Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma.

Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, in newly generated, clinically-relevant somatic female HCC mouse models, we identify cancer genetics' specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. Mitogen-activated protein kinase (MAPK)-activated, NrasG12D-driven tumors exhibit a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME. Mechanistically, we report a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbs the accumulation of these cells, reduces inflammation, induces cancer cell death and prolongs animal survival. Furthermore, GM-CSF neutralization synergizes with a vascular endothelial growth factor (VEGF) inhibitor to restrain HCC outgrowth. These findings underscore the profound alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.

CD4+ T cells produce IFN-I to license cDC1s for induction of cytotoxic T-cell activity in human tumors.

CD4+ T cells can "help" or "license" conventional type 1 dendritic cells (cDC1s) to induce CD8+ cytotoxic T lymphocyte (CTL) anticancer responses, as proven in mouse models. We recently identified cDC1s with a transcriptomic imprint of CD4+ T-cell help, specifically in T-cell-infiltrated human cancers, and these cells were associated with a good prognosis and response to PD-1-targeting immunotherapy. Here, we delineate the mechanism of cDC1 licensing by CD4+ T cells in humans. Activated CD4+ T cells produce IFNß via the STING pathway, which promotes MHC-I antigen (cross-)presentation by cDC1s and thereby improves their ability to induce CTL anticancer responses. In cooperation with CD40 ligand (L), IFNß also optimizes the costimulatory and other functions of cDC1s required for CTL response induction. IFN-I-producing CD4+ T cells are present in diverse T-cell-infiltrated cancers and likely deliver "help" signals to CTLs locally, according to their transcriptomic profile and colocalization with "helped/licensed" cDCs and tumor-reactive CD8+ T cells. In agreement with this scenario, the presence of IFN-I-producing CD4+ T cells in the TME is associated with overall survival and the response to PD-1 checkpoint blockade in cancer patients.

Chromosomal Rearrangements and Chromothripsis: The Alternative End Generation Model.

Chromothripsis defines a genetic phenomenon where up to hundreds of clustered chromosomal rearrangements can arise in a single catastrophic event. The phenomenon is associated with cancer and congenital diseases. Most current models on the origin of chromothripsis suggest that prior to chromatin reshuffling numerous DNA double-strand breaks (DSBs) have to exist, i.e., chromosomal shattering precedes rearrangements. However, the preference of a DNA end to rearrange in a proximal accessible region led us to propose chromothripsis as the reaction product of successive chromatin rearrangements. We previously coined this process Alternative End Generation (AEG), where a single DSB with a repair-blocking end initiates a domino effect of rearrangements. Accordingly, chromothripsis is the end product of this domino reaction taking place in a single catastrophic event.

Mammalian life depends on two distinct pathways of DNA damage tolerance.

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.

Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation.

The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.

Epigenetic dysregulation of enhancers in neurons is associated with Alzheimer's disease pathology and cognitive symptoms.

Epigenetic control of enhancers alters neuronal functions and may be involved in Alzheimer's disease (AD). Here, we identify enhancers in neurons contributing to AD by comprehensive fine-mapping of DNA methylation at enhancers, genome-wide. We examine 1.2 million CpG and CpH sites in enhancers in prefrontal cortex neurons of individuals with no/mild, moderate, and severe AD pathology (n = 101). We identify 1224 differentially methylated enhancer regions; most of which are hypomethylated at CpH sites in AD neurons. CpH methylation losses occur in normal aging neurons, but are accelerated in AD. Integration of epigenetic and transcriptomic data demonstrates a pro-apoptotic reactivation of the cell cycle in post-mitotic AD neurons. Furthermore, AD neurons have a large cluster of significantly hypomethylated enhancers in the DSCAML1 gene that targets BACE1. Hypomethylation of these enhancers in AD is associated with an upregulation of BACE1 transcripts and an increase in amyloid plaques, neurofibrillary tangles, and cognitive decline.

Circadian oscillations of cytosine modification in humans contribute to epigenetic variability, aging, and complex disease.

BACKGROUND: Maintenance of physiological circadian rhythm plays a crucial role in human health. Numerous studies have shown that disruption of circadian rhythm may increase risk for malignant, psychiatric, metabolic, and other diseases. RESULTS: Extending our recent findings of oscillating cytosine modifications (osc-modCs) in mice, in this study, we show that osc-modCs are also prevalent in human neutrophils. Osc-modCs may play a role in gene regulation, can explain parts of intra- and inter-individual epigenetic variation, and are signatures of aging. Finally, we show that osc-modCs are linked to three complex diseases and provide a new interpretation of cross-sectional epigenome-wide association studies. CONCLUSIONS: Our findings suggest that loss of balance between cytosine methylation and demethylation during the circadian cycle can be a potential mechanism for complex disease. Additional experiments, however, are required to investigate the possible involvement of confounding effects, such as hidden cellular heterogeneity. Circadian rhythmicity, one of the key adaptations of life forms on Earth, may contribute to frailty later in life.

Cell-Free DNA Modification Dynamics in Abiraterone Acetate-Treated Prostate Cancer Patients.

Purpose: Primary resistance to abiraterone acetate (AA), a key medication for the treatment of metastatic castration-resistant prostate cancer, occurs in 20% to 40% of patients. We aim to identify predictive biomarkers for AA-treatment response and understand the mechanisms related to treatment resistance.Experimental Design: We used the Infinium Human Methylation 450K BeadChip to monitor modification profiles of cell-free circulating DNA (cfDNA) in 108 plasma samples collected from 33 AA-treated patients.Results: Thirty cytosines showed significant modification differences (FDR Q < 0.05) between AA-sensitive and AA-resistant patients during the treatment, of which 21 cytosines were differentially modified prior to treatment. In addition, AA-sensitive patients, but not AA-resistant patients, lost interindividual variation of cfDNA modification shortly after starting AA treatment, but such variation returned to initial levels in the later phases of treatment.Conclusions: Our findings provide a list of potential biomarkers for predicting AA-treatment response, highlight the prognostic value of using cytosine modification variance as biomarkers, and shed new insights into the mechanisms of prostate cancer relapse in AA-sensitive patients. Clin Cancer Res; 24(14); 3317-24. ©2018 AACR.

Cytosine modifications exhibit circadian oscillations that are involved in epigenetic diversity and aging.

Circadian rhythmicity governs a remarkable array of fundamental biological functions and is mediated by cyclical transcriptomic and proteomic activities. Epigenetic factors are also involved in this circadian machinery; however, despite extensive efforts, detection and characterization of circadian cytosine modifications at the nucleotide level have remained elusive. In this study, we report that a large proportion of epigenetically variable cytosines show a circadian pattern in their modification status in mice. Importantly, the cytosines with circadian epigenetic oscillations significantly overlap with the cytosines exhibiting age-related changes in their modification status. Our findings suggest that evolutionary advantageous processes such as circadian rhythmicity can also contribute to an organism's deterioration.

Transcriptional heterogeneity in the lactase gene within cell-type is linked to the epigenome.

Transcriptional variation in histologically- and genetically- identical cells is a widespread phenomenon in tissues, yet the processes conferring this heterogeneity are not well understood. To identify contributing factors, we analyzed epigenetic profiles associated with the in vivo transcriptional gradient of the mouse lactase gene (Lct), which occurs in enterocytes along the proximal-to-distal axis of the small intestine. We found that epigenetic signatures at enhancer and promoter elements aligns with transcriptional variation of Lct in enterocytes. Age and phenotype-specific environmental cues (lactose exposure after weaning) induced changes to epigenetic modifications and CTCF binding at select regulatory elements, which corresponded to the alterations in the intestinal Lct mRNA gradient. Thus, epigenetic modifications in combination with CTCF binding at regulatory elements account for the transcriptional gradient in Lct in cells of the same type. Epigenetic divergence within enterocytes may contribute to the functional specialization of intestinal subregions.

Identification of the Common Origins of Osteoclasts, Macrophages, and Dendritic Cells in Human Hematopoiesis.

Osteoclasts (OCs) originate from the myeloid cell lineage, but the successive steps in their lineage commitment are ill-defined, especially in humans. To clarify OC origin, we sorted cell populations from pediatric bone marrow (BM) by flow cytometry and assessed their differentiation potential in vitro. Within the CD11b(-)CD34(+)c-KIT(+) BM cell population, OC-differentiation potential was restricted to FLT3(+) cells and enriched in an IL3 receptor (R)α(high) subset that constituted less than 0.5% of total BM. These IL3Rα(high) cells also generated macrophages (MΦs) and dendritic cells (DCs) but lacked granulocyte (GR)-differentiation potential, as demonstrated at the clonal level. The IL3Rα(low) subset was re-defined as common progenitor of GR, MΦ, OC, and DC (GMODP) and gave rise to the IL3Rα(high) subset that was identified as common progenitor of MΦ, OC, and DC (MODP). Unbiased transcriptome analysis of CD11b(-)CD34(+)c-KIT(+)FLT3(+) IL3Rα(low) and IL3Rα(high) subsets corroborated our definitions of the GMODP and MODP and their developmental relationship.