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Oxidative phosphorylation has emerged as a critical therapeutic vulnerability of M. tuberculosis (Mtb). However, it is unknown how intracellular bacterial pathogens such as Mtb maintain respiration during infection despite the chemical effectors of host immunity. Mtb synthesizes diisonitrile lipopeptides that tightly chelate copper, but the role of these chalkophores in host-pathogen interactions is also unknown. We demonstrate that M. tuberculosis chalkophores maintain the function of the heme-copper bcc:aa 3 respiratory oxidase under copper limitation. Chalkophore deficiency impairs Mtb survival, respiration to oxygen, and ATP production under copper deprivation in culture, effects that are exacerbated by loss of the heme dependent Cytochrome BD respiratory oxidase. Our genetic analyses indicate that maintenance of respiration is the only cellular target of chalkophore mediated copper acquisition. M. tuberculosis lacking chalkophore biosynthesis is attenuated in mice, a phenotype that is also severely exacerbated by loss of the CytBD respiratory oxidase. We find that the host immune pressure that attenuates chalkophore deficient Mtb is independent of adaptive immunity and neutrophils. These data demonstrate that chalkophores counter host inflicted copper deprivation and highlight a multilayered system by which M. tuberculosis maintains respiration during infection., Competing Interests: Conflicts of Interest MSG declares equity and consulting fees from Vedanta biosciences and consulting fees from Fimbrion therapeutics. DST declares no conflicts of interest pertaining to this work.

In the face of rapidly accumulating genomic data, our ability to accurately predict key mature RNA properties that underlie transcript function and regulation remains limited. Pre-trained genomic foundation models offer an avenue to adapt learned RNA representations to biological prediction tasks. However, existing genomic foundation models are trained using strategies borrowed from textual or visual domains that do not leverage biological domain knowledge. Here, we introduce Orthrus, a Mamba-based mature RNA foundation model pre-trained using a novel self-supervised contrastive learning objective with biological augmentations. Orthrus is trained by maximizing embedding similarity between curated pairs of RNA transcripts, where pairs are formed from splice isoforms of 10 model organisms and transcripts from orthologous genes in 400+ mammalian species from the Zoonomia Project. This training objective results in a latent representation that clusters RNA sequences with functional and evolutionary similarities. We find that the generalized mature RNA isoform representations learned by Orthrus significantly outperform existing genomic foundation models on five mRNA property prediction tasks, and requires only a fraction of fine-tuning data to do so. Finally, we show that Orthrus is capable of capturing divergent biological function of individual transcript isoforms.

Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. At-RS31, a plant-specific splicing factor in the Serine/Arginine (SR)-rich protein family, responds to light and the Target of Rapamycin (TOR) signaling pathway, yet its downstream targets and regulatory impact remain unknown.To identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing.iCLIP identified 4,034 At-RS31 binding sites across 1,421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the RS domain of At-RS31 may influence its binding specificity in planta , underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator.By regulating stress-response genes and genes in both TOR and abscisic acid (ABA) signaling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing., Competing Interests: COMPETING INTERESTS None declared.

We describe an effort ("Codebook") to determine the sequence specificity of 332 putative and largely uncharacterized human transcription factors (TFs), as well as 61 control TFs. Nearly 5,000 independent experiments across multiple in vitro and in vivo assays produced motifs for just over half of the putative TFs analyzed (177, or 53%), of which most are unique to a single TF. The data highlight the extensive contribution of transposable elements to TF evolution, both in cis and trans , and identify tens of thousands of conserved, base-level binding sites in the human genome. The use of multiple assays provides an unprecedented opportunity to benchmark and analyze TF sequence specificity, function, and evolution, as further explored in accompanying manuscripts. 1,421 human TFs are now associated with a DNA binding motif. Extrapolation from the Codebook benchmarking, however, suggests that many of the currently known binding motifs for well-studied TFs may inaccurately describe the TF's true sequence preferences., Competing Interests: DECLARATION OF COMPETING INTERESTS O.F. is employed by Roche.

A long-standing challenge in human regulatory genomics is that transcription factor (TF) DNA-binding motifs are short and degenerate, while the genome is large. Motif scans therefore produce many false-positive binding site predictions. By surveying 179 TFs across 25 families using >1,500 cyclic in vitro selection experiments with fragmented, naked, and unmodified genomic DNA - a method we term GHT-SELEX (Genomic HT-SELEX) - we find that many human TFs possess much higher sequence specificity than anticipated. Moreover, genomic binding regions from GHT-SELEX are often surprisingly similar to those obtained in vivo (i.e. ChIP-seq peaks). We find that comparable specificity can also be obtained from motif scans, but performance is highly dependent on derivation and use of the motifs, including accounting for multiple local matches in the scans. We also observe alternative engagement of multiple DNA-binding domains within the same protein: long C2H2 zinc finger proteins often utilize modular DNA recognition, engaging different subsets of their DNA binding domain (DBD) arrays to recognize multiple types of distinct target sites, frequently evolving via internal duplication and divergence of one or more DBDs. Thus, contrary to conventional wisdom, it is common for TFs to possess sufficient intrinsic specificity to independently delineate cellular targets.

Biological insights often depend on comparing conditions such as disease and health, yet we lack effective computational tools for integrating single-cell genomics data across conditions or characterizing transitions from normal to deviant cell states. Here, we present Decipher, a deep generative model that characterizes derailed cell-state trajectories. Decipher jointly models and visualizes gene expression and cell state from normal and perturbed single-cell RNA-seq data, revealing shared and disrupted dynamics. We demonstrate its superior performance across diverse contexts, including in pancreatitis with oncogene mutation, acute myeloid leukemia, and gastric cancer., Competing Interests: D.P. is on the scientific advisory board of Insitro. R.L.L. is on the supervisory board of Qiagen and on the board of directors of Ajax Therapeutics, for which he receives compensation and equity support. He is or has recently been a scientific advisor to Imago, Mission Bio, and Syndax. Zentalis, Ajax, Bakx, Auron, Prelude, C4 Therapeutics, and Isoplexis, for which he receives equity support. He also has research support from Ajax and AbbVie, consulted for Janssen, and received honoraria from Astra Zeneca and Kura for invited lectures.

Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP., Competing Interests: Declaration of interests The authors declare no competing interests.

Neurocognitive impairment is a prevalent and important co-morbidity in virologically suppressed people living with HIV (PLWH), yet the underlying mechanisms remain elusive and treatments lacking. Here, we explored for the first time, use of participant-derived directly induced neurons (iNs) to model neuronal biology and injury in PLWH. iNs retain age- and disease-related features of the donors, providing unique opportunities to reveal novel aspects of neurological disorders. We obtained primary dermal fibroblasts from six virologically suppressed PLWH (range: 27 - 64 years, median: 53); 83% Male; 50% White) and seven matched people without HIV (PWOH) (range: 27 - 66, median: 55); 71% Male; 57% White). iNs were generated using transcription factors NGN2 and ASCL1, and validated by immunocytochemistry and single-cell-RNAseq. Transcriptomic analysis using bulk-RNAseq identified 29 significantly differentially expressed genes between iNs from PLWH and PWOH. Of these, 16 genes were downregulated and 13 upregulated in PLWH iNs. Protein-protein interaction network mapping indicates that iNs from PLWH exhibit differences in extracellular matrix organization and synaptic transmission. IFI27 was upregulated in iNs from PLWH, which complements independent post-mortem studies demonstrating elevated IFI27 expression in PLWH-derived brain tissue, indicating that iN generation reconstitutes this pathway. Finally, we observed that expression of the FOXL2NB-FOXL2-LINC01391 genome locus is reduced in iNs from PLWH and negatively correlates with neurocognitive impairment. Thus, we have identified an iN gene signature of HIV through direct reprogramming of skin fibroblasts into neurons revealing novel mechanisms of neurocognitive impairment in PLWH.

RNA-binding proteins (RBPs) are key regulators of gene expression. Here, we introduce EuPRI (Eukaryotic Protein-RNA Interactions) - a freely available resource of RNA motifs for 34,736 RBPs from 690 eukaryotes. EuPRI includes in vitro binding data for 504 RBPs, including newly collected RNAcompete data for 174 RBPs, along with thousands of reconstructed motifs. We reconstruct these motifs with a new computational platform - Joint Protein-Ligand Embedding (JPLE) - which can detect distant homology relationships and map specificity-determining peptides. EuPRI quadruples the number of known RBP motifs, expanding the motif repertoire across all major eukaryotic clades, and assigning motifs to the majority of human RBPs. EuPRI drastically improves knowledge of RBP motifs in flowering plants. For example, it increases the number of Arabidopsis thaliana RBP motifs 7-fold, from 14 to 105. EuPRI also has broad utility for inferring post-transcriptional function and evolutionary relationships. We demonstrate this by predicting a role for 12 Arabidopsis thaliana RBPs in RNA stability and identifying rapid and recent evolution of post-transcriptional regulatory networks in worms and plants. In contrast, the vertebrate RNA motif set has remained relatively stable after its drastic expansion between the metazoan and vertebrate ancestors. EuPRI represents a powerful resource for the study of gene regulation across eukaryotes., Competing Interests: Declaration of interests The authors declare no competing interests.

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

The heterogeneity of epithelial-to-mesenchymal transition (EMT) programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers. In these cells, TGF-β induces growth inhibition accompanied by a full EMT response that subsequently transitions into an atypical mesenchymal state of round morphology and lacking actin stress fibers. TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy., Competing Interests: Declaration of interests J.M. owns company stock in Scholar Rock.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. Unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites that transitioned to a longer-lived, focused pause in metazoans. This event coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve., Competing Interests: Competing Interests Statement The authors declare no competing interests.

Recent studies have identified a family of rod-shaped proteins which includes VPS13 and ATG2 and are thought to mediate unidirectional lipid transport at intracellular membrane contacts by a bridge-like mechanism. Here, we show that one such protein, BLTP3A/UHRF1BP1, associates with VAMP7-positive vesicles via its C-terminal region and anchors them to lysosomes via the binding of its chorein domain containing N-terminal region to Rab7. Upon damage of lysosomal membranes and resulting mATG8 recruitment to their surface by CASM, BLTP3A first dissociates from lysosomes but then reassociates with them via an interaction of its LIR motif with mATG8. Such interaction is mutually exclusive to the binding of BLTP3A to vesicles and leaves its N-terminal chorein domain, i.e. the proposed entry site of lipids into this family of proteins, available for binding to another membrane, possibly the ER. Our findings reveal that BLTP3A is an effector CASM, potentially as part of a mechanism to help repair or minimize lysosome damage by delivering lipids.

Myeloid phagocytes are essential for antifungal immunity against pulmonary Aspergillus fumigatus and systemic Candida albicans infections. However, the molecular mechanisms underlying fungal clearance by phagocytes remain incompletely understood. In this study, we investigated the role of perforin-2 ( Mpeg1 ) in antifungal immunity. We found that Mpeg1 -/- mice generated on a mixed C57BL/6J-DBA/2 background exhibited enhanced survival, reduced lung fungal burden, and greater neutrophil fungal killing activity compared to wild-type C57BL/6J (B6) mice, suggesting that perforin-2 may impair antifungal immune responses. However, when we compared Mpeg1 -/- mice with co-housed Mpeg +/+ littermate controls, these differences were no longer observed, indicating that initial findings were likely influenced by differences in the murine genetic background or the microbiota composition. Furthermore, perforin-2 was dispensable for antifungal immunity during C. albicans bloodstream infection. These results suggest that perforin-2 is not essential for host defense against fungal infections in otherwise immune competent mice and highlight the importance of generating co-housed littermate controls to minimize murine genetic and microbiota-related factors in studies of host defense mechanisms.

MicroRNAs are essential post-transcriptional regulators of gene expression and involved in many biological processes; however, our understanding of their genetic regulation and role in brain illnesses is limited. Here, we mapped brain microRNA expression quantitative trait loci (miR-QTLs) using genome-wide small RNA sequencing profiles from dorsolateral prefrontal cortex (dlPFC) samples of 604 older adult donors of European ancestry. miR-QTLs were identified for 224 miRNAs (48% of 470 tested miRNAs) at false discovery rate < 1%. We found that miR-QTLs were enriched in brain promoters and enhancers, and that intragenic miRNAs often did not share QTLs with their host gene. Additionally, we integrated the brain miR-QTLs with results from 16 GWAS of psychiatric and neurodegenerative diseases using multiple independent integration approaches and identified four miRNAs that contribute to the pathogenesis of bipolar disorder, major depression, post-traumatic stress disorder, schizophrenia, and Parkinson's disease. This study provides novel insights into the contribution of miRNAs to the complex biological networks that link genetic variation to disease.

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/β-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1 ; Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Recent advances in machine learning (ML) are reshaping drug discovery. Structure-based ML methods use physically-inspired models to predict binding affinities from protein:ligand complexes. These methods promise to enable the integration of data for many related targets, which addresses issues related to data scarcity for single targets and could enable generalizable predictions for a broad range of targets, including mutants. In this work, we report our experiences in building KinoML, a novel framework for ML in target-based small molecule drug discovery with an emphasis on structure-enabled methods. KinoML focuses currently on kinases as the relative structural conservation of this protein superfamily, particularly in the kinase domain, means it is possible to leverage data from the entire superfamily to make structure-informed predictions about binding affinities, selectivities, and drug resistance. Some key lessons learned in building KinoML include: the importance of reproducible data collection and deposition, the harmonization of molecular data and featurization, and the choice of the right data format to ensure reusability and reproducibility of ML models. As a result, KinoML allows users to easily achieve three tasks: accessing and curating molecular data; featurizing this data with representations suitable for ML applications; and running reproducible ML experiments that require access to ligand, protein, and assay information to predict ligand affinity. Despite KinoML focusing on kinases, this framework can be applied to other proteins. The lessons reported here can help guide the development of platforms for structure-enabled ML in other areas of drug discovery.

The metastatic spread of a cancer can be reconstructed from DNA sequencing of primary and metastatic tumours, but doing so requires solving a challenging combinatorial optimization problem. This problem often has multiple solutions that cannot be distinguished based on current maximum parsimony principles alone. Current algorithms use ad hoc criteria to select among these solutions, and decide, a priori, what patterns of metastatic spread are more likely, which is itself a key question posed by studies of metastasis seeking to use these tools. Here we introduce Metient, a freely available open-source tool which proposes multiple possible hypotheses of metastatic spread in a cohort of patients and rescores these hypotheses using independent data on genetic distance of metastasizing clones and organotropism. Metient is more accurate and is up to 50x faster than current state-of-the-art. Given a cohort of patients, Metient can calibrate its parsimony criteria, thereby identifying shared patterns of metastatic dissemination in the cohort. Reanalyzing metastasis in 169 patients based on 490 tumors, Metient automatically identifies cancer type-specific trends of metastatic dissemination in melanoma, high-risk neuroblastoma and non-small cell lung cancer. Metient's reconstructions usually agree with semi-manual expert analysis, however, in many patients, Metient identifies more plausible migration histories than experts, and further finds that polyclonal seeding of metastases is more common than previously reported. By removing the need for hard constraints on what patterns of metastatic spread are most likely, Metient introduces a way to further our understanding of cancer type-specific metastatic spread.

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a noncanonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC-motif and Drosha's PAZ-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP., Competing Interests: Declaration of interest Authors declare no competing financial interest.

DNA transposable elements and transposase-derived genes are present in most living organisms, including vertebrates, but their function is largely unknown. PiggyBac Transposable Element Derived 5 (PGBD5) is an evolutionarily conserved vertebrate DNA transposase-derived gene with retained nuclease activity in human cells. Vertebrate brain development is known to be associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. Here, we show that PGBD5 contributes to normal brain development in mice and humans, where its deficiency causes disorder of intellectual disability, movement, and seizures. In mice, Pgbd5 is required for the developmental induction of post-mitotic DNA breaks and recurrent somatic genome rearrangements. In the brain cortex, loss of Pgbd5 leads to aberrant differentiation and gene expression of distinct neuronal populations, including specific types of glutamatergic neurons, which explains the features of PGBD5 deficiency in humans. Thus, PGBD5 might be a transposase-derived enzyme required for brain development in mammals., Competing Interests: Competing interests: Authors declare that they have no competing interests. AK is a consultant for Novartis, Rgenta, Blueprint, and Syndax. RR is a founder and a member of the SAB of Genotwin, and a member of the SAB of Diatech Pharmacogenetics. None of these activities are related to the work described in this manuscript.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients., Competing Interests: Competing interests. FG has been a paid consultant (no equity) to Third Rock Ventures from 2018 to 2020. Sequencing costs and analysis in this study were covered in part by a SRA between Third Rock venture and MSKCC. This work led to patents PCT/US2022/037893/WO2023004054A1 ‘Methods and compositions for the treatment of alzheimer’s disease’ by MSKCC and PCT/US2018/047964 ‘Kinase mutation-associated neurodegenerative disorders by MSKCC’.

Langerhans cell Histiocytosis (LCH) and Erdheim-Chester disease (ECD) are clonal myeloid disorders, associated with MAP-Kinase activating mutations and an increased risk of neurodegeneration. Surprisingly, we found pervasive PU.1 + microglia mutant clones across the brain of LCH and ECD patients with and without neurological symptoms, associated with microgliosis, reactive astrocytosis, and neuronal loss. The disease predominated in the grey nuclei of the rhombencephalon, a topography attributable to a local proliferative advantage of mutant microglia. Presence of clinical symptoms was associated with a longer evolution of the disease and a larger size of PU.1 + clones (p= 0.0003). Genetic lineage tracing of PU.1 + clones suggest a resident macrophage lineage or a bone marrow precursor origin depending on patients. Finally, a CSF1R-inhibitor depleted mutant microglia and limited neuronal loss in mice suggesting an alternative to MAPK inhibitors. These studies characterize a progressive neurodegenerative disease, caused by clonal proliferation of inflammatory microglia (CPIM), with a decade(s)-long preclinical stage of incipient disease that represent a therapeutic window for prevention of neuronal death., Competing Interests: Conflict of Interest. FG has performed consulting for Third Rock venture in the past. Targeted Sequencing was funded in part by a grant from Third Rock venture. FG and RV are inventors in MSKCC’s United States application or PCT international application number PCT/US2018/047964 filed on 8/24/2018 (KINASE MUTATION-ASSOCIATED NEURODEGENERATIVE DISORDERS)

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4/5 deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/5 deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development., Competing Interests: Competing interests Authors declare that they have no competing interests.

G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and transmit critical signals from the extracellular to the intracellular spaces. Transcriptomic data of resected breast tumors shows that low mRNA expression of the orphan GPCR GPR52 correlates with reduced overall survival in breast cancer patients, leading to the hypothesis that loss of GPR52 supports breast cancer progression. CRISPR-Cas9 was used to knockout GPR52 in human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line, MCF10A. Loss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells. To determine the in vivo impact of GPR52 loss, MDA-MB-468 cells were injected into zebrafish and loss of GPR52 was associated with a greater total cancer area compared to control cells. RNA-sequencing and proteomic analyses of GPR52-null breast cancer cells reveal an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates production of cAMP, induces some phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some of the GPR52 KO phenotypes. Overall, our results reveal GPR52 loss as a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option in breast cancer., Competing Interests: The authors declare no potential conflicts of interest.

Haploinsufficiency for GATA6 is associated with congenital heart disease (CHD) with variable comorbidity of pancreatic or diaphragm defects, although the etiology of disease is not well understood. Here, we used cardiac directed differentiation from human embryonic stem cells (hESCs) as a platform to study GATA6 function during early cardiogenesis. GATA6 loss-of-function hESCs had a profound impairment in cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation due to early defects during the mesendoderm and lateral mesoderm patterning stages. Profiling by RNA-seq and CUT&RUN identified genes of the WNT and BMP programs regulated by GATA6 during early mesoderm patterning. Furthermore, interactome analysis detected GATA6 binding with developmental transcription factors and chromatin remodelers suggesting cooperative regulation of cardiac lineage gene accessibility. We show that modulating WNT and BMP inputs during the first 48 hours of cardiac differentiation is sufficient to partially rescue CPC and CM defects in GATA6 heterozygous and homozygous mutant hESCs. This study provides evidence of the regulatory functions for GATA6 directing human precardiac mesoderm patterning during the earliest stages of cardiogenesis to further our understanding of haploinsufficiency causing CHD and the co-occurrence of cardiac and other organ defects caused by human GATA6 mutations., Competing Interests: Competing interests T.E. is a co-founder of OncoBeat, LLC.

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations., Competing Interests: Competing interests All authors declare to have no actual or potential conflict of interest including any financial, personal, or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Drug discovery is stochastic. The effectiveness of candidate compounds in satisfying design objectives is unknown ahead of time, and the tools used for prioritization-predictive models and assays-are inaccurate and noisy. In a typical discovery campaign, thousands of compounds may be synthesized and tested before design objectives are achieved, with many others ideated but deprioritized. These challenges are well-documented, but assessing potential remedies has been difficult. We introduce DrugGym , a framework for modeling the stochastic process of drug discovery. Emulating biochemical assays with realistic surrogate models, we simulate the progression from weak hits to sub-micromolar leads with viable ADME. We use this testbed to examine how different ideation, scoring, and decision-making strategies impact statistical measures of utility, such as the probability of program success within predefined budgets and the expected costs to achieve target candidate profile (TCP) goals. We also assess the influence of affinity model inaccuracy, chemical creativity, batch size, and multi-step reasoning. Our findings suggest that reducing affinity model inaccuracy from 2 to 0.5 pIC50 units improves budget-constrained success rates tenfold. DrugGym represents a realistic testbed for machine learning methods applied to the hit-to-lead phase. Source code is available at www.drug-gym.org.

The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut- derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications., Competing Interests: Declaration of interests The authors declare no competing interests

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the FXR1 network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause Fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. These findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as organizer of signaling reactions., Competing Interests: Declaration of Interests Christine Mayr is a member of the Cell Advisory Board. The authors declare no other competing interests.

Natural Killer (NK) cells can recognize and kill Mtb -infected cells in vitro, however their role after natural human exposure has not been well-studied. To identify Mtb -responsive NK cell populations, we analyzed the peripheral blood of healthy household contacts of active Tuberculosis (TB) cases and source community donors in an endemic region of Port-au-Prince, Haiti by flow cytometry. We observed higher CD8α expression on NK cells in putative resistors (IGRA- contacts) with a progressive loss of these circulating cells during household-associated latent infection and disease. In vitro assays and CITE-seq analysis of CD8α + NK cells demonstrated enhanced maturity, cytotoxic gene expression, and response to cytokine stimulation relative to CD8α - NK cells. CD8α + NK cells also displayed dynamic surface expression dependent on MHC I in contrast to conventional CD8 + T cells. Together, these results support a specialized role for CD8α + NK cell populations during Mtb infection correlating with disease resistance., Competing Interests: Competing interests: Authors declare that they have no competing interests.

The majority of human breast cancers are dependent on hormone-stimulated estrogen receptor alpha (ER) and are sensitive to its inhibition. Treatment resistance arises in most advanced cancers due to genetic alterations that promote ligand independent activation of ER itself or ER target genes. Whereas re-targeting of the ER ligand binding domain (LBD) with newer ER antagonists can work in some cases, these drugs are largely ineffective in many genetic backgrounds including ER fusions that lose the LBD or in cancers that hyperactivate ER targets. By identifying the mechanism of ER translation, we herein present an alternative strategy to target ER and difficult to treat ER variants. We find that ER translation is cap-independent and mTOR inhibitor insensitive, but dependent on 5' UTR elements and sensitive to pharmacologic inhibition of the translation initiation factor eIF4A, an mRNA helicase. EIF4A inhibition rapidly reduces expression of ER and short-lived targets of ER such as cyclin D1 and other components of the cyclin D-CDK complex in breast cancer cells. These effects translate into suppression of growth of a variety of ligand-independent breast cancer models including those driven by ER fusion proteins that lack the ligand binding site. The efficacy of eIF4A inhibition is enhanced when it is combined with fulvestrant-an ER degrader. Concomitant inhibition of ER synthesis and induction of its degradation causes synergistic and durable inhibition of ER expression and tumor growth. The clinical importance of these findings is confirmed by results of an early clinical trial (NCT04092673) of the selective eIF4A inhibitor zotatifin in patients with estrogen receptor positive metastatic breast cancer. Multiple clinical responses have been observed on combination therapy including durable regressions. These data suggest that eIF4A inhibition could be a useful new strategy for treating advanced ER+ breast cancer., Competing Interests: Declaration of Interests G.Y. and O.O. are listed as inventors in patents that were filed by MSKCC. O.O. receives royalties from some of the licensed patents. O.O. is an unpaid member of the SAB and owns shares of Angiogenex Therapeutics Inc. All are not related to this work. FP is a member of the scientific advisory board of MultiplexDX. In addition, FP serves on the diagnostic advisory board and reports receiving consultancy fees from AstraZeneca. S. Chandarlapaty reports personal fees from AstraZeneca, Daiichi-Sankyo, Effector, Nuvalent, Neogenomics, Genesis, Casdin Capital, Blueprint, grants from Daiichi-Sankyo and AstraZeneca and equity in Effector, Odyssey Bio, and Totus outside the submitted work. N.R. is on the scientific advisory board (SAB) and owns equity in Beigene, Zai Labs, MapKure, Ribon and Effector. N.R. is also on the SAB of Astra Zeneca and Chugai and a past SAB member of Novartis, Millennium-Takeda, Kura, and Araxes. N.R. is a consultant to RevMed, Tarveda, Array-Pfizer, Boehringer-Ingelheim and Eli Lilly. He receives research funding from Revmed, AstraZeneca, Array, Pfizer and Boehringer-Ingelheim and owns equity in Kura Oncology and Fortress.

Despite the potential of targeted epigenetic therapies, most cancers do not respond to current epigenetic drugs. The Polycomb repressive complex EZH2 inhibitor tazemetostat was recently approved for the treatment of SMARCB1 -deficient epithelioid sarcomas, based on the functional antagonism between PRC2 and loss of SMARCB1. Through the analysis of tazemetostat-treated patient tumors, we recently defined key principles of their response and resistance to EZH2 epigenetic therapy. Here, using transcriptomic inference from SMARCB1 -deficient tumor cells, we nominate the DNA damage repair kinase ATR as a target for rational combination EZH2 epigenetic therapy. We show that EZH2 inhibition promotes DNA damage in epithelioid and rhabdoid tumor cells, at least in part via its induction of the transposase-derived PGBD5. We leverage this collateral synthetic lethal dependency to target PGBD5-dependent DNA damage by inhibition of ATR but not CHK1 using elimusertib. Consequently, combined EZH2 and ATR inhibition improves therapeutic responses in diverse patient-derived epithelioid and rhabdoid tumors in vivo . This advances a combination epigenetic therapy based on EZH2-PGBD5 synthetic lethal dependency suitable for immediate translation to clinical trials for patients., Competing Interests: Conflict of Interest The authors declare no potential conflicts of interest. AK is a consultant for Novartis, Rgenta, Blueprint, and Syndax.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2 P95H /+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2 P95H -induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2 P95H /+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2 P95H /+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2 P95H mutant MDS and AML., Competing Interests: Conflict of Interest Statement: PSK is collaborating with Blueprint Medicines under a sponsored research agreement unrelated to the current manuscript. OA-W has served as a consultant for H3B Biomedicine, Foundation Medicine Inc, Merck, Prelude Therapeutics, and Janssen, and is on the Scientific Advisory Board of Envisagenics Inc, AIChemy, Harmonic Discovery Inc, and Pfizer Boulder; OA-W has received prior research funding from H3B Biomedicine, Nurix Therapeutics, Minovia Therapeutics, and LOXO Oncology unrelated to the current manuscript. DCW is on the Scientific Advisory Boards of Pano Therapeutics and Medical Excellence Capital.

BRAF V600E mutation occurs in 46% of melanomas and drives high levels of ERK activity and ERK-dependent proliferation. However, BRAF V600E is insufficient to drive melanoma in GEMM models, and 82% of human benign nevi harbor BRAF V600E mutations. We show here that BRAF V600E inhibits mesenchymal migration by causing feedback inhibition of RAC1 activity. ERK pathway inhibition induces RAC1 activation and restores migration and invasion. In cells with BRAF V600E , mutant RAC1, overexpression of PREX1, PREX2, or PTEN inactivation restore RAC1 activity and cell motility. Together, these lesions occur in 48% of BRAF V600E melanomas. Thus, although BRAF V600E activation of ERK deregulates cell proliferation, it prevents full malignant transformation by causing feedback inhibition of cell migration. Secondary mutations are, therefore, required for tumorigenesis. One mechanism underlying tumor evolution may be the selection of lesions that rescue the deleterious effects of oncogenic drivers., Competing Interests: Declaration of interests (Authors declare no potential conflicts of interest)

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)--a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis (TMA) on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated, but also found that the expression of molecules targeted therapeutically, namely PSMA , STEAP1 , STEAP2 , TROP2, CEACAM5 , and DLL3 , varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer (SCLC) subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to novel antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types., Competing Interests: Competing Interests: P.S.N. has received consulting fees from Janssen, Merck and Bristol Myers Squibb and research support from Janssen for work unrelated to the present studies. S.Z. has received consulting fees from Guidepoint and GLG consulting. M.C.H served as a paid consultant/received honoraria from Pfizer and has received research funding from Merck, Novartis, Genentech, Promicell and Bristol Myers Squibb. C.L.S is on the board of directors of Novartis, is a co-founder of ORIC Pharmaceuticals, and is a co-inventor of the prostate cancer drugs enzalutamide and apalutamide, covered by U.S. patents 7,709,517, 8,183,274, 9,126,941, 8,445,507, 8,802,689, and 9,388,159 filed by the University of California. C.L.S. is on the scientific advisory boards of the following biotechnology companies: Beigene, Blueprint, Cellcarta, Column Group, Foghorn, Housey Pharma, Nextech, PMV.

Lineage plasticity is a recognized hallmark of cancer progression that can shape therapy outcomes. The underlying cellular and molecular mechanisms mediating lineage plasticity remain poorly understood. Here, we describe a versatile in vivo platform to identify and interrogate the molecular determinants of neuroendocrine lineage transformation at different stages of prostate cancer progression. Adenocarcinomas reliably develop following orthotopic transplantation of primary mouse prostate organoids acutely engineered with human-relevant driver alterations (e.g., Rb1 - / - ; Trp53 - / - ; cMyc + or Pten - / - ; Trp53 - / - ; cMyc + ), but only those with Rb1 deletion progress to ASCL1+ neuroendocrine prostate cancer (NEPC), a highly aggressive, androgen receptor signaling inhibitor (ARSI)-resistant tumor. Importantly, we show this lineage transition requires a native in vivo microenvironment not replicated by conventional organoid culture. By integrating multiplexed immunofluorescence, spatial transcriptomics and PrismSpot to identify cell type-specific spatial gene modules, we reveal that ASCL1+ cells arise from KRT8+ luminal epithelial cells that progressively acquire transcriptional heterogeneity, producing large ASCL1 + ;KRT8 - NEPC clusters. Ascl1 loss in established NEPC results in transient tumor regression followed by recurrence; however, Ascl1 deletion prior to transplantation completely abrogates lineage plasticity, yielding adenocarcinomas with elevated AR expression and marked sensitivity to castration. The dynamic feature of this model reveals the importance of timing of therapies focused on lineage plasticity and offers a platform for identification of additional lineage plasticity drivers.

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1 ), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that then positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.

Driver gene mutations can increase the metastatic potential of the primary tumor 1-3 , but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGFβ signaling - which is a hallmark of multiple gastrointestinal malignancies 4,5 . SMAD4 inactivation mediates TGFβ's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis 6-14 . To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4 's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease., Competing Interests: COMPETING INTERESTS S.W.L. is a consultant for Fate Therapeutics, and is a consultant and holds equity in Blueprint Medicines, ORIC Pharmaceuticals, Mirimus, PMV Pharmaceuticals, Faeth Therapeutics, and Senescea Therapeutics. R.P.K. is a co-founder of and consultant for Econic Biosciences. D.P. is on the scientific advisory board of Insitro. J.M. holds equity in Scholar Rock. All other authors have no competing interests. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this report.

Whether neurodegenerative diseases linked to misfolding of the same protein share genetic risk drivers or whether different protein-aggregation pathologies in neurodegeneration are mechanistically related remains uncertain. Conventional genetic analyses are underpowered to address these questions. Through careful selection of patients based on protein aggregation phenotype (rather than clinical diagnosis) we can increase statistical power to detect associated variants in a targeted set of genes that modify proteotoxicities. Genetic modifiers of alpha-synuclein (ɑS) and beta-amyloid (Aβ) cytotoxicity in yeast are enriched in risk factors for Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. Here, along with known AD/PD risk genes, we deeply sequenced exomes of 430 ɑS/Aβ modifier genes in patients across alpha-synucleinopathies (PD, Lewy body dementia and multiple system atrophy). Beyond known PD genes GBA1 and LRRK2 , rare variants AD genes ( CD33 , CR1 and PSEN2 ) and Aβ toxicity modifiers involved in RhoA/actin cytoskeleton regulation ( ARGHEF1, ARHGEF28, MICAL3, PASK, PKN2, PSEN2 ) were shared risk factors across synucleinopathies. Actin pathology occurred in iPSC synucleinopathy models and RhoA downregulation exacerbated ɑS pathology. Even in sporadic PD, the expression of these genes was altered across CNS cell types. Genome-wide CRISPR screens revealed the essentiality of PSEN2 in both human cortical and dopaminergic neurons, and PSEN2 mutation carriers exhibited diffuse brainstem and cortical synucleinopathy independent of AD pathology. PSEN2 contributes to a common-risk signal in PD GWAS and regulates ɑS expression in neurons. Our results identify convergent mechanisms across synucleinopathies, some shared with AD.

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG , the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG . To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage in Drosophila melanogaster . We report that translesion synthesis, rather than template switching, is the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Despite rapid evolution across eutherian mammals, the X-linked miR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes ( Slitrk2 and Fmr1 ) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked miR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of the miR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked miR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the miR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis., Competing Interests: Competing Interest Statement: The authors declare no conflict of interest.

The cGAS-STING pathway detects cytosolic DNA and activates a signaling cascade that results in a type I interferon (IFN) response. The endoplasmic reticulum (ER)-associated exonuclease TREX1 suppresses cGAS-STING by eliminating DNA from the cytosol. Mutations that compromise TREX1 function are linked to autoinflammatory disorders, including systemic lupus erythematosus (SLE) and Aicardi-Goutières syndrome (AGS). Despite key roles in regulating cGAS-STING and suppressing excessive inflammation, the impact of many disease-associated TREX1 mutations - particularly those outside of the core catalytic domains - remains poorly understood. Here, we characterize a recessive AGS-linked TREX1 P61Q mutation occurring within the poorly characterized polyproline helix (PPII) motif. In keeping with its position outside of the catalytic core or ER targeting motifs, neither the P61Q mutation, nor aggregate proline-to-alanine PPII mutation, disrupt TREX1 exonuclease activity, subcellular localization, or cGAS-STING regulation in overexpression systems. Introducing targeted mutations into the endogenous TREX1 locus revealed that PPII mutations destabilize the protein, resulting in impaired exonuclease activity and unrestrained cGAS-STING activation. Overall, these results demonstrate that TREX1 PPII mutations, including P61Q, impair proper immune regulation and lead to autoimmune disease through TREX1 destabilization., Competing Interests: DECLARATION OF INTERESTS The authors declare no conflicts of interest.

Identifying predictive biomarkers of patient outcomes from high-throughput microbiome data is of high interest, while existing computational methods do not satisfactorily account for complex survival endpoints, longitudinal samples, and taxa-specific sequencing biases. We present FLORAL (https://vdblab.github.io/FLORAL/), an open-source computational tool to perform scalable log-ratio lasso regression and microbial feature selection for continuous, binary, time-to-event, and competing risk outcomes, with compatibility of longitudinal microbiome data as time-dependent covariates. The proposed method adapts the augmented Lagrangian algorithm for a zero-sum constraint optimization problem while enabling a two-stage screening process for extended false-positive control. In extensive simulation and real-data analyses, FLORAL achieved consistently better false-positive control compared to other lasso-based approaches, and better sensitivity over popular differential abundance testing methods for datasets with smaller sample size. In a survival analysis in allogeneic hematopoietic-cell transplant, we further demonstrated considerable improvement by FLORAL in microbial feature selection by utilizing longitudinal microbiome data over only using baseline microbiome data., Competing Interests: Authors’ Disclosures J.U. Peled reports research funding, intellectual property fees, and travel reimbursement from Seres Therapeutics, and consulting fees from DaVolterra, CSL Behring, Crestone Inc, and from MaaT Pharma. He serves on an Advisory board of and holds equity in Postbiotics Plus Research. He has filed intellectual property applications related to the microbiome (reference numbers #62/843,849, #62/977,908, and #15/756,845). D.M. Ponce has served as advisory board member for Evive Biotechnology (Shanghai) Ltd (formerly Generon [Shanghai] Corporation Ltd), she served as advisory board member or consultant of Sanofi Corporation, CareDx, Ceramedix, Incyte, and receives research funding from Takeda Corporation and Incyte. M.-A. Perales reports honoraria from Adicet, Allovir, Caribou Biosciences, Celgene, Bristol-Myers Squibb, Equilium, Exevir, Incyte, Karyopharm, Kite/Gilead, Merck, Miltenyi Biotec, MorphoSys, Nektar Therapeutics, Novartis, Omeros, OrcaBio, Syncopation, VectivBio AG, and Vor Biopharma. He serves on DSMBs for Cidara Therapeutics, Medigene, and Sellas Life Sciences, and the scientific advisory board of NexImmune. He has ownership interests in NexImmune and Omeros. He has received institutional research support for clinical trials from Incyte, Kite/Gilead, Miltenyi Biotec, Nektar Therapeutics, and Novartis. M.R.M. van den Brink has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he has received royalties from Wolters Kluwer; has consulted, received honorarium from or participated in advisory boards for Seres Therapeutics, Vor Biopharma, Rheos Medicines, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Ceramedix, Lygenesis, Pluto Therapeutics, GlaskoSmithKline, Da Volterra, Thymofox, Garuda, Novartis (Spouse), Synthekine (Spouse), Beigene (Spouse), Kite (Spouse); he has IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). Memorial Sloan Kettering Cancer Center (MSK) has institutional financial interests relative to Seres Therapeutics.

Antiviral immune mediators, including interferons and their downstream effectors, are critical for host defense yet can become detrimental when uncontrolled. Here, we identify a macrophage-mediated anti-inflammatory mechanism that limits type I interferon (IFN-I) responses. Specifically, we found that cellular stress and pathogen recognition induce Oncostatin M (OSM) production by macrophages. OSM-deficient mice succumbed to challenge with influenza or a viral mimic due to heightened IFN-I activation. Macrophage-derived OSM restricted excessive IFN-I production by lung epithelial cells following viral stimulation. Furthermore, reconstitution of OSM in the respiratory tract was sufficient to protect mice lacking macrophage-derived OSM against morbidity, indicating the importance of local OSM production. This work reveals a host strategy to dampen inflammation in the lung through the negative regulation of IFN-I by macrophages.

Understanding diverse responses of individual cells to the same perturbation is central to many biological and biomedical problems. Current methods, however, do not precisely quantify the strength of perturbation responses and, more importantly, reveal new biological insights from heterogeneity in responses. Here we introduce the perturbation-response score (PS), based on constrained quadratic optimization, to quantify diverse perturbation responses at a single-cell level. Applied to single-cell transcriptomes of large-scale genetic perturbation datasets (e.g., Perturb-seq), PS outperforms existing methods for quantifying partial gene perturbation responses. In addition, PS presents two major advances. First, PS enables large-scale, single-cell-resolution dosage analysis of perturbation, without the need to titrate perturbation strength. By analyzing the dose-response patterns of over 2,000 essential genes in Perturb-seq, we identify two distinct patterns, depending on whether a moderate reduction in their expression induces strong downstream expression alterations. Second, PS identifies intrinsic and extrinsic biological determinants of perturbation responses. We demonstrate the application of PS in contexts such as T cell stimulation, latent HIV-1 expression, and pancreatic cell differentiation. Notably, PS unveiled a previously unrecognized, cell-type-specific role of coiled-coil domain containing 6 (CCDC6) in guiding liver and pancreatic lineage decisions, where CCDC6 knockouts drive the endoderm cell differentiation towards liver lineage, rather than pancreatic lineage. The PS approach provides an innovative method for dose-to-function analysis and will enable new biological discoveries from single-cell perturbation datasets., Competing Interests: Competing interests T.B. is a co-founder and Managing Director of Myllia Biotechnology. A.K., A.V., N.U. and A.L. are employees of Myllia Biotechnology. Other authors declare that they have no competing interest.

Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shown Drosophila cofilin ( DmCFL ) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology., Competing Interests: Competing Interests The authors declare no competing or financial interests.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease 1-6 . Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium 7 . We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics., Competing Interests: Conflict of Interest Statement Z.A. is employed by Google DeepMind. J.C.U. is an employee of Illumina, Inc. D.R.K. is employed by Calico Life Sciences LLC. Z.W. co-founded Rgenta Therapeutics, and she serves as a scientific advisor for the company and is a member of its board. W.J.G. is an inventor on IP licensed by 10x Genomics. A.Kundaje is on the scientific advisory board of PatchBio, SerImmune and OpenTargets, was a consultant with Illumina, and owns shares in DeepGenomics, ImmunAI and Freenome. J.M.E. is a consultant and equity holder in Martingale Labs, Inc. and has received materials from 10x Genomics unrelated to this study.