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We report a novel single-cell whole-genome amplification method (LCS-WGA) that can efficiently capture spontaneous DNA damage existing in single cells. We refer to these damage-associated single-nucleotide variants as "damSNVs," and the whole-genome distribution of damSNVs as the damagenome. We observed that in single human neurons, the damagenome distribution was significantly correlated with three-dimensional genome structures. This nonuniform distribution indicates different degrees of DNA damage effects on different genes. Next, we identified the functionals that were significantly enriched in the high-damage genes. Similar functionals were also enriched in the differentially expressed genes (DEGs) detected by single-cell transcriptome of both Alzheimer's disease (AD) and autism spectrum disorder (ASD). This result can be explained by the significant enrichment of high-damage genes in the DEGs of neurons for both AD and ASD. The discovery of high-damage genes sheds new lights on the important roles of DNA damage in human diseases and disorders.

Cancer-induced immune responses affect tumor progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages, which define “immune subtypes” of triple negative breast cancer (TNBC) including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumor-intrinsic pathways and mutual regulation between macrophages/monocytes and neutrophils contribute to the development of dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells (gMDSCs), is resistant to ICB, and contains a minority of macrophages that appear to be unaffected by CCR2 knockout. A MES-to-NES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality, and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.

Significance Our findings identify a type of oncogene addiction process driven by a lipid that controls prosurvival effector protein secretion. The crosstalk between functionally redundant phosphatidylinositol (PI) 4-kinases maintains addiction and can be targeted with small molecule kinase inhibitors that might be applicable to a genetically defined subset of cancers for which there are no effective targeted therapies., A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes phosphatidylinositol (PI)-4 kinase IIIβ (PI4KIIIβ), a key regulator of secretory vesicle biogenesis and trafficking. Chromosome 1q21.3–amplified lung adenocarcinoma (1q-LUAD) cells rely on PI4KIIIβ for Golgi-resident PI-4-phosphate (PI4P) synthesis, prosurvival effector protein secretion, and cell viability. Here, we show that 1q-LUAD cells subjected to prolonged PI4KIIIβ antagonist treatment acquire tolerance by activating an miR-218-5p–dependent competing endogenous RNA network that up-regulates PI4KIIα, which provides an alternative source of Golgi-resident PI4P that maintains prosurvival effector protein secretion and cell viability. These findings demonstrate an addiction to Golgi-resident PI4P synthesis in a genetically defined subset of cancers.

ARID1A is one of the most frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and promoters. These broad and complex effects make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Here, we identified the anti-senescence effect of Arid1a deficiency in the progression of pancreatic intraepithelial neoplasia (PanIN) by profiling the transcriptome of individual PanINs in a mouse model. In a human cell line model, we found that ARID1A deficiency upregulates the expression of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which plays an essential role in attenuating the senescence induced by oncogenic KRAS through scavenging reactive oxygen species. As a subunit of the SWI/SNF chromatin remodeling complex, our ATAC sequencing data showed that ARID1A deficiency increases the accessibility of the enhancer region of ALDH1A1. This study provides the first evidence that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence through the upregulation of ALDH1A1 expression.

ARID1A is one of the most frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and promoters. These broad and complex effects make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Here we identified the anti-senescence effect of ARID1A deficiency in the progression of pancreatic intraepithelial neoplasia (PanIN) by profiling the transcriptome of individual PanINs in the mouse model. Interestingly, we found that ARID1A deficiency upregulates the expression of Aldehyde dehydrogenase ALDH1A1, which plays an essential role in attenuating the senescence induced by oncogenic KRAS. Despite that ALDH proteins have been commonly used as cancer stem cell markers, their effect in promoting attenuation of senescence is not known before. Therefore, ALDH proteins could be considered a potential adjuvant drug target in treating pancreatic cancer.

ARID1A is one of the most frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and promoters. These broad and complex effects make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Here, we identified the anti-senescence effect of Arid1a deficiency in the progression of pancreatic intraepithelial neoplasia (PanIN) by profiling the transcriptome of individual PanINs in a mouse model. In a human cell line model, we found that ARID1A deficiency upregulates the expression of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which plays an essential role in attenuating the senescence induced by oncogenic KRAS through scavenging reactive oxygen species. As a subunit of the SWI/SNF chromatin remodeling complex, our ATAC sequencing data showed that ARID1A deficiency increases the accessibility of the enhancer region of ALDH1A1. This study provides the first evidence that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence through the upregulation of ALDH1A1 expression.

Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblasts (CAFs) are distinguishable on the basis of gene expression signatures they acquire in co-culture with epithelial- or mesenchymal-like lung adenocarcinoma (LUAD) cells. High expression of the EMT activator ZEB1 endows LUAD cells with the capacity to activate a soluble factor exchange that leads to CAF reprogramming, to generate CAF-led invasive projections in multicellular aggregates, and to respond to pro-metastatic signals from CAFs in mice. Thus, ZEB1-expressing LUAD cells are positioned at the apex of a signaling hierarchy in the tumor microenvironment.

In the studies of single-cell genomics, the large endeavor has been focused on the detection of the permanent changes in the genome. On the other hand, spontaneous DNA damage frequently occurs and results in transient single-stranded changes to the genome until they are repaired. So far, successful profiling of these dynamic changes has not been demonstrated by single-cell whole-genome amplification methods. Here we reported a novel single-cell WGA method: Linearly Produced Semiamplicon based Split Amplification Reaction (LPSSAR), which allows, for the first time, the genome-wide detection of the DNA damage associated single nucleotide variants (dSNVs) in single human cells. The sequence-based detection of dSNVs allows the direct characterization of the major damage signature that occurred in human cells. In the analysis of the abundance of dSNVs along the genome, we observed two modules of dSNV abundance, instead of a homogeneous abundance of dSNVs. Interestingly, we found that the two modules are associated with the A/B topological compartments of the genome. This result suggests that the genome topology directly influences genome stability. Furthermore, with the detection of a large number of dSNVs in single cells, we showed that only under a stringent filtering condition, can we distinguish the de novo mutations from the dSNVs and achieve a reliable estimation of the total level of de novo mutations in a single cell.

SUMMARY Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis., In brief Bota-Rabassedas et al. show that EMT in lung adenocarcinoma cells activates a secretory process that governs CAF heterogeneity and, in turn, sensitizes lung adenocarcinoma cells to pro-metastatic signals from CAFs. Thus, EMT positions lung adenocarcinoma cells at the apex of a signaling hierarchy in the tumor microenvironment., Graphical Abstract

Cellular senescence has been viewed as a tumor suppression mechanism and also as a contributor to individual aging. Widespread shortening of 3′ untranslated regions (3′ UTRs) in messenger RNAs (mRNAs) by alternative polyadenylation (APA) has recently been discovered in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. Here, we found that hundreds of genes in senescent cells tended to use distal poly(A) (pA) sites, leading to a global lengthening of 3′ UTRs and reduced gene expression. Genes that harbor longer 3′ UTRs in senescent cells were enriched in senescence-related pathways. Rras2, a member of the Ras superfamily that participates in multiple signal transduction pathways, preferred longer 3′ UTR usage and exhibited decreased expression in senescent cells. Depletion of Rras2 promoted senescence, while rescue of Rras2 reversed senescence-associated phenotypes. Mechanistically, splicing factor TRA2B bound to a core “AGAA” motif located in the alternative 3′ UTR of Rras2, thereby reducing the RRAS2 protein level and causing senescence. Both proximal and distal poly(A) signals showed strong sequence conservation, highlighting the vital role of APA regulation during evolution. Our results revealed APA as a novel mechanism in regulating cellular senescence.

The quantification of transcriptional variation in single cells, particularly within the same cell population, is currently limited by the low sensitivity and high technical noise of single-cell RNA-seq assays. We report multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), a highly sensitive and quantitative method for single-cell sequencing of total RNA. By systematically determining technical noise, we show that MATQ-seq captures genuine biological variation between whole transcriptomes of single cells.