Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity.

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells.

Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.

Fibrosis induced by resident macrophages has divergent roles in pancreas inflammatory injury and PDAC.

Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages. Whether TRMs and monocyte-derived macrophages have district roles under differing pathologies is not understood. Here, we showed that a substantial portion of the macrophages that accumulated during pancreatitis and pancreatic cancer in mice had expanded from TRMs. Pancreas TRMs had an extracellular matrix remodeling phenotype that was important for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and death, due to impaired acinar cell survival and recovery. During pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

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. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.

N6-methyladenine DNA Modification in Glioblastoma.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

Identification of the Human Skeletal Stem Cell.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

Tissue remodeling by an opportunistic pathogen triggers allergic inflammation.

Different effector arms of the immune system are optimized to protect from different classes of pathogens. In some cases, pathogens manipulate the host immune system to promote the wrong type of effector response-a phenomenon known as immune deviation. Typically, immune deviation helps pathogens to avoid destructive immune responses. Here, we report on a type of immune deviation whereby an opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa), induces the type 2 immune response resulting in mucin production that is used as an energy source by the pathogen. Specifically, P. aeruginosa-secreted toxin, LasB, processed and activated epithelial amphiregulin to induce type 2 inflammation and mucin production. This "niche remodeling" by P. aeruginosa promoted colonization and, as a by-product, allergic sensitization. Our study thus reveals a type of bacterial immune deviation by increasing nutrient supply. It also uncovers a mechanism of allergic sensitization by a bacterial virulence factor.

Ionized gas extends over 40 kpc in an odd radio circle host galaxy.

A new class of extragalactic astronomical sources discovered in 2021, named odd radio circles (ORCs)1, are large rings of faint, diffuse radio continuum emission spanning approximately 1 arcminute on the sky. Galaxies at the centres of several ORCs have photometric redshifts of z ≃ 0.3-0.6, implying physical scales of several 100 kpc in diameter for the radio emission, the origin of which is unknown. Here we report spectroscopic data on an ORC including strong [O II] emission tracing ionized gas in the central galaxy of ORC4 at z = 0.4512. The physical extent of the [O II] emission is approximately 40 kpc in diameter, larger than expected for a typical early-type galaxy2 but an order of magnitude smaller than the large-scale radio continuum emission. We detect an approximately 200 km s-1 velocity gradient across the [O II] nebula, as well as a high velocity dispersion of approximately 180 km s-1. The [O II] equivalent width (approximately 50 Å) is extremely high for a quiescent galaxy. The morphology, kinematics and strength of the [O II] emission are consistent with the infall of shock ionized gas near the galaxy, following a larger, outward-moving shock. Both the extended optical and radio emission, although observed on very different scales, may therefore result from the same dramatic event.

A warm Neptune's methane reveals core mass and vigorous atmospheric mixing.

Observations of transiting gas giant exoplanets have revealed a pervasive depletion of methane1-4, which has only recently been identified atmospherically5,6. The depletion is thought to be maintained by disequilibrium processes such as photochemistry or mixing from a hotter interior7-9. However, the interiors are largely unconstrained along with the vertical mixing strength and only upper limits on the CH4 depletion have been available. The warm Neptune WASP-107b stands out among exoplanets with an unusually low density, reported low core mass10, and temperatures amenable to CH4, though previous observations have yet to find the molecule2,4. Here we present a JWST-NIRSpec transmission spectrum of WASP-107b that shows features from both SO2 and CH4 along with H2O, CO2, and CO. We detect methane with 4.2σ significance at an abundance of 1.0 ± 0.5 ppm, which is depleted by 3 orders of magnitude relative to equilibrium expectations. Our results are highly constraining for the atmosphere and interior, which indicate the envelope has a super-solar metallicity of 43 ± 8 × solar, a hot interior with an intrinsic temperature of Tint = 460 ± 40 K, and vigorous vertical mixing which depletes CH4 with a diffusion coefficient of Kzz = 1011.6±0.1 cm2 s-1. Photochemistry has a negligible effect on the CH4 abundance but is needed to account for the SO2. We infer a core mass of 11.5 - 3.6 + 3.0 M ⊕ , which is much higher than previous upper limits10, releasing a tension with core-accretion models11.

Tumour evolution and microenvironment interactions in 2D and 3D space.

To study the spatial interactions among cancer and non-cancer cells1, we here examined a cohort of 131 tumour sections from 78 cases across 6 cancer types by Visium spatial transcriptomics (ST). This was combined with 48 matched single-nucleus RNA sequencing samples and 22 matched co-detection by indexing (CODEX) samples. To describe tumour structures and habitats, we defined 'tumour microregions' as spatially distinct cancer cell clusters separated by stromal components. They varied in size and density among cancer types, with the largest microregions observed in metastatic samples. We further grouped microregions with shared genetic alterations into 'spatial subclones'. Thirty five tumour sections exhibited subclonal structures. Spatial subclones with distinct copy number variations and mutations displayed differential oncogenic activities. We identified increased metabolic activity at the centre and increased antigen presentation along the leading edges of microregions. We also observed variable T cell infiltrations within microregions and macrophages predominantly residing at tumour boundaries. We reconstructed 3D tumour structures by co-registering 48 serial ST sections from 16 samples, which provided insights into the spatial organization and heterogeneity of tumours. Additionally, using an unsupervised deep-learning algorithm and integrating ST and CODEX data, we identified both immune hot and cold neighbourhoods and enhanced immune exhaustion markers surrounding the 3D subclones. These findings contribute to the understanding of spatial tumour evolution through interactions with the local microenvironment in 2D and 3D space, providing valuable insights into tumour biology.

Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.

Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.

Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.

Emergent interface vibrational structure of oxide superlattices.

As the length scales of materials decrease, the heterogeneities associated with interfaces become almost as important as the surrounding materials. This has led to extensive studies of emergent electronic and magnetic interface properties in superlattices1-9. However, the interfacial vibrations that affect the phonon-mediated properties, such as thermal conductivity10,11, are measured using macroscopic techniques that lack spatial resolution. Although it is accepted that intrinsic phonons change near boundaries12,13, the physical mechanisms and length scales through which interfacial effects influence materials remain unclear. Here we demonstrate the localized vibrational response of interfaces in strontium titanate-calcium titanate superlattices by combining advanced scanning transmission electron microscopy imaging and spectroscopy, density functional theory calculations and ultrafast optical spectroscopy. Structurally diffuse interfaces that bridge the bounding materials are observed and this local structure creates phonon modes that determine the global response of the superlattice once the spacing of the interfaces approaches the phonon spatial extent. Our results provide direct visualization of the progression of the local atomic structure and interface vibrations as they come to determine the vibrational response of an entire superlattice. Direct observation of such local atomic and vibrational phenomena demonstrates that their spatial extent needs to be quantified to understand macroscopic behaviour. Tailoring interfaces, and knowing their local vibrational response, provides a means of pursuing designer solids with emergent infrared and thermal responses.

Modeling of spatiotemporal dynamics of ligand-coated particle flow in targeted drug delivery processes.

Nanoparticles tethered with vasculature-binding epitopes have been used to deliver the drug into injured or diseased tissues via the bloodstream. However, the extent that blood flow dynamics affects nanoparticle retention at the target site after adhesion needs to be better understood. This knowledge gap potentially underlies significantly different therapeutic efficacies between animal models and humans. An experimentally validated mathematical model that accurately simulates the effects of blood flow on nanoparticle adhesion and retention, thus circumventing the limitations of conventional trial-and-error-based drug design in animal models, is lacking. This paper addresses this technical bottleneck and presents an integrated mathematical method that derives heavily from a unique combination of a mechanics-based dispersion model for nanoparticle transport and diffusion in the boundary layers, an asperity model to account for surface roughness of endothelium, and an experimentally calibrated stochastic nanoparticle-cell adhesion model to describe nanoparticle adhesion and subsequent retention at the target site under external flow. PLGA-b-HA nanoparticles tethered with VHSPNKK peptides that specifically bind to vascular cell adhesion molecules on the inflamed vascular wall were investigated. The computational model revealed that larger particles perform better in adhesion and retention at the endothelium for the particle sizes suitable for drug delivery applications and within physiologically relevant shear rates. The computational model corresponded closely to the in vitro experiments which demonstrates the impact that model-based simulations can have on optimizing nanocarriers in vascular microenvironments, thereby substantially reducing in vivo experimentation as well as the development costs.

Water striders are impervious to raindrop collision forces and submerged by collapsing craters.

Water striders are abundant in areas with high humidity and rainfall. Raindrops can weigh more than 40 times the adult water strider and some pelagic species spend their entire lives at sea, never contacting ground. Until now, researchers have not systematically investigated the survival of water striders when impacted by raindrops. In this experimental study, we use high-speed videography to film drop impacts on water striders. Drops force the insects subsurface upon direct contact. As the ensuing crater rebounds upward, the water strider is propelled airborne by a Worthington jet, herein called the first jet. We show the water strider's locomotive responses, low density, resistance to wetting when briefly submerged, and ability to regain a super-surface rest state, rendering it impervious to the initial impact. When pulled subsurface during a second crater formation caused by the collapsing first jet, water striders face the possibility of ejection above the surface or submersion below the surface, a fate determined by their position in the second crater. We identify a critical crater collapse acceleration threshold ∼ 5.7 gravities for the collapsing second crater which determines the ejection and submersion of passive water striders. Entrapment by submersion makes the water strider poised to penetrate the air-water interface from below, which appears impossible without the aid of a plastron and proper locomotive techniques. Our study is likely the first to consider second crater dynamics and our results translate to the submersion dynamics of other passively floating particles such as millimetric microplastics atop the world's oceans.

Growth deficiency in a mouse model of Kabuki syndrome 2 bears mechanistic similarities to Kabuki syndrome 1.

Growth deficiency is a characteristic feature of both Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), Mendelian disorders of the epigenetic machinery with similar phenotypes but distinct genetic etiologies. We previously described skeletal growth deficiency in a mouse model of KS1 and further established that a Kmt2d-/- chondrocyte model of KS1 exhibits precocious differentiation. Here we characterized growth deficiency in a mouse model of KS2, Kdm6atm1d/+. We show that Kdm6atm1d/+ mice have decreased femur and tibia length compared to controls and exhibit abnormalities in cortical and trabecular bone structure. Kdm6atm1d/+ growth plates are also shorter, due to decreases in hypertrophic chondrocyte size and hypertrophic zone height. Given these disturbances in the growth plate, we generated Kdm6a-/- chondrogenic cell lines. Similar to our prior in vitro model of KS1, we found that Kdm6a-/- cells undergo premature, enhanced differentiation towards chondrocytes compared to Kdm6a+/+ controls. RNA-seq showed that Kdm6a-/- cells have a distinct transcriptomic profile that indicates dysregulation of cartilage development. Finally, we performed RNA-seq simultaneously on Kmt2d-/-, Kdm6a-/-, and control lines at Days 7 and 14 of differentiation. This revealed surprising resemblance in gene expression between Kmt2d-/- and Kdm6a-/- at both time points and indicates that the similarity in phenotype between KS1 and KS2 also exists at the transcriptional level.

Glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer.

Glioblastoma ranks among the most lethal of all human cancers. Glioblastomas display striking cellular heterogeneity, with stem-like glioblastoma stem cells (GSCs) at the apex. Although the original identification of GSCs dates back more than a decade, the purification and characterization of GSCs remains challenging. Despite these challenges, the evidence that GSCs play important roles in tumor growth and response to therapy has grown. Like normal stem cells, GSCs are functionally defined and distinguished from their differentiated tumor progeny at core transcriptional, epigenetic, and metabolic regulatory levels, suggesting that no single therapeutic modality will be universally effective against a heterogenous GSC population. Glioblastomas induce a systemic immunosuppression with mixed responses to oncoimmunologic modalities, suggesting the potential for augmentation of response with a deeper consideration of GSCs. Unfortunately, the GSC literature has been complicated by frequent use of inferior cell lines and a lack of proper functional analyses. Collectively, glioblastoma offers a reliable cancer to study cancer stem cells to better model the human disease and inform improved biologic understanding and design of novel therapeutics.

The multifaceted role of autophagy in cancer.

Autophagy is a cellular degradative pathway that plays diverse roles in maintaining cellular homeostasis. Cellular stress caused by starvation, organelle damage, or proteotoxic aggregates can increase autophagy, which uses the degradative capacity of lysosomal enzymes to mitigate intracellular stresses. Early studies have shown a role for autophagy in the suppression of tumorigenesis. However, work in genetically engineered mouse models and in vitro cell studies have now shown that autophagy can be either cancer-promoting or inhibiting. Here, we summarize the effects of autophagy on cancer initiation, progression, immune infiltration, and metabolism. We also discuss the efforts to pharmacologically target autophagy in the clinic and highlight future areas for exploration.