IL-4 drives exhaustion of CD8+ CART cells.

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here, we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion, RNA and ATAC sequencing on baseline and exhausted CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further, IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely, IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore, we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy.

RNA shielding of P65 is required to potentiate oncogenic inflammation in TET2 mutated clonal hematopoiesis.

TET2 mutations (mTET2) are common genetic events in myeloid malignancies and clonal hematopoiesis (CH). These mutations arise in the founding clone and are implicated in many clinical sequelae associated with oncogenic feedforward inflammatory circuits. However, the direct downstream effector of mTET2 responsible for the potentiation of this inflammatory circuit is unknown. To address this, we performed scRNA and scATAC-seq in COVID-19 patients with and without TET2-mutated CH reasoning that the inflammation from COVID-19 may highlight critical downstream transcriptional targets of mTET2. Using this approach, we identified MALAT1, a therapeutically tractable lncRNA, as a central downstream effector of mTET2 that is both necessary and sufficient to induce the oncogenic pro-inflammatory features of mTET2 in vivo. We also elucidate the mechanism by which mTET2 upregulate MALAT1 and describe an interaction between MALAT1 and P65 which leads to RNA "shielding" from PP2A dephosphorylation thus preventing resolution of inflammatory signaling.

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Empirical modeling of agricultural climate risk.

Effective policies for adaptation to climate change require understanding how impacts are related to exposures and vulnerability, the dimensions of the climate system that will change most and where human impacts will be most draconian, and the institutions best suited to respond. Here, we propose a simple method for more credibly pairing empirical statistical damage estimates derived from recent weather and outcome observations with projected future climate changes and proposed responses. We first analyze agricultural production and loan repayment data from Brazil to understand vulnerability to historical variation in the more predictable components of temperature and rainfall (trend and seasonality) as well as to shocks (both local and over larger spatial scales). This decomposed weather variation over the past two decades explains over 50% of the yield variation in major Brazilian crops and, critically, can be constructed in the same way for future climate projections. Combining our estimates with bias-corrected downscaled climate simulations for Brazil, we find increased variation in yields and revenues (including more bad years and worse outcomes) and higher agricultural loan default at midcentury. Results in this context point to two particularly acute dimensions of vulnerability: Intensified seasonality and local idiosyncratic shocks both contribute to worsening outcomes, along with a reduced capacity for spatially correlated ("covariate") shocks to ameliorate these effects through prices. These findings suggest that resilience strategies should focus on institutions such as water storage, financial services, and reinsurance.

Microfluidic Organoid Cultures Derived from Pancreatic Cancer Biopsies for Personalized Testing of Chemotherapy and Immunotherapy.

Patient-derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non-resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold-standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Climate and fertility amid a public health crisis.

One line of enquiry in demographic research assesses whether climate affects fertility. We extend this literature by examining the ramifications of climate conditions on fertility over a period of public health crisis in a highly unequal, urban middle-income country. We use monthly data for Brazil's 5,564 municipalities and apply spatial fixed-effects models to account for unobserved municipal heterogeneity and spatial dependence. Findings suggest that increases in temperature and precipitation are associated with declines in births. We also show that changes in response to climate conditions became greater during the Zika epidemic, particularly in urban areas. Combined, findings highlight the value of understanding the intersections between climate and fertility across geographic boundaries and during this public health crisis. Epidemics have become more important in people's lives with the recurring emergence of novel infectious disease threats, such as Zika and Covid-19.

Characterization and Optimization of Multiomic Single-Cell Epigenomic Profiling.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

Targeting BET Proteins Downregulates miR-33a To Promote Synergy with PIM Inhibitors in CMML.

PURPOSE: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. EXPERIMENTAL DESIGN: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. RESULTS: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. CONCLUSIONS: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.

Can the Content of Social Networks Explain Epidemic Outbreaks?

People share and seek information online that reflects a variety of social phenomena, including concerns about health conditions. We analyze how the contents of social networks provide real-time information to monitor and anticipate policies aimed at controlling or mitigating public health outbreaks. In November 2020, we collected tweets on the COVID-19 pandemic with content ranging from safety measures, vaccination, health, to politics. We then tested different specifications of spatial econometrics models to relate the frequency of selected keywords with administrative data on COVID-19 cases and deaths. Our results highlight how mentions of selected keywords can significantly explain future COVID-19 cases and deaths in one locality. We discuss two main mechanisms potentially explaining the links we find between Twitter contents and COVID-19 diffusion: risk perception and health behavior.

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined 'macro-Bound Enhancers', that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia.

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of ß cell function.

Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic ß cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, ß cell function, transcriptional profile, and reestablishment of proline and acidic amino acid­rich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of ß cell function and corresponding beneficial effects of tRF in prevention of T2DM.

Improving production and quality of life for smallholder farmers through a climate resilience program: An experience in the Brazilian Sertão.

We use a combination of economic and wellbeing metrics to evaluate the impacts of a climate resilience program designed for family farmers in the semiarid region of Brazil. Most family farmers in the region are on the verge of income and food insufficiency, both of which are exacerbated in prolonged periods of droughts. The program assisted farmers in their milk and sheepmeat production, implementing a set of climate-smart production practices and locally-adapted technologies. We find that the program under evaluation had substantive and significant impacts on production practices, land management, and quality of life in general, using several different quasi-experimental strategies to estimate the average treatment effect on the treated farmers. We highlight the strengths and limitations of each evaluation strategy and how the set of analyses and outcome indicators complement each other. The evaluation provides valuable insights into the economic and environmental sustainability of family farming in semiarid regions, which are under growing pressure from climate change and environmental degradation worldwide.

Migration and Democratization in Brazil: The Case of Electoral Participation and Competition.

Deepening democratization in Brazil has coincided with sustained flows of domestic migration, which raises an important question of whether migration deepens or depresses democratic development in migrant-sending regions. Whereas earlier perspectives have viewed migration as a political "brain drain," we contend that out-migration can generate resources that promote democratic processes back home. We investigate the role of migration in two aspects of democratization: electoral participation and competition. The analyses are based on spatial panel data models of mayoral election results across all municipalities between 1996 and 2012. The results show that migration increases electoral participation and competition in migrant-sending localities in Brazil. This study also identifies the sociopolitical context that conditions the impact of migration: the effect is most often present in the context of rural-urban migration and is more pronounced in sending localities with less democratic political structures. Moreover, using spatial network models, we find evidence for the transmission of political remittances from migration destination municipalities to origin municipalities. The present study extends the research on the migration-development nexus to the political arena, thus demonstrating the value of integrating demographic processes into explanations of political change.

The short-term impacts of coronavirus quarantine in São Paulo: The health-economy trade-offs.

We analyze the trade-offs between health and the economy during the period of social distancing in São Paulo, the state hardest hit by the COVID-19 pandemic in Brazil. We use longitudinal data with municipal-level information and check the robustness of our estimates to several sources of bias, including spatial dependence, reverse causality, and time-variant omitted variables. We use exogenous climate shocks as instruments for social distancing since people are more likely to stay home in wetter and colder periods. Our findings suggest that the health benefits of social distancing differ by levels of municipal development and may have vanished if the COVID-19 spread was not controlled in neighboring municipalities. In turn, we did not find evidence that municipalities with tougher social distancing performed worse economically. Our results also highlight that estimates that do not account for endogeneity may largely underestimate the benefits of social distancing on reducing the spread of COVID-19.

Cancer-specific CTCF binding facilitates oncogenic transcriptional dysregulation.

BACKGROUND: The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers. RESULTS: To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena. CONCLUSIONS: Specific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.

The transcription factor GLI1 cooperates with the chromatin remodeler SMARCA2 to regulate chromatin accessibility at distal DNA regulatory elements.

The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.