Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties
. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs
...have induced remarkable responses in patients with refractory leukemia
and lymphoma
and were recently approved by the US Food and Drug Administration
. These CARs program highly performing effector functions that mediate potent tumor elimination
despite the limited persistence they confer on T cells
. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion
, which may be accentuated by the redundancy of CD28 and CD3ζ signaling
as well as the spatiotemporal constraints imparted by the structure of second-generation CARs
. Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.
Molecularly targeted therapies aim to obstruct cell autonomous programs required for tumor growth. We show that mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 4/6 inhibitors act ...in combination to suppress the proliferation of KRAS-mutant lung cancer cells while simultaneously provoking a natural killer (NK) cell surveillance program leading to tumor cell death. The drug combination, but neither agent alone, promotes retinoblastoma (RB) protein-mediated cellular senescence and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). SASP components tumor necrosis factor-α and intercellular adhesion molecule-1 are required for NK cell surveillance of drug-treated tumor cells, which contributes to tumor regressions and prolonged survival in a KRAS-mutant lung cancer mouse model. Therefore, molecularly targeted agents capable of inducing senescence can produce tumor control through non-cell autonomous mechanisms involving NK cell surveillance.
Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage ...events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.
Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment
. Physiologically, senescence serves as a tumour-suppressive ...mechanism that prevents the expansion of premalignant cells
and has a beneficial role in wound-healing responses
. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis
. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity
. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)
as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.
Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. ...A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated, nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single Escherichia coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1- to 2-Mb resolution. MIDAS can potentially further the characterization of genomic diversity in many heterogeneous cell populations.
Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that ...average over bulk tissue extracts. However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present a
ingle-cell RNA-seq
nalysis and
lustering
valuation (SAKE), a robust method for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range of cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors (BRAFi). Single-cell RNA-seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor-resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. These experimentally validated markers of BRAFi resistance share overlap with previous analyses in different melanoma cell lines, demonstrating the generality of these findings and highlighting the utility of single-cell analysis to elucidate mechanisms of BRAFi resistance.
It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely ...acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease.
Although p53 inactivation promotes genomic instability
and presents a route to malignancy for more than half of all human cancers
, the patterns through which heterogenous TP53 (encoding human p53) ...mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours.
When adopted in black hole spacetimes, geometric-optics approximations imply a mapping between the quasinormal mode (QNM) spectrum of black holes in the eikonal limit and black hole images. In ...particular, the real part and the imaginary part of eikonal QNM frequencies are associated with the apparent size and the detailed structure of the ring images, respectively. This correspondence could be violated when going beyond general relativity. We propose a novel method to test the eikonal correspondence via the comparison of two sets of observables from a nonrotating black hole, one extracted from QNM spectra and the other from the lensed photon rings on the image plane. Specifically, the photon ring observables robustly capture the information of the black hole spacetime itself regardless of the surrounding emission models. Therefore, the proposed test of eikonal correspondence can be validated in quite broad scenarios.
The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We generated a high-resolution Hi-C spatial ...organization map of the G1-arrested mouse pro-B cell genome and used high-throughput genome-wide translocation sequencing to map translocations from target DNA double-strand breaks (DSBs) within it. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high-frequency DSBs at these loci and their colocalization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and subchromosomal domains in a manner directly related to pre-existing spatial proximity. By combining two high-throughput genomic methods in a genetically tractable system, we provide a new lens for viewing cancer genomes.
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► A high-resolution Hi-C spatial organization map of the G1-arrested mouse cell genome ► Genomic spatial heterogeneity contributes to recurrent chromosomal translocations ► Spatial organization of chromosomes promotes dominant translocations in cis ► Translocations are guided genome-wide by pre-existing spatial genome organization
The first high-resolution Hi-C map of the murine G1 nucleus combined with high-throughput genome-wide translocation sequencing reveals how chromatin spatial organization contributes to DSB-driven translocations.
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