An expanded universe of cancer targets.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

Nucleolar-based Dux repression is essential for embryonic two-cell stage exit.

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

Systematic identification of barriers to human iPSC generation.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. To elucidate endogenous barriers limiting this process, we systematically dissected human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation, and mechanistic investigation of relevant pathways and networks. We identify reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins inhibit reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGF-ß signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel, or feedforward loop architectures to antagonize reprogramming. These results provide a global view of barriers to human cellular reprogramming.

miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility.

Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

Examining the evidence for extracellular RNA function in mammals.

The presence of RNAs in the extracellular milieu has sparked the hypothesis that RNA may play a role in mammalian cell-cell communication. As functional nucleic acids transfer from cell to cell in plants and nematodes, the idea that mammalian cells also transfer functional extracellular RNA (exRNA) is enticing. However, untangling the role of mammalian exRNAs poses considerable experimental challenges. This Review discusses the evidence for and against functional exRNAs in mammals and their proposed roles in health and disease, such as cancer and cardiovascular disease. We conclude with a discussion of the forward-looking prospects for studying the potential of mammalian exRNAs as mediators of cell-cell communication.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals.

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

MicroRNAs 24 and 27 Suppress Allergic Inflammation and Target a Network of Regulators of T Helper 2 Cell-Associated Cytokine Production.

MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.

BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Dynamics of Chromatin Accessibility During Hematopoietic Stem Cell Differentiation Into Progressively Lineage-Committed Progeny.

Epigenetic mechanisms regulate the multilineage differentiation capacity of hematopoietic stem cells (HSCs) into a variety of blood and immune cells. Mapping the chromatin dynamics of functionally defined cell populations will shed mechanistic insight into 2 major, unanswered questions in stem cell biology: how does epigenetic identity contribute to a cell type's lineage potential, and how do cascades of chromatin remodeling dictate ensuing fate decisions? Our recent work revealed evidence of multilineage gene priming in HSCs, where open cis-regulatory elements (CREs) exclusively shared between HSCs and unipotent lineage cells were enriched for DNA binding motifs of known lineage-specific transcription factors. Oligopotent progenitor populations operating between the HSCs and unipotent cells play essential roles in effecting hematopoietic homeostasis. To test the hypothesis that selective HSC-primed lineage-specific CREs remain accessible throughout differentiation, we used ATAC-seq to map the temporal dynamics of chromatin remodeling during progenitor differentiation. We observed epigenetic-driven clustering of oligopotent and unipotent progenitors into distinct erythromyeloid and lymphoid branches, with multipotent HSCs and MPPs associating with the erythromyeloid lineage. We mapped the dynamics of lineage-primed CREs throughout hematopoiesis and identified both unique and shared CREs as potential lineage reinforcement mechanisms at fate branch points. Additionally, quantification of genome-wide peak count and size revealed overall greater chromatin accessibility in HSCs, allowing us to identify HSC-unique peaks as putative regulators of self-renewal and multilineage potential. Finally, CRISPRi-mediated targeting of ATACseq-identified putative CREs in HSCs allowed us to demonstrate the functional role of selective CREs in lineage-specific gene expression. These findings provide insight into the regulation of stem cell multipotency and lineage commitment throughout hematopoiesis and serve as a resource to test functional drivers of hematopoietic lineage fate.

A National Survey of Pediatric Post-Acute Care Facilities.

OBJECTIVES: To identify and geolocate pediatric post-acute care (PAC) facilities in the United States. DESIGN: Cross-sectional survey using both online resources and telephone inquiry. SETTING: All 50 U.S. states surveyed from June 2022 to May 2023. Care sites identified via state regulatory agencies and the Centers for Medicare & Medicaid Services. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Number, size, and type of facility, scope of practice, and type of care provided. One thousand three hundred fifty-five facilities were surveyed; of these, 18.6% (252/1355) were pediatric-specific units or adult facilities accepting some pediatric patients. There were 109 pediatric-specific facilities identified within 39 U.S. states. Of these, 38 were freestanding with all accepting children with tracheostomies, 97.4% (37/38) accepting those requiring mechanical ventilation via tracheostomy, and 81.6% (31/38) accepting those requiring parenteral nutrition. The remaining 71 facilities were adult facilities with embedded pediatric units or children's hospitals with 88.7% (63/71), 54.9% (39/71), and 54.9% (39/71), accepting tracheostomies, mechanical ventilation via tracheostomy, and parenteral nutrition, respectively. Eleven states lacked any pediatric-specific PAC units or facilities. CONCLUSIONS: The distribution of pediatric PAC is sparse and uneven across the United States. We present an interactive map and database describing these facilities. These data offer a starting point for exploring the consequences of pediatric PAC supply.

Erratum: In situ Signature of Cyclotron Resonant Heating in the Solar Wind [Phys. Rev. Lett. 129, 165101 (2022)].

This corrects the article DOI: 10.1103/PhysRevLett.129.165101.

Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR.

The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools--specifically CRISPR-based technologies--have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: "Which technology should I use in my experiment?" This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.

Decreasing trends in total suspended solids and cumulative effects of nonpoint source projects in the Cuyahoga River Watershed, OH.

Using the US EPA's Grants Reporting and Tracking System (GRTS), we test if completion of best management practices (BMPs) through the Clean Water Act Section (§)319 National Nonpoint Source Program was associated with a decreasing trend in total suspended solids (TSS) load (metric tons/year). The study area chosen had 21 completed projects in the Cuyahoga River watershed in northeastern Ohio from 2000 to 2018. The §319 projects ranged from dam removal, floodplain/wetland restoration to stormwater projects. There was an overall decreasing trend in TSS loads. We identified three phases of project implementation and completion, where phase 1 had ongoing projects, but none completed (2000-2004). The steepest decrease in loads, identified as phase 2 (2005-2011), was associated with completion of low-head dam modification and removal projects on the mainstem of the Cuyahoga River. A likely decreasing trend was associated with projects completed in the tributaries, such as natural channel design restoration and stormwater green infrastructure (phase 3). Pairing sediment reduction estimates from projects with the river's flow normalized TSS loading trend, we estimated that the §319 effort may account for a small fraction of the TSS load reduction. Other stream restoration projects (non-§319) have also been done in the Cuyahoga watershed by other organizations. However, trying to compile these other projects is challenging in larger watersheds having multiple municipalities, agencies, and nonprofits doing restoration without better coordinated record keeping and monitoring. While a decreasing trend in a pollutant load is a desirable water quality outcome, determining what contributed to that trend remains difficult.

In Situ Signature of Cyclotron Resonant Heating in the Solar Wind.

The dissipation of magnetized turbulence is an important paradigm for describing heating and energy transfer in astrophysical environments such as the solar corona and wind; however, the specific collisionless processes behind dissipation and heating remain relatively unconstrained by measurements. Remote sensing observations have suggested the presence of strong temperature anisotropy in the solar corona consistent with cyclotron resonant heating. In the solar wind, in situ magnetic field measurements reveal the presence of cyclotron waves, while measured ion velocity distribution functions have hinted at the active presence of cyclotron resonance. Here, we present Parker Solar Probe observations that connect the presence of ion-cyclotron waves directly to signatures of resonant damping in observed proton-velocity distributions using the framework of quasilinear theory. We show that the quasilinear evolution of the observed distribution functions should absorb the observed cyclotron wave population with a heating rate of 10^{-14} W/m^{3}, indicating significant heating of the solar wind.

Impact of Allocation on Survival During Intermittent Chemotherapy Shortages: A Modeling Analysis.

BACKGROUND: Intermittent shortages of chemotherapeutics used to treat curable malignancies are a worldwide problem that increases patient mortality. Although multiple strategies have been proposed for managing these shortages (eg, prioritizing patients by age, scarce treatment efficacy per volume, alternative treatment efficacy difference), critical clinical dilemmas arise when selecting a management strategy and understanding its impact. PATIENTS AND METHODS: We developed a model to compare the impact of different allocation strategies on overall survival during intermittent chemotherapy shortages and tested it using vincristine, which was recently scarce for 9 months in the United States. Demographic and treatment data were abstracted from 1,689 previously treated patients in our tertiary-care system; alternatives were abstracted from NCCN Clinical Practice Guidelines in Oncology for each disease and survival probabilities from the studies cited therein. Modeled survival was validated using SEER data. Nine-month shortages were modeled for all possible supply levels. Pairwise differences in 3-year survival and risk reductions were calculated for each strategy compared with standard practice (first-come, first-served) for each 50-mg supply increment, as were supply thresholds above which each strategy maintained survival similar to scenarios without shortages. RESULTS: A strategy prioritizing by higher vincristine efficacy per volume and greater alternative treatment efficacy difference performed best, improving survival significantly (P<.01) across 86.5% of possible shortages (relative risk reduction, 8.3%; 99% CI, 8.0-8.5) compared with standard practice. This strategy also maintained survival rates similar to a model without shortages until supply fell below 72.2% of the amount required to treat all patients, compared with 94.3% for standard practice. CONCLUSIONS: During modeled vincristine shortages, prioritizing patients by higher efficacy per volume and alternative treatment efficacy difference significantly improved survival over standard practice. This approach can help optimize allocation as intermittent chemotherapy shortages continue to arise.