Search

Supplementary Table 3

Supplementary Table 8

Supplementary Table 7

Supplementary Table 1

Supplementary Table 9

Supplementary Table 6

Supplementary Figure 5

Supplementary Figure 3

Supplementary Table 2

Supplementary Table 5

Supplementary Figure 2

Supplementary Table 4

Supplementary Figure 1

Cancer evolution determines molecular and morphologic intratumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histologic features remains elusive. Here, we generated multiomics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intratumor spatial heterogeneity, tracing nongenetic routes of cancer evolution.Significance:Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized nongenetic mechanisms underlying intratumor patterns and molecular markers predicting patient prognosis. Intratumor patterns determined diverse immune microenvironments, warranting their study in the context of current immunotherapies.This article is highlighted in the In This Issue feature, p. 1307

Whole-genome doubling (WGD) is a recurrent event in human cancers and it promotes chromosomal instability and acquisition of aneuploidies1–8. However, the three-dimensional organization of chromatin in WGD cells and its contribution to oncogenic phenotypes are currently unknown. Here we show that in p53-deficient cells, WGD induces loss of chromatin segregation (LCS). This event is characterized by reduced segregation between short and long chromosomes, A and B subcompartments and adjacent chromatin domains. LCS is driven by the downregulation of CTCF and H3K9me3 in cells that bypassed activation of the tetraploid checkpoint. Longitudinal analyses revealed that LCS primes genomic regions for subcompartment repositioning in WGD cells. This results in chromatin and epigenetic changes associated with oncogene activation in tumours ensuing from WGD cells. Notably, subcompartment repositioning events were largely independent of chromosomal alterations, which indicates that these were complementary mechanisms contributing to tumour development and progression. Overall, LCS initiates chromatin conformation changes that ultimately result in oncogenic epigenetic and transcriptional modifications, which suggests that chromatin evolution is a hallmark of WGD-driven cancer.

Tissues are organized in niches where cell types interact to implement specific functions. Spatial -omics technologies allow to decode the molecular features and spatial interactions that determine such niches. However, computational approaches to process and interpret spatial molecular profiles are challenged by the scale and diversity of this data. Here, we present CellCharter, an algorithmic framework for the identification, characterization, and comparison of cellular niches from heterogeneous spatial transcriptomics and proteomics datasets comprising multiple samples. CellCharter outperformed existing methods, identified biologically meaningful cellular niches in different contexts, and discovered spatial cancer cell states, characterized by cell-intrinsic features and spatial interactions between tumor and immune cells. In non-small cell lung cancer, CellCharter revealed a cellular niche composed of neutrophils and tumor cells expressing markers of hypoxia and cell migration. Expression of these markers determined a spatial gradient associated with cancer cell state transition and neutrophil infiltration. Moreover, CellCharter showed that similar compositions of immune cell types can exhibit remarkably different spatial organizations in different tumors, highlighting the need for exploring spatial cell interactions to decipher intratumor heterogeneity. Overall, CellCharter is a flexible and scalable framework to explore and compare the spatial organization of normal and malignant tissues.

Data related to paper Lymphoma tissue fragments to anticipate response to targeted therapies Folder structure ├── 10xVisium # spatial transcriptomics data │ ├── LP03 │ │ ├── CTRL │ │ └── Parental │ └── LP08 │ ├── CTRL │ ├── IBR │ ├── IDL │ └── Parental └── scRNAseq # processed single RNA seq data ├── Spleen 1 │ └── qc_data.h5ad └── Spleen 2 └── qc_data.h5ad Each spatial transcriptomics data folders contains ├── NucleiPerSpot.txt ├── bayesprism_updatedReference.rdata ├── filtered_feature_bc_matrix │ ├── barcodes.tsv.gz │ ├── features.tsv.gz │ └── matrix.mtx.gz └── spatial ├── aligned_fiducials.jpg ├── detected_tissue_image.jpg ├── scalefactors_json.json ├── tissue_hires_image.png ├── tissue_lowres_image.png └── tissue_positions_list.csv

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often the HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes (such as E6/E7) is well known, the cis-regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. Here, we investigate the genome-wide patterns of HPV integrations and associated host gene expression changes in the context of chromatin states and topologically associating domains (TADs). We find that HPV integrations are significantly enriched and depleted in active and inactive chromatin regions, respectively. Interestingly, regardless of the chromatin state, the genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, the upregulation (both local and long-range) is mostly confined to the TADs with integration and does not affect the adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (such asMYC, PVT1, TP63andERBB2), regardless of the proximity. To further understand the long-range effect, we performed HiC and 4C-seq analyses in HeLa and observed chromatin looping interaction between the integrated HPV andMYC/PVT1regions (situated ∼500 kb apart), leading to allele-specific overexpression of these genes. Again, these chromatin interactions involving integrated HPV are mostly observed within the same TAD. Together, these results suggest the cis-regulatory potential of integrated HPV DNA that drives host gene upregulation at intra-TAD level in cervical cancer. Based on this, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

BackgroundSpatial interactions and insulation of chromatin regions are associated with transcriptional regulation. Domains of frequent chromatin contacts are proposed as functional units, favoring and delimiting gene regulatory interactions. However, contrasting evidence supports the association between chromatin domains and transcription.ResultHere, we assess gene co-regulation in chromatin domains across multiple human cancers, which exhibit great transcriptional heterogeneity. Across all datasets, gene co-regulation is observed only within a small yet significant number of chromatin domains. We design an algorithmic approach to identify differentially active domains (DADo) between two conditions and show that these provide complementary information to differentially expressed genes. Domains comprising co-regulated genes are enriched in the less active B sub-compartments and for genes with similar function. Notably, differential activation of chromatin domains is not associated with major changes of domain boundaries, but rather with changes of sub-compartments and intra-domain contacts.ConclusionOverall, gene co-regulation is observed only in a minority of chromatin domains, whose systematic identification will help unravel the relationship between chromatin structure and transcription.

Cancer cells retain genomic alterations that provide a selective advantage. The prediction and validation of advantageous alterations are major challenges in cancer genomics. Moreover, it is crucial to understand how the coexistence of specific alterations alters response to genetic and therapeutic perturbations. In the present study, we inferred functional alterations and preferentially selected combinations of events in >9,000 human tumors. Using a Bayesian inference framework, we validated computational predictions with high-throughput readouts from genetic and pharmacological screenings on 2,000 cancer cell lines. Mutually exclusive and co-occurring cancer alterations reflected, respectively, functional redundancies able to rescue the phenotype of individual target inhibition, or synergistic interactions, increasing oncogene addiction. Among the top scoring dependencies, co-alteration of the phosphoinositide 3-kinase (PI3K) subunit PIK3CA and the nuclear factor NFE2L2 was a synergistic evolutionary trajectory in squamous cell carcinomas. By integrating computational, experimental and clinical evidence, we provide a framework to study the combinatorial functional effects of cancer genomic alterations. Computational analysis of over 9,000 cancer genomes, coupled with functional validation in cell lines, highlights combinations of mutations required for tumor progression. This integrated approach provides a framework to stratify patients on the basis of interdependent genetic aberrations.

Whole genome doubling promotes tumorigenesis through loss of chromatin segregation and compartment repositioning PART 2 hic_maps_K562.zip:Hi-C maps (.hic format) of K562 samples hic_features.zip hic_features/compartment_domains:contains, for each of the hic matrices, the compartmentdomains imputed by Calder in BED format hic_features/compartment_domain_boundaries:boundaries between adjacent compartment domains in Control andWGD samples from RPE TP53-/- cell line hic_features/compartment_scores:for each compartment domain in Control and WGD samples fromRPE TP53-/- cell line it reports its C-score hic_features/insulation_scores:for each of the Hi-C matrices, the insulation scores computed at50 Kb resolution, in bigwig format ihic_features/nsulation_boundaries:TAD boundaries detected using the insulation score, for each Hi-Csample chipseq.zip chipseq/input_normalized: contains bigwig files of ChIP-seq experiments generatedin this study. Files are log2 input normalized tracks. The "bw_signals_attributes.txt" file can be used to addmetadata to the tracks for visualization in IGV scRNAseq.zip differential_expression: contains the output of the differential expression analysis. A README file is present explaining the meaning of each column in the table expression: gene expression matrix (cell x gene) containing normalized expression value. A table containing metadata of each cell (like sample of origin, uuid) is also available infercnv: copy numbers inferred by INFERCNV. Linkage matrix providing the clustering output of the infercnv data is also provided. copy_numbers.zip ControlFREEC_log2_coverage_ratio_10Kb.seg: .seg file (to be loaded in IGV) containing coverage log2 fold-change between samples and control as computed by ControlFREEC for the detection of CNVs filtered_cnvs.seg: significant CNVs detected by ControlFREEC RPE_cnvs_20wTumors_merged.bed: consensus set of CNVs in RPE TP53-/- 20w post-WGD tumors mutations.zip RPE_somatic_all.maf:MAF file of mutations detected by Mutect2 on RPE TP53-/- sample RPE_somatic_all_FILTER_PASS.tsv: mutations passing the quality filters &nbsp

Chromatin folding in the 3D space of the nucleus can be explored through high-throughput chromosome conformation capture (Hi-C) approaches. These experiments quantify the number of interactions between any pair of genomic loci in the genome and, thus, allow building genome-scale maps of intra- and inter-chromosomal contacts (contact maps). Statistical and algorithmic analyses of Hi-C data consist in extracting information from these contact maps. One of the most striking patterns observed in intra-chromosomal Hi-C contact maps emerged from genomic regions that exhibit dense intra-region but sparse inter-region contacts. These have been termed topologically associating domains (TADs). The identification of TADs from Hi-C contact maps is of great interest as they have been shown to act as unit of chromosome organization and, potentially, functional activity. Several approaches have been developed to identify TADs (TAD callers). However, results from these methods are often dependent on data resolution and poorly concordant. In this chapter, we present four TAD callers and we provide detailed protocols for their use. In addition, we show how to compare TADs identified by different callers and how to assess the enrichment for TAD-associated biological features. TAD calling has become a key step in the study of chromatin 3D organization in different cellular contexts. Here we provide guidelines to improve the robustness and quality of these analyses.

Chromatin folding in the 3D space of the nucleus can be explored through high-throughput chromosome conformation capture (Hi-C) approaches. These experiments quantify the number of interactions between any pair of genomic loci in the genome and, thus, allow building genome-scale maps of intra- and inter-chromosomal contacts (contact maps). Statistical and algorithmic analyses of Hi-C data consist in extracting information from these contact maps. One of the most striking patterns observed in intra-chromosomal Hi-C contact maps emerged from genomic regions that exhibit dense intra-region but sparse inter-region contacts. These have been termed topologically associating domains (TADs). The identification of TADs from Hi-C contact maps is of great interest as they have been shown to act as unit of chromosome organization and, potentially, functional activity. Several approaches have been developed to identify TADs (TAD callers). However, results from these methods are often dependent on data resolution and poorly concordant. In this chapter, we present four TAD callers and we provide detailed protocols for their use. In addition, we show how to compare TADs identified by different callers and how to assess the enrichment for TAD-associated biological features. TAD calling has become a key step in the study of chromatin 3D organization in different cellular contexts. Here we provide guidelines to improve the robustness and quality of these analyses.

In cancer cells, enhancer hijacking mediated by chromosomal alterations and/or increased deposition of acetylated histone H3 lysine 27 (H3K27ac) can support oncogene expression. However, how the chromatin conformation of enhancer-promoter interactions is affected by these events is unclear. In the present study, by comparing chromatin structure and H3K27ac levels in normal and lymphoma B cells, we show that enhancer-promoter-interacting regions assume different conformations according to the local abundance of H3K27ac. Genetic or pharmacological depletion of H3K27ac decreases the frequency and the spreading of these interactions, altering oncogene expression. Moreover, enhancer hijacking mediated by chromosomal translocations influences the epigenetic status of the regions flanking the breakpoint, prompting the formation of distinct intrachromosomal interactions in the two homologous chromosomes. These interactions are accompanied by allele-specific gene expression changes. Overall, our work indicates that H3K27ac dynamics modulates interaction frequency between regulatory regions and can lead to allele-specific chromatin configurations to sustain oncogene expression. Enhancer-promoter three-dimensional interactions at oncogenic loci are modulated by H3K27ac dynamics. Enhancer hijacking mediated by chromosomal translocations leads to distinct chromatin states, intrachromosomal interactions and allele-specific gene expression.

Ma et al. uncovers an essential role of AR signaling in melanoma cell expansion and tumorigenesis, with loss of AR activity inducing cellular senescence, genomic DNA breakage, a STING-dependent inflammatory cascade, and immune cell recruitment, providing an attractive venue for new combination approaches to disease management., Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II–associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex., Graphical Abstract

Cancer evolution determines molecular and morphologic intratumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histologic features remains elusive. Here, we generated multiomics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intratumor spatial heterogeneity, tracing nongenetic routes of cancer evolution., SIGNIFICANCE: Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized nongenetic mechanisms underlying intratumor patterns and molecular markers predicting patient prognosis. Intratumor patterns determined diverse immune microenvironments, warranting their study in the context of current immunotherapies.

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF. CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B-cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease., publishedVersion

Background Chromatin folding gives rise to structural elements among which are clusters of densely interacting DNA regions termed topologically associating domains (TADs). TADs have been characterized across multiple species, tissue types, and differentiation stages, sometimes in association with regulation of biological functions. The reliability and reproducibility of these findings are intrinsically related with the correct identification of these domains from high-throughput chromatin conformation capture (Hi-C) experiments. Results Here, we test and compare 22 computational methods to identify TADs across 20 different conditions. We find that TAD sizes and numbers vary significantly among callers and data resolutions, challenging the definition of an average TAD size, but strengthening the hypothesis that TADs are hierarchically organized domains, rather than disjoint structural elements. Performances of these methods differ based on data resolution and normalization strategy, but a core set of TAD callers consistently retrieve reproducible domains, even at low sequencing depths, that are enriched for TAD-associated biological features. Conclusions This study provides a reference for the analysis of chromatin domains from Hi-C experiments and useful guidelines for choosing a suitable approach based on the experimental design, available data, and biological question of interest. Electronic supplementary material The online version of this article (10.1186/s13059-018-1596-9) contains supplementary material, which is available to authorized users.

Genomic instability is a major driver of intra-tumor heterogeneity. However, unstable genomes often exhibit different molecular and clinical phenotypes, which are associated with distinct mutational processes. Here, we algorithmically inferred the clonal phylogenies of ~6,000 human tumors from 32 tumor types to explore how intra-tumor heterogeneity depends on different implementations of genomic instability. We found that extremely unstable tumors associated with DNA repair deficiencies or high chromosomal instability are not the most intrinsically heterogeneous. Conversely, intra-tumor heterogeneity is greatest in tumors exhibiting relatively high numbers of both mutations and copy number alterations, a feature often observed in cancers associated with exogenous mutagens. Independently of the type of instability, tumors with high number of clones invariably evolved through branching phylogenies that could be stratified based on the extent of clonal (early) and subclonal (late) instability. Interestingly, tumors with high number of subclonal mutations frequently exhibited chromosomal instability, TP53 mutations, and APOBEC-related mutational signatures. Vice versa, mutations of chromatin remodeling genes often characterized tumors with few subclonal but multiple clonal mutations. Understanding how intra-tumor heterogeneity depends on genomic instability is critical to identify markers predictive of the tumor complexity and envision therapeutic strategies able to exploit this association., Author summary Cancer is characterized by cells accumulating molecular alterations promoting specific phenotypic features, such as uncontrolled proliferation and survival. Cancer cells sometimes exhibit a high number of such alterations, often driven by defects of the DNA repair pathway or by external mutagens, such as tobacco smoking or UV-radiation. Highly altered cells are termed genomically unstable. A major consequence of genomic instability is that a single tumor is often composed by cells that have accumulated distinct alterations. This diversity is termed intra-tumor heterogeneity and represents a critical clinical challenge. In this study, we examined how different forms of genomic instability are associated with intra-tumor heterogeneity. We inferred intra-tumor heterogeneity in ~6000 human tumors and found that tumors with extreme mutational or chromosomal instability were not the tumors with the highest number of clones. Instead, tumors harboring both mutational and chromosomal alterations were the most diverse. Furthermore, we identified specific genetic fingerprints that are associated with early and/or late genomic instability. These results show that cancer genomic instability does not necessarily lead to high intra-tumor heterogeneity and, importantly, they provide markers to recognize when it does.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

Cancer evolves through the emergence and selection of molecular alterations. Cancer genome profiling has revealed that specific events are more or less likely to be co-selected, suggesting that the selection of one event depends on the others. However, the nature of these evolutionary dependencies and their impact remain unclear. Here, we designed SELECT, an algorithmic approach to systematically identify evolutionary dependencies from alteration patterns. By analyzing 6,456 genomes from multiple tumor types, we constructed a map of oncogenic dependencies associated with cellular pathways, transcriptional readouts, and therapeutic response. Finally, modeling of cancer evolution shows that alteration dependencies emerge only under conditional selection. These results provide a framework for the design of strategies to predict cancer progression and therapeutic response.

Reference EPFL-CONF-232278View record in Web of Science Record created on 2017-11-08, modified on 2017-11-08

Chromatin is organized into topologically associating domains (TADs) enriched in distinct histone marks. In cancer, gain-of-function mutations in the gene encoding the enhancer of zeste homolog 2 protein (EZH2) lead to a genome-wide increase in histone-3 Lys27 trimethylation (H3K27me3) associated with transcriptional repression. However, the effects of these epigenetic changes on the structure and function of chromatin domains have not been explored. Here, we found a functional interplay between TADs and epigenetic and transcriptional changes mediated by mutated EZH2. Altered EZH2 (p.Tyr646* (EZH2Y646X)) led to silencing of entire domains, synergistically inactivating multiple tumor suppressors. Intra-TAD gene silencing was coupled with changes of interactions between gene promoter regions. Notably, gene expression and chromatin interactions were restored by pharmacological inhibition of EZH2Y646X. Our results indicate that EZH2Y646X alters the topology and function of chromatin domains to promote synergistic oncogenic programs.

Chromatin compartmentalization reflects biological activity. However, inference of chromatin sub-compartments and compartment domains from chromosome conformation capture (Hi-C) experiments is limited by data resolution. As a result, these have been characterized only in a few cell types and systematic comparisons across multiple tissues and conditions are missing. Here, we present Calder, an algorithmic approach that enables the identification of multi-scale sub-compartments at variable data resolution. Calder allows to infer and compare chromatin sub-compartments and compartment domains in >100 cell lines. Our results reveal sub-compartments enriched for poised chromatin states and undergoing spatial repositioning during lineage differentiation and oncogenic transformation., Computational algorithms to infer chromatin sub-compartments and compartment domains require high-resolution Hi-C maps. Here the authors present Calder, an algorithm that can infer sub-compartments and compartment domains with variable resolution Hi-C data, and they apply it to more than a hundred Hi-C experiments to study sub-compartment repositioning.