TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer.

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

An epigenetically distinct HSC subset supports thymic reconstitution.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). The Kit lo subset of HSCs is enriched for multipotential precursors, 1, 2 but their T-cell lineage potential has not been well-characterized. We therefore studied the thymic reconstituting and T-cell potential of Kit lo HSCs. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs support better thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. We find the frequency of the Kit lo subset declines with age, providing one explanation for the reduced frequency of T-competent HSCs and reduced T-lymphopoietic potential in BM precursors of aged mice. 3, 4, 5 Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs with enhanced lymphoid potential have a distinct underlying epigenetic program.

ERG activates a stem-like proliferation-differentiation program in prostate epithelial cells with mixed basal-luminal identity.

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in all prostate epithelial cells, proliferation was enriched in a small, stem-like population with mixed-luminal basal identity (called intermediate cells). Through a series of lineage tracing and primary prostate tissue transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that co-express the luminal genes Tmprss2 and Nkx3.1 (called BasalLum) but not in the larger population of classical Krt8+ luminal cells. Upon ERG activation, BasalLum cells give rise to the highly proliferative intermediate state, which subsequently transitions to the larger population of Krt8+ luminal cells characteristic of ERG-positive human cancers. Furthermore, this proliferative population is characterized by an ERG-specific chromatin state enriched for NFkB, AP-1, STAT and NFAT binding, with implications for TF cooperativity. The fact that the proliferative potential of ERG is enriched in a small stem-like population implicates the chromatin context of these cells as a critical variable for unmasking its oncogenic activity.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Microenvironment-Driven Dynamic Chromatin Changes in Glioblastoma Recapitulate Early Neural Development at Single-Cell Resolution.

The tumor microenvironment is necessary for recapitulating the intratumoral heterogeneity and cell state plasticity found in human primary glioblastoma (GBM). Conventional models do not accurately recapitulate the spectrum of GBM cellular states, hindering elucidation of the underlying transcriptional regulation of these states. Using our glioblastoma cerebral organoid model, we profiled the chromatin accessibility of 28,040 single cells in five patient-derived glioma stem cell lines. Integration of paired epigenomes and transcriptomes within the context of tumor-normal host cell interactions was used to probe the gene-regulatory networks underlying individual GBM cellular states in a way not readily possible in other in vitro models. These analyses identified the epigenetic underpinnings of GBM cellular states and characterized dynamic chromatin changes reminiscent of early neural development that underlie GBM cell state transitions. Despite large differences between tumors, a shared cellular compartment made up of neural progenitor-like cells and outer radial glia-like cells was observed. Together, these results shed light on the transcriptional regulation program in GBM and offer novel therapeutic targets across a broad range of genetically heterogenous GBMs. SIGNIFICANCE: Single-cell analyses elucidate the chromatin landscape and transcriptional regulation of glioblastoma cellular states and identify a radial glia-like population, providing potential targets to disrupt cell states and improve therapeutic efficacy.

Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering.

Cell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9-mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering.

SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis.

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.

TP53 mutations and RNA-binding protein MUSASHI-2 drive resistance to PRMT5-targeted therapy in B-cell lymphoma.

To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.

Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that although clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8+ T cells that negatively correlated with patient prognosis, chromophobe RCC (chRCC) had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

Programme of self-reactive innate-like T cell-mediated cancer immunity.

Cellular transformation induces phenotypically diverse populations of tumour-infiltrating T cells1-5, and immune checkpoint blockade therapies preferentially target T cells that recognize cancer cell neoantigens6,7. Yet, how other classes of tumour-infiltrating T cells contribute to cancer immunosurveillance remains elusive. Here, in a survey of T cells in mouse and human malignancies, we identified a population of αß T cell receptor (TCR)-positive FCER1G-expressing innate-like T cells with high cytotoxic potential8 (ILTCKs). These cells were broadly reactive to unmutated self-antigens, arose from distinct thymic progenitors following early encounter with cognate antigens, and were continuously replenished by thymic progenitors during tumour progression. Notably, expansion and effector differentiation of intratumoural ILTCKs depended on interleukin-15 (IL-15) expression in cancer cells, and inducible activation of IL-15 signalling in adoptively transferred ILTCK progenitors suppressed tumour growth. Thus, the antigen receptor self-reactivity, unique ontogeny, and distinct cancer cell-sensing mechanism distinguish ILTCKs from conventional cytotoxic T cells, and define a new class of tumour-elicited immune response.

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-ß (TGF-ß) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

Globin vector regulatory elements are active in early hematopoietic progenitor cells.

The globin genes are archetypal tissue-specific genes that are silent in most tissues but for late-stage erythroblasts upon terminal erythroid differentiation. The transcriptional activation of the ß-globin gene is under the control of proximal and distal regulatory elements located on chromosome 11p15.4, including the ß-globin locus control region (LCR). The incorporation of selected LCR elements in lentiviral vectors encoding ß and ß-like globin genes has enabled successful genetic treatment of the ß-thalassemias and sickle cell disease. However, recent occurrences of benign clonal expansions in thalassemic patients and myelodysplastic syndrome in patients with sickle cell disease call attention to the non-erythroid functions of these powerful vectors. Here we demonstrate that lentivirally encoded LCR elements, in particular HS1 and HS2, can be activated in early hematopoietic cells including hematopoietic stem cells and myeloid progenitors. This activity is position-dependent and results in the transcriptional activation of a nearby reporter gene in these progenitor cell populations. We further show that flanking a globin vector with an insulator can effectively restrain this non-erythroid activity without impairing therapeutic globin expression. Globin lentiviral vectors harboring powerful LCR HS elements may thus expose to the risk of trans-activating cancer-related genes, which can be mitigated by a suitable insulator.

SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes.

SETD2 is a histone H3 lysine 36 (H3K36) trimethyltransferase that is mutated with high prevalence (13%) in clear cell renal cell carcinoma (ccRCC). Genomic profiling of primary ccRCC tumors reveals a positive correlation between SETD2 mutations and metastasis. However, whether and how SETD2 loss promotes metastasis remains unclear. In this study, we used a SETD2-mutant (SETD2MT) metastatic ccRCC human-derived cell line and xenograft models and showed that H3K36me3 restoration greatly reduced distant metastases of ccRCC in mice in a matrix metalloproteinase 1 (MMP1)-dependent manner. An integrated multiomics analysis using assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) established a tumor suppressor model in which loss of SETD2-mediated H3K36me3 activates enhancers to drive oncogenic transcriptional output through regulation of chromatin accessibility. Furthermore, we uncovered mechanism-based therapeutic strategies for SETD2-deficient cancer through the targeting of specific histone chaperone complexes, including ASF1A/ASF1B and SPT16. Overall, SETD2 loss creates a permissive epigenetic landscape for cooperating oncogenic drivers to amplify transcriptional output, providing unique therapeutic opportunities.

Single-Cell Transcriptional Profiling Reveals Signatures of Helper, Effector, and Regulatory MAIT Cells during Homeostasis and Activation.

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

Glycolytic ATP fuels phosphoinositide 3-kinase signaling to support effector T helper 17 cell responses.

Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Single-cell sequencing links multiregional immune landscapes and tissue-resident T cells in ccRCC to tumor topology and therapy efficacy.

Clear cell renal cell carcinomas (ccRCCs) are highly immune infiltrated, but the effect of immune heterogeneity on clinical outcome in ccRCC has not been fully characterized. Here we perform paired single-cell RNA (scRNA) and T cell receptor (TCR) sequencing of 167,283 cells from multiple tumor regions, lymph node, normal kidney, and peripheral blood of two immune checkpoint blockade (ICB)-naïve and four ICB-treated patients to map the ccRCC immune landscape. We detect extensive heterogeneity within and between patients, with enrichment of CD8A+ tissue-resident T cells in a patient responsive to ICB and tumor-associated macrophages (TAMs) in a resistant patient. A TCR trajectory framework suggests distinct T cell differentiation pathways between patients responding and resistant to ICB. Finally, scRNA-derived signatures of tissue-resident T cells and TAMs are associated with response to ICB and targeted therapies across multiple independent cohorts. Our study establishes a multimodal interrogation of the cellular programs underlying therapeutic efficacy in ccRCC.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.