The next horizon in precision oncology: Proteogenomics to inform cancer diagnosis and treatment.

When it comes to precision oncology, proteogenomics may provide better prospects to the clinical characterization of tumors, help make a more accurate diagnosis of cancer, and improve treatment for patients with cancer. This perspective describes the significant contributions of The Cancer Genome Atlas and the Clinical Proteomic Tumor Analysis Consortium to precision oncology and makes the case that proteogenomics needs to be fully integrated into clinical trials and patient care in order for precision oncology to deliver the right cancer treatment to the right patient at the right dose and at the right time.

Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series.

Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

Timing the Landmark Events in the Evolution of Clear Cell Renal Cell Cancer: TRACERx Renal.

Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention.

Mechanisms of Autophagy Initiation.

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

Cell-cell interfaces as specialized compartments directing cell function.

Cell-cell interfaces are found throughout multicellular organisms, from transient interactions between motile immune cells to long-lived cell-cell contacts in epithelia. Studies of immune cell interactions, epithelial cell barriers, neuronal contacts and sites of cell-cell fusion have identified a core set of features shared by cell-cell interfaces that critically control their function. Data from diverse cell types also show that cells actively and passively regulate the localization, strength, duration and cytoskeletal coupling of receptor interactions governing cell-cell signalling and physical connections between cells, indicating that cell-cell interfaces have a unique membrane organization that emerges from local molecular and cellular mechanics. In this Review, we discuss recent findings that support the emerging view of cell-cell interfaces as specialized compartments that biophysically constrain the arrangement and activity of their protein, lipid and glycan components. We also review how these biophysical features of cell-cell interfaces allow cells to respond with high selectivity and sensitivity to multiple inputs, serving as the basis for wide-ranging cellular functions. Finally, we consider how the unique properties of cell-cell interfaces present opportunities for therapeutic intervention.

Friction at the BAR Leads to Membrane Breakup.

A long-standing question in cell biology is how endocytic vesicles and tubules detach from the plasma membrane in the absence of constriction by dynamin. In this issue of Cell, Simunovic et al. describe an elegant biophysical model in which friction between lipids and BAR-domain proteins drives the scission of elongating membrane tubules.

On the Design of Combination Cancer Therapy.

Combination therapy programs are the hallmark of the successful treatment of all forms of human malignancies. In this issue of Cell, Palmer and Sorger present data suggesting that cell culture results indicative of synergistic anticancer drug interactions rarely translate clinically and that the results of combination therapies in mouse models or human clinical trials, even if successful, are best explained by the independent activities of the individually administered drugs.

Rules of river avulsion change downstream.

Avulsing rivers create new pathways on the floodplain and the associated flooding can profoundly affect society1-4. River avulsions are thought to occur when the water column becomes perched above the floodplain5 or when the slope down the flanks of the channel provides a steeper descent than the existing river channel6,7. We test these classical ideas by quantifying the topography around avulsing rivers and show that these mechanisms, historically invoked separately, work together. Near coasts, rivers avulse when the slope away from the channel is steeper, not because they are perched. The opposite is true near mountain fronts; on fans, the alternative paths are similarly steep to the downstream path, so rivers avulse when they are perched above the surrounding landscape. We reconcile these findings and present a new theoretical framework that identifies which rivers are vulnerable to avulsion and predicts the path of an avulsing river. These first-order rules of avulsion suggest that avulsion risks are underestimated in many coastal environments8 and that probabilistic predictions of avulsion pathfinding can efficiently map hazards with minimal information. Applying these principles for risk assessment could particularly benefit the Global South, which is disproportionately affected by avulsions.

A lanthanide-rich kilonova in the aftermath of a long gamma-ray burst.

Observationally, kilonovae are astrophysical transients powered by the radioactive decay of nuclei heavier than iron, thought to be synthesized in the merger of two compact objects1-4. Over the first few days, the kilonova evolution is dominated by a large number of radioactive isotopes contributing to the heating rate2,5. On timescales of weeks to months, its behaviour is predicted to differ depending on the ejecta composition and the merger remnant6-8. Previous work has shown that the kilonova associated with gamma-ray burst 230307A is similar to kilonova AT2017gfo (ref. 9), and mid-infrared spectra revealed an emission line at 2.15 micrometres that was attributed to tellurium. Here we report a multi-wavelength analysis, including publicly available James Webb Space Telescope data9 and our own Hubble Space Telescope data, for the same gamma-ray burst. We model its evolution up to two months after the burst and show that, at these late times, the recession of the photospheric radius and the rapidly decaying bolometric luminosity (Lbol ∝ t-2.7±0.4, where t is time) support the recombination of lanthanide-rich ejecta as they cool.

Reverse-topology membrane scission by the ESCRT proteins.

The narrow membrane necks formed during viral, exosomal and intra-endosomal budding from membranes, as well as during cytokinesis and related processes, have interiors that are contiguous with the cytosol. Severing these necks involves action from the opposite face of the membrane as occurs during the well-characterized formation of coated vesicles. This 'reverse' (or 'inverse')-topology membrane scission is carried out by the endosomal sorting complex required for transport (ESCRT) proteins, which form filaments, flat spirals, tubes and conical funnels that are thought to direct membrane remodelling and scission. Their assembly, and their disassembly by the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively studied, but the mechanism of scission has been elusive. New insights from cryo-electron microscopy and various types of spectroscopy may finally be close to rectifying this situation.

Atomistic autophagy: the structures of cellular self-digestion.

Autophagy is directed by numerous distinct autophagy-related (Atg) proteins. These transmit starvation-induced signals to lipids and regulatory proteins and assemble a double-membrane autophagosome sequestering bulk cytoplasm and/or selected cargos destined for degradation upon autophagosome fusion with a vacuole or lysosome. This Review discusses the structural mechanisms by which Atg proteins sense membrane curvature, mediate a PI(3)P-signaling cascade, and utilize autophagy-specific ubiquitin-like protein cascades to tether proteins to autophagosomal membranes. Recent elucidation of molecular interactions enabling vesicle nucleation, elongation, and cargo recruitment provides insights into how dynamic protein-protein and protein-membrane interactions may dictate size, shape, and contents of autophagosomes.

Identification of a circadian output circuit for rest:activity rhythms in Drosophila.

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain and report here that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis.

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1.

AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the ß1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.

Clathrin-associated AP-1 controls termination of STING signalling.

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Delayed use of bioenergy crops might threaten climate and food security.

The potential of mitigation actions to limit global warming within 2 °C (ref. 1) might rely on the abundant supply of biomass for large-scale bioenergy with carbon capture and storage (BECCS) that is assumed to scale up markedly in the future2-5. However, the detrimental effects of climate change on crop yields may reduce the capacity of BECCS and threaten food security6-8, thus creating an unrecognized positive feedback loop on global warming. We quantified the strength of this feedback by implementing the responses of crop yields to increases in growing-season temperature, atmospheric CO2 concentration and intensity of nitrogen (N) fertilization in a compact Earth system model9. Exceeding a threshold of climate change would cause transformative changes in social-ecological systems by jeopardizing climate stability and threatening food security. If global mitigation alongside large-scale BECCS is delayed to 2060 when global warming exceeds about 2.5 °C, then the yields of agricultural residues for BECCS would be too low to meet the Paris goal of 2 °C by 2200. This risk of failure is amplified by the sustained demand for food, leading to an expansion of cropland or intensification of N fertilization to compensate for climate-induced yield losses. Our findings thereby reinforce the urgency of early mitigation, preferably by 2040, to avoid irreversible climate change and serious food crises unless other negative-emission technologies become available in the near future to compensate for the reduced capacity of BECCS.