Lesion-remote astrocytes govern microglia-mediated white matter repair.

Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.

Single-cell and spatial profiling identify three response trajectories to pembrolizumab and radiation therapy in triple negative breast cancer.

Strategies are needed to better identify patients that will benefit from immunotherapy alone or who may require additional therapies like chemotherapy or radiotherapy to overcome resistance. Here we employ single-cell transcriptomics and spatial proteomics to profile triple negative breast cancer biopsies taken at baseline, after one cycle of pembrolizumab, and after a second cycle of pembrolizumab given with radiotherapy. Non-responders lack immune infiltrate before and after therapy and exhibit minimal therapy-induced immune changes. Responding tumors form two groups that are distinguishable by a classifier prior to therapy, with one showing high major histocompatibility complex expression, evidence of tertiary lymphoid structures, and displaying anti-tumor immunity before treatment. The other responder group resembles non-responders at baseline and mounts a maximal immune response, characterized by cytotoxic T cell and antigen presenting myeloid cell interactions, only after combination therapy, which is mirrored in a murine model of triple negative breast cancer.

Single-cell profiling of murine bladder cancer identifies sex-specific transcriptional signatures with prognostic relevance.

Bladder cancer (BLCA) is more common in men but more aggressive in women. Sex-based differences in cancer biology are commonly studied using a murine model with BLCA generated by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). While tumors in the BBN model have been profiled, these profiles provide limited information on the tumor microenvironment. Here, we applied single-cell RNA sequencing to characterize cell-type specific transcriptional differences between male and female BBN-induced tumors. We found proportional and gene expression differences in epithelial and non-epithelial subpopulations between male and female tumors. Expression of several genes predicted sex-specific survival in several human BLCA datasets. We identified novel and clinically relevant sex-specific transcriptional signatures including immune cells in the tumor microenvironment and it validated the relevance of the BBN model for studying sex differences in human BLCA. This work highlights the importance of considering sex as a biological variable in the development of new and accurate cancer markers.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

The molecular consequences of androgen activity in the human breast.

Estrogen and progesterone have been extensively studied in the mammary gland, but the molecular effects of androgen remain largely unexplored. Transgender men are recorded as female at birth but identify as male and may undergo gender-affirming androgen therapy to align their physical characteristics and gender identity. Here we perform single-cell-resolution transcriptome, chromatin, and spatial profiling of breast tissues from transgender men following androgen therapy. We find canonical androgen receptor gene targets are upregulated in cells expressing the androgen receptor and that paracrine signaling likely drives sex-relevant androgenic effects in other cell types. We also observe involution of the epithelium and a spatial reconfiguration of immune, fibroblast, and vascular cells, and identify a gene regulatory network associated with androgen-induced fat loss. This work elucidates the molecular consequences of androgen activity in the human breast at single-cell resolution.

MRI/RNA-Seq-Based Radiogenomics and Artificial Intelligence for More Accurate Staging of Muscle-Invasive Bladder Cancer.

Accurate staging of bladder cancer assists in identifying optimal treatment (e.g., transurethral resection vs. radical cystectomy vs. bladder preservation). However, currently, about one-third of patients are over-staged and one-third are under-staged. There is a pressing need for a more accurate staging modality to evaluate patients with bladder cancer to assist clinical decision-making. We hypothesize that MRI/RNA-seq-based radiogenomics and artificial intelligence can more accurately stage bladder cancer. A total of 40 magnetic resonance imaging (MRI) and matched formalin-fixed paraffin-embedded (FFPE) tissues were available for analysis. Twenty-eight (28) MRI and their matched FFPE tissues were available for training analysis, and 12 matched MRI and FFPE tissues were used for validation. FFPE samples were subjected to bulk RNA-seq, followed by bioinformatics analysis. In the radiomics, several hundred image-based features from bladder tumors in MRI were extracted and analyzed. Overall, the model obtained mean sensitivity, specificity, and accuracy of 94%, 88%, and 92%, respectively, in differentiating intra- vs. extra-bladder cancer. The proposed model demonstrated improvement in the three matrices by 17%, 33%, and 25% and 17%, 16%, and 17% as compared to the genetic- and radiomic-based models alone, respectively. The radiogenomics of bladder cancer provides insight into discriminative features capable of more accurately staging bladder cancer. Additional studies are underway.

Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumor cell (DTC) dormancy in this organ is limited. Here using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micrometastatic state as the rate-limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein, thereby sequestering it from the nucleus and preventing its prometastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.

Local non-pituitary growth hormone is induced with aging and facilitates epithelial damage.

Microenvironmental factors modulating age-related DNA damage are unclear. Non-pituitary growth hormone (npGH) is induced in human colon, non-transformed human colon cells, and fibroblasts, and in 3-dimensional intestinal organoids with age-associated DNA damage. Autocrine/paracrine npGH suppresses p53 and attenuates DNA damage response (DDR) by inducing TRIM29 and reducing ATM phosphorylation, leading to reduced DNA repair and DNA damage accumulation. Organoids cultured up to 4 months exhibit aging markers, p16, and SA-ß-galactosidase and decreased telomere length, as well as DNA damage accumulation, with increased npGH, suppressed p53, and attenuated DDR. Suppressing GH in aged organoids increases p53 and decreases DNA damage. WT mice exhibit age-dependent colon DNA damage accumulation, while in aged mice devoid of colon GH signaling, DNA damage remains low, with elevated p53. As age-associated npGH induction enables a pro-proliferative microenvironment, abrogating npGH signaling could be targeted as anti-aging therapy by impeding DNA damage and age-related pathologies.

TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer.

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.

An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer.

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

Characterization of universal features of partially methylated domains across tissues and species.

BACKGROUND: Partially methylated domains (PMDs) are a hallmark of epigenomes in reproducible and specific biological contexts, including cancer cells, the placenta, and cultured cell lines. Existing methods for deciding whether PMDs exist in a sample, as well as their identification, are few, often tailored to specific biological questions, and require high coverage samples for accurate identification. RESULTS: In this study, we outline a set of axioms that take a step towards a functional definition for PMDs, describe an improved method for comparable PMD detection across samples with substantially differing sequencing depths, and refine the decision criteria for whether a sample contains PMDs using a data-driven approach. Applying our method to 267 methylomes from 7 species, we corroborated recent results regarding the general association between replication timing and PMD state, and report identification of several reproducibly "escapee" genes within late-replicating domains that escape the reduced expression and hypomethylation of their immediate genomic neighborhood. We also explored the discordant PMD state of orthologous genes between human and mouse, and observed a directional association of PMD state with gene expression and local gene density. CONCLUSIONS: Our improved method makes low sequencing depth, population-level studies of PMD variation possible and our results further refine the model of PMD formation as one where sequence context and regional epigenomic features both play a role in gradual genome-wide hypomethylation.

Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans.

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.

Lymph node metastases develop through a wider evolutionary bottleneck than distant metastases.

Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms.

Natural killer cell activity and prostate cancer risk in veteran men undergoing prostate biopsy.

BACKGROUND: A previous pilot study found that men with a positive prostate biopsy had low numbers of circulating natural killer (NK) cells, compared to biopsy negative men. METHODS: To confirm these data, we analyzed differences in NK cells from 94 men undergoing prostate biopsy to determine whether NK cells could predict for a positive biopsy. NK cells activity (NKA) was measured by an in vitro diagnostic system, with a pre-defined cut-off value for NKA at 200 pg/mL. Logistic regression and receiver operator characteristics (Area Under the Curve (AUC)) analyses were used to test the diagnostic value of NKA. RESULTS: The NKA test performance showed specificity of 88%, positive predictive value of 84%, sensitivity of 34%, and a negative predictive value of 41%. Among the 94 men analyzed, NKA was not significantly linked with age, race, digital rectal examination (DRE), prostate volume, PSA or biopsy grade group (all P ≥ 0.14). In multivariable logistic regression analysis, the odds ratio (OR) of low NKA (<200 pg/mL) for the detection of PC was 4.89, 95%CI 1.34-17.8, with a ROC area under the curve of 0.79 in all participants and increasing to 0.83 and 0.85 for the detection of PC and high-grade PC, respectively, among men with a normal DRE. CONCLUSIONS: Men with a low NKA value had five-times higher odds of PC at biopsy. The implementation of this NKA assay in the clinic together with PSA may help to advise patients with the highest risk of PC whether, or not, to undergo a prostate biopsy.

lncRNA Spehd Regulates Hematopoietic Stem and Progenitor Cells and Is Required for Multilineage Differentiation.

Long non-coding RNAs (lncRNAs) show patterns of tissue- and cell type-specific expression that are very similar to those of protein coding genes and consequently have the potential to control stem and progenitor cell fate decisions along a differentiation trajectory. To understand the roles that lncRNAs may play in hematopoiesis, we selected a subset of mouse lncRNAs with potentially relevant expression patterns and refined our candidate list using evidence of conserved expression in human blood lineages. For each candidate, we assessed its possible role in hematopoietic differentiation in vivo using competitive transplantation. Our studies identified two lncRNAs that were required for hematopoiesis. One of these, Spehd, showed defective multilineage differentiation, and its silencing yielded common myeloid progenitors that are deficient in their oxidative phosphorylation pathway. This effort not only suggests that lncRNAs can contribute to differentiation decisions during hematopoiesis but also provides a path toward the identification of functional lncRNAs in other differentiation hierarchies.

Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors.

Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.

Erratum: Asparagine bioavailability governs metastasis in a model of breast cancer.

This corrects the article DOI: 10.1038/nature25465.

Asparagine bioavailability governs metastasis in a model of breast cancer.

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors.

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling-dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.