M. Ryan Corces

A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells

Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases.Xenotransplantation models provide powerful tools for the investigation of human normal and malignant hematopoiesis. However, current models do not extensively recapitulate human bone marrow (BM) microenvironment components and exhibit limited engraftment of many human leukemias and other disorders. Here, we describe a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment formed by in situ differentiation of human BM-derived mesenchymal stromal cells. In these humanized ossicles, we observed robust engraftment of normal human hematopoietic stem and progenitor cells, and detected extensive engraftment of diverse primary acute myeloid leukemia samples at levels much greater than in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in substantially higher leukemia-initiating cell frequencies. We also observed robust engraftment of acute promyelocytic leukemia and myelofibrosis leading to the identification of leukemia-initiating cells in hematopoietic stem cells in myelofibrosis. This humanized ossicle xenotransplantation approach provides a novel system to model human hematologic disease.

Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation

Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

The three-dimensional cancer genome

The past decade of cancer research has ushered in a comprehensive understanding of the way that the sequence of the genome can be co-opted during the process of tumorigenesis. However, only recently has the epigenome, and in particular the three-dimensional topology of chromatin, been implicated in cancer progression. Here we review recent findings of how the cancer genome is regulated and dysregulated to effect changes in 3D genome topology. We discuss the impact of the spatial organization of the genome on the frequency of tumorigenic chromosomal translocations and the effects of disruption of the proteins responsible for the establishment of chromatin loops. Alteration of the three-dimensional cancer genome is a rapidly emerging hallmark of multiple cancer subtypes.

Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation

Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

Superenhancer Analysis Defines Novel Epigenomic Subtypes of Non-APL AML, Including an RARα Dependency Targetable by SY-1425, a Potent and Selective RARα Agonist

We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136-53. ©2017 AACR.See related commentary by Wang and Aifantis, p. 1065.This article is highlighted in the In This Issue feature, p. 1047.

Preleukemic Hematopoietic Stem Cells in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by an uncontrolled proliferation of undifferentiated myeloid lineage cells. Decades of research have demonstrated that AML evolves from the sequential acquisition of genetic alterations within a single lineage of hematopoietic cells. More recently, the advent of high-throughput sequencing has enabled the identification of a premalignant phase of AML termed preleukemia. Multiple studies have demonstrated that AML can arise from the accumulation of mutations within hematopoietic stem cells (HSCs). These HSCs have been termed “preleukemic HSCs” as they represent the evolutionary ancestors of the leukemia. Through examination of the biological and clinical characteristics of these preleukemic HSCs, this review aims to shed light on some of the unexplored questions in the field. We note that some of the material discussed is speculative in nature and is presented in order to motivate future work.

The chromatin accessibility landscape of primary human cancers

Cancer chromatin accessibility landscape The Cancer Genome Atlas (TCGA) provides a high-quality resource of molecular data on a large variety of human cancers. Corces et al. used a recently modified assay to profile chromatin accessibility to determine the accessible chromatin landscape in 410 TCGA samples from 23 cancer types (see the Perspective by Taipale). When the data were integrated with other omics data available for the same tumor samples, inherited risk loci for cancer predisposition were revealed, transcription factors and enhancers driving molecular subtypes of cancer with patient survival differences were identified, and noncoding mutations associated with clinical prognosis were discovered. Science, this issue p. eaav1898; see also p. 401 Chromatin accessibility profiling identifies principles of epigenetic regulation in 23 primary human cancers. INTRODUCTION Cancer is one of the leading causes of death worldwide. Although the 2% of the human genome that encodes proteins has been extensively studied, much remains to be learned about the noncoding genome and gene regulation in cancer. Genes are turned on and off in the proper cell types and cell states by transcription factor (TF) proteins acting on DNA regulatory elements that are scattered over the vast noncoding genome and exert long-range influences. The Cancer Genome Atlas (TCGA) is a global consortium that aims to accelerate the understanding of the molecular basis of cancer. TCGA has systematically collected DNA mutation, methylation, RNA expression, and other comprehensive datasets from primary human cancer tissue. TCGA has served as an invaluable resource for the identification of genomic aberrations, altered transcriptional networks, and cancer subtypes. Nonetheless, the gene regulatory landscapes of these tumors have largely been inferred through indirect means. RATIONALE A hallmark of active DNA regulatory elements is chromatin accessibility. Eukaryotic genomes are compacted in chromatin, a complex of DNA and proteins, and only the active regulatory elements are accessible by the cell’s machinery such as TFs. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) quantifies DNA accessibility through the use of transposase enzymes that insert sequencing adapters at these accessible chromatin sites. ATAC-seq enables the genome-wide profiling of TF binding events that orchestrate gene expression programs and give a cell its identity. RESULTS We generated high-quality ATAC-seq data in 410 tumor samples from TCGA, identifying diverse regulatory landscapes across 23 cancer types. These chromatin accessibility profiles identify cancer- and tissue-specific DNA regulatory elements that enable classification of tumor subtypes with newly recognized prognostic importance. We identify distinct TF activities in cancer based on differences in the inferred patterns of TF-DNA interaction and gene expression. Genome-wide correlation of gene expression and chromatin accessibility predicts tens of thousands of putative interactions between distal regulatory elements and gene promoters, including key oncogenes and targets in cancer immunotherapy, such as MYC, SRC, BCL2, and PDL1. Moreover, these regulatory interactions inform known genetic risk loci linked to cancer predisposition, nominating biochemical mechanisms and target genes for many cancer-linked genetic variants. Lastly, integration with mutation profiling by whole-genome sequencing identifies cancer-relevant noncoding mutations that are associated with altered gene expression. A single-base mutation located 12 kilobases upstream of the FGD4 gene, a regulator of the actin cytoskeleton, generates a putative de novo binding site for an NKX TF and is associated with an increase in chromatin accessibility and a concomitant increase in FGD4 gene expression. CONCLUSION The accessible genome of primary human cancers provides a wealth of information on the susceptibility, mechanisms, prognosis, and potential therapeutic strategies of diverse cancer types. Prediction of interactions between DNA regulatory elements and gene promoters sets the stage for future integrative gene regulatory network analyses. The discovery of hundreds of noncoding somatic mutations that exhibit allele-specific regulatory effects suggests a pervasive mechanism for cancer cells to manipulate gene expression and increase cellular fitness. These data may serve as a foundational resource for the cancer research community. Cancer gene regulatory landscape. Chromatin accessibility profiling of 23 human cancer types (left) in 410 tumor samples from TCGA revealed 562,709 DNA regulatory elements. The activity of these DNA elements organized cancer subtypes, identified TF proteins and regulatory elements controlling cancer gene expression, and suggested molecular mechanisms for cancer-associated inherited variants and somatic mutations in the noncoding genome. See main article for abbreviations of cancer types. Ref., reference; Var., variant. We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.

Abstract 1511: AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner

Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS. Citation Format: Michael R. McKeown, Matthew L. Eaton, Chris Fiore, Emily Lee, Katie Austgen, Darren Smith, M. Ryan Corces, Ravindra Majeti, Christian C. Fritz. AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2017-1511