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The small GTPase Rac1 is thought to play an important role in cell migration and invasion. We have previously identified synaptojanin 2, a phosphoinositide phosphatase, as an effector of Rac1. Here, we show that small interfering RNA-mediated depletion of either Rac1 or synaptojanin 2 inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and rat brain slices. Depletion of Rac1 or synaptojanin 2 also inhibits migration of SNB19 and U87MG cells on glioma-derived extracellular matrix. In addition, we found that depletion of Rac1 or synaptojanin 2 inhibits the formation of lamellipodia and invadopodia, specialized membrane structures that are thought to be involved in extracellular matrix degradation. These results suggest that synaptojanin 2 contributes to the role of Rac1 in cell invasion and migration by regulating the formation of invadopodia and lamellipodia. This study also identifies synaptojanin 2 as a novel potential target for therapeutic intervention in malignant tumors.

Supplementary Figure 1 from Role of Synaptojanin 2 in Glioma Cell Migration and Invasion

Supplementary Figure 1 Legend from Role of Synaptojanin 2 in Glioma Cell Migration and Invasion

Single-cell T cell repertoire sequencing can pair both T cell receptor (TCR) and gene expression sequence data, providing an enriched view of T cell behavior. This powerful tool can identify and characterize specific clonotypes and phenotypes as well as track their changes in response to therapy, such as immune checkpoint blockade (ICB). We present a novel information theoretic framework called TCRi for characterizing single cell T cell repertoires by formalizing the relationship between clonotype and phenotype in a joint probability distribution. Our strategy allows for the identification of subpopulations of T cells and jointly quantifies their TCR and expression profiles in response to stimuli, in addition the framework tracks the phenotypic changes in individual T cell clones over time. We applied this framework to four datasets of T cells sequenced from cancer patients treated with anti-PD-(L)1 ICB immunotherapies and examined evolution of T cell responses pre- and post-treatment. Quantitative of phenotypic and clonotypic entropy analysis with TCRi demonstrated improvements in characterization of the transcriptional signature of clonotypes. Furthermore, TCRi highlighted the importance of phenotypic flux and specific T-cell phenotypes as determinants of therapeutic response.

Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. In this work, we show in four single cell RNA-seq datasets that GeneVector was able to capture phenotypespecific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1–4 patterned by distinct mutational processes5,6, tumour heterogeneity7–9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11–13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFβ signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability patterned by distinct mutational processes, intratumoral heterogeneity and intraperitoneal spread. We investigated determinants of immune recognition and evasion in HGSOC to elucidate co- evolutionary processes underlying malignant progression and tumor immunity. Mutational processes and anatomic sites of tumor foci were key determinants of tumor microenvironment cellular phenotypes, inferred from whole genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumor sites from 42 treatment-naive HGSOC patients. Homologous recombination-deficient (HRD)-Dup (BRCA1 mutant-like) and HRD- Del (BRCA2 mutant-like) tumors harbored increased neoantigen burden, inflammatory signaling and ongoing immunoediting, reflected in loss of HLA diversity and tumor infiltration with highly- differentiated dysfunctional CD8+ T cells. Foldback inversion (FBI, non-HRD) tumors exhibited elevated TGFβ signaling and immune exclusion, with predominantly naive/stem-like and memory T cells. Our findings implicate distinct immune resistance mechanisms across HGSOC subtypes which can inform future immunotherapeutic strategies.HIGHLIGHTSMulti-region, multi-modal profiling of malignant and immune cell phenotypes in ovarian cancerAnatomic site specificity is a determinant of cancer cell and intratumoral immune phenotypesTumor mutational processes impact mechanisms of immune control and immune evasionSpatial topology of HR-deficient tumors is defined by immune interactions absent from immune inert HR-proficient subtypes

Cancer genomes exhibit extensive chromosomal copy number changes and structural variation, yet how allele specific alterations drive cancer genome evolution remains unclear. Here, through application of a new computational approach we report allele specific copy number alterations in 11,097 single cell whole genomes from genetically engineered mammary epithelial cells and 21,852 cells from high grade serous ovarian and triple negative breast cancers. Resolving single cell copy number profiles to individual alleles uncovered genomic background distributions of gains, losses and loss of heterozygosity, yielding evidence of positive selection of specific chromosomal alterations. In addition specific genomic loci in maternal and paternal alleles were commonly found to be altered in parallel with convergent phenotypic transcriptional effects. Finally we show that haplotype specific alterations trace the cyclical etiology of high level amplifications and reveal clonal haplotype decomposition of complex structures. Together, our results illuminate how allele and haplotype specific alterations, here determined across thousands of single cell cancer genomes, impact the etiology and evolution of structural variations in human tumours.

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD throughBRCA1andBRCA2inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

How cell-to-cell copy number alterations that underpin genomic instability

Tumour fitness landscapes underpin selection in cancer, impacting etiology, evolution and response to treatment. Progress in defining fitness landscapes has been impeded by a lack of timeseries perturbation experiments over realistic intervals at single cell resolution. We studied the nature of clonal dynamics induced by genetic and pharmacologic perturbation with a quantitative fitness model developed to ascribe quantitative selective coefficients to individual cancer clones, enable prediction of clone-specific growth potential, and forecast competitive clonal dynamics over time. We applied the model to serial single cell genome (>60,000 cells) and transcriptome (>58,000 cells) experiments ranging from 10 months to 2.5 years in duration. We found that genetic perturbation ofTP53in epithelial cell lines induces multiple forms of copy number alteration that confer increased fitness to clonal populations with measurable consequences on gene expression. In patient derived xenografts, predicted selective coefficients accurately forecasted clonal competition dynamics, that were validated with timeseries sampling of experimentally engineered mixtures of low and high fitness clones. In cisplatin-treated patient derived xenografts, the fitness landscape was inverted in a time-dependent manner, whereby a drug resistant clone emerged from a phylogenetic lineage of low fitness clones, and high fitness clones were eradicated. Moreover, clonal selection mediated reversible drug response early in the selection process, whereas late dynamics in genomically fixed clones were associated with transcriptional plasticity on a fixed clonal genotype. Together, our findings outline causal mechanisms with implication for interpreting how mutations and multi-faceted drug resistance mechanisms shape the etiology and cellular fitness of human cancers.

A new generation of scalable single cell whole genome sequencing (scWGS) methods allows unprecedented high resolution measurement of the evolutionary dynamics of cancer cell populations. Phylogenetic reconstruction is central to identifying sub-populations and distinguishing the mutational processes that gave rise to them. Existing phylogenetic tree building models do not scale to the tens of thousands of high resolution genomes achievable with current scWGS methods. We constructed a phylogenetic model and associated Bayesian inference procedure, sitka, specifically for scWGS data. The method is based on a novel phylogenetic encoding of copy number (CN) data, the sitka transformation, that simplifies the site dependencies induced by rearrangements while still forming a sound foundation to phylogenetic inference. The sitka transformation allows us to design novel scalable Markov chain Monte Carlo (MCMC) algorithms. Moreover, we introduce a novel point mutation calling method that incorporates the CN data and the underlying phylogenetic tree to overcome the low per-cell coverage of scWGS. We demonstrate our method on three single cell datasets, including a novel PDX series, and analyse the topological properties of the inferred trees. Sitka is freely available athttps://github.com/UBC-Stat-ML/sitkatree.git.

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh

The elimination of all cross-talk in a signaling pathway turns a yeast cell into a model cell that can sense and report on metabolites.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education., Natural killer (NK) cells are functionally calibrated against self MHC during a process termed education. Here the authors show that NK cell education is associated with the accumulation of dense-core secretory lysosomes for expedited release of granzyme B and Ca2+ flux upon target recognition and NK cell activation.

We used a deeply sequenced dataset of 910 individuals, all of African descent, to construct a set of DNA sequences present in these individuals but missing from the reference human genome. We aligned 1.19 trillion reads from the 910 individuals to the reference genome (GRCh38), collected all reads that failed to align, and assembled these reads into contiguous sequences (contigs). We then compared all contigs to one another to identify a set of unique sequences representing regions of the African pan-genome missing from the reference genome. Our analysis revealed 296,485,284 bp in 125,715 distinct contigs present in the African-descended populations, demonstrating that the African pan-genome contains ~10% more DNA than the current human reference genome. Although the functional significance of nearly all of this sequence is unknown, 387 of the novel contigs fall within 315 distinct protein-coding genes while the rest appear to be intergenic., Editorial Summary: Assembly of a pan-genome from 910 humans of African descent identifies 296.5 Mb of novel DNA mapping to 125,715 distinct contigs. This African pan-genome contains ~10% more DNA than the current human reference genome.

The UK Biobank project is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals from across the United Kingdom, aged between 40 and 69 at recruitment. The open resource is unique in its size and scope. A rich variety of phenotypic and health-related information is available on each participant, including biological measurements, lifestyle indicators, biomarkers in blood and urine, and imaging of the body and brain. Follow-up information is provided by linking health and medical records. Genome-wide genotype data have been collected on all participants, providing many opportunities for the discovery of new genetic associations and the genetic bases of complex traits. Here we describe the centralized analysis of the genetic data, including genotype quality, properties of population structure and relatedness of the genetic data, and efficient phasing and genotype imputation that increases the number of testable variants to around 96 million. Classical allelic variation at 11 human leukocyte antigen genes was imputed, resulting in the recovery of signals with known associations between human leukocyte antigen alleles and many diseases., Deep phenotype and genome-wide genetic data from 500,000 individuals from the UK Biobank, describing population structure and relatedness in the cohort, and imputation to increase the number of testable variants to 96 million.

Acquisition of RNA into CRISPR arrays allows the recording of transcriptional dynamics in cells.

Independent efforts shine light on the 3D genome structure by looking at multiple contacts along an allele or equalizing the distance between restriction sites for higher-resolution Hi-C maps.

Tagging and sequencing RNAs that are in close spatial proximity in the nucleus shows how the transcriptome is partitioned.

Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS are not only smaller than naturally-occurring CRISPR-Cas programmable RNA binding systems, but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, while the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.

A library of degrons allows tuning of half-life and expression levels of proteins in genetic circuits.

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices., Editor’s summary The accuracy of nanopore DNA sequencing is substantially improved by application of a variable-voltage sequencing mode.

Lineage tracing provides key insights into the fate of individual cells in complex organisms. While effective genetic labeling approaches are available in model systems, in intact humans most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mitochondrial DNA (mtDNA) can be tracked by single cell RNA or ATAC sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo, and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.

Comparisons of genome organization in many individual cells with high-throughput FISH show extensive variation.

Cellular engineering that allows budding yeast to survive with the four core human histones opens the door to exploring the function of histone variants and their modifications.

Ultradeep sequencing of Hi-C data from mouse cells in different developmental stages can address outstanding questions about 3D chromatin function.

In vitro Cas9-binding assays show the effects of mutations in the target sequence on binding kinetics.

Thin slices through nuclei provide an unbiased view of the complex 3D organization of mammalian genomes.

A three-component system of epigenetic writers and readers preserves memory over many generations of cells.

Clustered, regularly interspaced, short palindromic repeat (CRISPR)–CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro–transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here, we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that is similar to canonical immune-stimulating ligands. IVT gRNAs are recognized in the cytosol through the retinoic acid–inducible gene I (RIG-I) pathway but not the melanoma differentiation–associated gene 5 (MDA5) pathway, thereby triggering a type I interferon response. Removal of the 5’-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells., Author summary Clustered, regularly interspaced, short palindromic repeat (CRISPR)–CRISPR-associated 9 (Cas9) genome editing is transforming fundamental research, as it allows researchers to make targeted changes to the genome of cells. For efficient editing, the Cas9 protein (a DNA nuclease) and a guide RNA (gRNA), which leads the nuclease to the correct location in the genome, have to be introduced into cells. One recent advancement is the delivery of a Cas9 protein and an in vitro–transcribed (IVT) gRNA as a precomplexed ribonucleoprotein (RNP). This approach allows editing of more sensitive cell types such as immune cells and hematopoietic stem cells. However, the consequences of introducing foreign Cas9 nuclease and gRNA into mammalian cells are not fully understood. Here, we show that in many cell types, the IVT gRNAs trigger a potent innate immune response—a natural defense mechanism against RNA viruses. We show that the innate immune response causes cell death in primary hematopoietic stem cells but that removal of the 5’-triphosphate from gRNAs by phosphatase treatment can ameliorate the immune response and prevent the loss of viability. Hence, CRISPR-Cas9 RNP editing has the potential to induce a potent antiviral response, and we suggest that care must be taken when designing therapeutic strategies to edit primary cells.