX3X, which is essential for TLOC1-induced transformation and affected protein translation. SKIL induced invasion through upregulation of SLUG (SNAI2) expression. Together, these studies identify TLOC1 and SKIL as driver genes at 3q26 and more broadly suggest that cooperating genes may be coamplified in other regions with somatic copy number gain. SIGNIFICANCE: These studies identify TLOC1 and SKIL as driver genes in 3q26. These observations provide evidence that regions of somatic copy number gain may harbor cooperating genes of different but complementary functions.

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.