Targeting the dependence on PIK3C3-mTORC1 signaling in dormancy-prone breast cancer cells blunts metastasis initiation.

Unlabelled: Halting breast cancer metastatic relapses following primary tumor removal and the clinical dormant phase, remains challenging, due to a lack of specific vulnerabilities to target during dormancy. To address this, we conducted genome-wide CRISPR screens on two breast cancer cell lines with distinct dormancy properties: 4T1 (short-term dormancy) and 4T07 (prolonged dormancy). We discovered that loss of class-III PI3K, Pik3c3, revealed a unique vulnerability in 4T07 cells.

Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSC) that persist after therapy and seed treatment-refractory recurrent tumors. GBM tumors display a high degree of intra- and intertumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury response.

Mechanical nanosurgery of chemoresistant glioblastoma using magnetically controlled carbon nanotubes.

Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Despite multimodal treatment including surgery, radiotherapy, and chemotherapy, median patient survival has remained at ~15 months for decades. This situation demands an outside-the-box treatment approach.

Identification of Drug Resistance Mechanisms Using Genome-Wide CRISPR-Cas9 Screens.

CRISPR-Cas9 genome editing provides a means for simple and scalable production of gene knockouts in mammalian cell lines. The development of guide RNA (gRNA) libraries targeting tens of thousands of genes has allowed researchers to produce pools of cells, each containing a single gene knockout for use in genetic screens. In addition to assessing the effect of gene knockout on cell proliferation, CRISPR-Cas9 genetic screens can be used to assess gene-drug interactions.

Gradient of Developmental and Injury Response transcriptional states defines functional vulnerabilities underpinning glioblastoma heterogeneity.

Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations.

A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to βcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.

Single-cell chromatin accessibility profiling of glioblastoma identifies an invasive cancer stem cell population associated with lower survival.

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions.

Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer.

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets.

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27).

Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin.

Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells.

Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth.

Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial.

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355).

SAPCD2 Controls Spindle Orientation and Asymmetric Divisions by Negatively Regulating the Gαi-LGN-NuMA Ternary Complex.

Control of cell-division orientation is integral to epithelial morphogenesis and asymmetric cell division. Proper spatiotemporal localization of the evolutionarily conserved Gαi-LGN-NuMA protein complex is critical for mitotic spindle orientation, but how this is achieved remains unclear. Here we identify Suppressor APC domain containing 2 (SAPCD2) as a previously unreported LGN-interacting protein.

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed.

Positive regulation of TRAF6-dependent innate immune responses by protein phosphatase PP1-γ.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events.

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis.

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates.

The application of proteomic approaches to the study of mammalian spermatogenesis and sperm function.

Spermatogenesis is the process by which terminally differentiated sperm are produced from male germline stem cells. This complex developmental process requires the coordination of both somatic and germ cells through phases of proliferation, meiosis, and morphological differentiation, to produce the cell responsible for the delivery of the paternal genome. With infertility affecting ~ 15% of all couples, furthering our understanding of spermatogenesis and sperm function is vital for improving the diagnosis and treatment of male factor infertility.

Tandem affinity purification in transgenic mouse embryonic stem cells identifies DDOST as a novel PPP1CC2 interacting protein.

Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males.

Identification of potentially damaging amino acid substitutions leading to human male infertility.

There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility.