Changes In Chromatin Accessibility Research Articles

Tracking single-cell evolution using clock-like chromatin accessibility loci

AbstractSingle-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurementallowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Neuron-specific chromatin disruption at CpG islands and aging-related regions in Kabuki syndrome mice.

Many Mendelian developmental disorders caused by coding variants in epigenetic regulators have now been discovered. Epigenetic regulators are broadly expressed, and each of these disorders typically exhibits phenotypic manifestations from many different organ systems. An open question is whether the chromatin disruption - the root of the pathogenesis - is similar in the different disease-relevant cell types. This is possible in principle, since all these cell types are subject to effects from the same causative gene, that has the same kind of function (e.g. methylates histones) and is disrupted by the same germline variant. We focus on mouse models for Kabuki syndrome types 1 and 2, and find that the chromatin accessibility changes in neurons are mostly distinct from changes in B or T cells. This is not because the neuronal accessibility changes occur at regulatory elements that are only active in neurons. Neurons, but not B or T cells, show preferential chromatin disruption at CpG islands and at regulatory elements linked to aging. A sensitive analysis reveals that regulatory elements disrupted in B/T cells do exhibit chromatin accessibility changes in neurons, but these are very subtle and of uncertain functional significance. Finally, we are able to identify a small set of regulatory elements disrupted in all three cell types. Our findings reveal the cellular-context-specific effect of variants in epigenetic regulators and suggest that blood-derived episignatures, while useful diagnostically, may not be well-suited for understanding the mechanistic basis of neurodevelopment in Mendelian disorders of the epigenetic machinery.

Chromatin accessibility during human first-trimester neurodevelopment.

The human brain develops through a tightly organized cascade of patterning events, induced by transcription factor expression and changes in chromatin accessibility. Although gene expression across the developing brain has been described at single-cell resolution1, similar atlases of chromatin accessibility have been primarily focused on the forebrain2-4. Here we describe chromatin accessibility and paired gene expression across the entire developing human brain during the first trimester (6-13 weeks after conception). We defined 135 clusters and used multiomic measurements to link candidate cis-regulatory elements to gene expression. The number of accessible regions increased both with age and along neuronal differentiation. Using a convolutional neural network, we identified putative functional transcription factor-binding sites in enhancers characterizing neuronal subtypes. We applied this model to cis-regulatory elements linked to ESRRB to elucidate its activation mechanism in the Purkinje cell lineage. Finally, by linking disease-associated single nucleotide polymorphisms to cis-regulatory elements, we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder-related mutations. Our findings provide a more detailed view of key gene regulatory mechanisms underlying the emergence of brain cell types during the first trimester and a comprehensive reference for future studies related to human neurodevelopment.

Investigation of inherited noncoding genetic variation impacting the pharmacogenomics of childhood acute lymphoblastic leukemia treatment.

Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.

Consequences of Transient YAP Signaling in the Liver

The transcriptional co-activator YAP plays a complex role in liver homeostasis and disease development. In response to liver injuries and hepatotoxins, YAP is transiently activated to promote liver regeneration by coordinating cell proliferation and metabolism. By contrast, chronic activation of YAP in experimental models has been associated with liver diseases and cancer. These primary responses to YAP are well established; however, it is presently unknown how acute YAP signaling contributes to the eventual development of liver diseases. Here, we developed an in vivo model of dynamic YAP signaling using a doxycycline-inducible system to control the transient expression of a constitutively active YAP mutant in hepatocytes. We found that while YAP-dependent effects on liver growth and cell proliferation are reversible, there are metabolic changes in the liver that remain stable even after YAP inactivation. By coupling RNAseq and ATACseq analyses, we unraveled persistent changes in gene expression and chromatin accessibility, particularly those involved in lipid metabolism. Furthermore, we demonstrated that transient YAP signaling sensitizes the liver to develop more severe liver injuries than normally induced in an unprimed liver. These data suggest that transient YAP signaling imparts a form of lipid metabolic memory that promotes susceptibility to liver diseases. Overall, our current work reveals long-term consequences of acute YAP signaling that may influence the development of liver diseases. 5R01HL128850-07 (FC) and 5T32DK007477-40 (KA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3′ UTR of FAIM2

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Single-cell analyses reveal transient retinal progenitor cells in the ciliary margin of developing human retina

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids.

Alternative splicing and its regulation in the malaria vector Anopheles gambiae

IntroductionAlternative splicing (AS) is a highly conserved mechanism that allows for the expansion of the coding capacity of the genome, through modifications of the way that multiple isoforms are expressed or used to generate different phenotypes. Despite its importance in physiology and disease, genome-wide studies of AS are lacking in most insects, including mosquitoes. Even for model organisms, chromatin associated processes involved in the regulation AS are poorly known.MethodsIn this study, we investigated AS in the mosquito Anopheles gambiae in the context of tissue-specific gene expression and mosquito responses to a Plasmodium falciparum infection, as well as the relationship between patterns of differential isoform expression and usage with chromatin accessibility changes. For this, we combined RNA-seq and ATAC-seq data from A. gambiae midguts and salivary glands, infected and non-infected.ResultsWe report differences between tissues in the expression of 392 isoforms and in the use of 247 isoforms. Secondly, we find a clear and significant association between chromatin accessibility states and tissue-specific patterns of AS. The analysis of differential accessible regions located at splicing sites led to the identification of several motifs resembling the binding sites of Drosophila transcription factors. Finally, the genome-wide analysis of tissue-dependent enhancer activity revealed that approximately 20% of A. gambiae transcriptional enhancers annotate to a differentially expressed or used isoform, and that their activation status is linked to AS differences between tissues.ConclusionThis research elucidates the role of AS in mosquito vector gene expression and identifies regulatory regions potentially involved in AS regulation, which could be important in the development of novel strategies for vector control.

Repetitive element transcript accumulation is associated with inflammaging in humans.

Chronic, low-grade inflammation increases with aging, contributing to functional declines and diseases that reduce healthspan. Growing evidence suggests that transcripts from repetitive elements (RE) in the genome contribute to this "inflammaging" by stimulating innate immune activation, but evidence of RE-associated inflammation with aging in humans is limited. Here, we present transcriptomic and clinical data showing that RE transcript levels are positively related to gene expression of innate immune sensors, and to serum interleukin 6 (a marker of systemic inflammation), in a large group of middle-aged and older adults. We also: (1) use transcriptomics and whole-genome bisulfite (methylation) sequencing to show that many RE may be hypomethylated with aging, and that aerobic exercise, a healthspan-extending intervention, reduces RE transcript levels and increases RE methylation in older adults; and (2) extend our findings in a secondary dataset demonstrating age-related changes in RE chromatin accessibility. Collectively, our data support the idea that age-related RE transcript accumulation may play a role in inflammaging in humans, and that RE dysregulation with aging may be due in part to upstream epigenetic changes.

Multiome in the Same Cell Reveals the Impact of Osmotic Stress on Arabidopsis Root Tip Development at Single-Cell Level.

Cell-specific transcriptional regulatory networks (TRNs) play vital roles in plant development and response to environmental stresses. However, traditional single-cell mono-omics techniques are unable to directly capture the relationships and dynamics between different layers of molecular information within the same cells. While advanced algorithm facilitates merging scRNA-seq and scATAC-seq datasets, accurate data integration remains a challenge, particularly when investigating cell-type-specific TRNs. By examining gene expression and chromatin accessibility simultaneously in 16,670 Arabidopsis root tip nuclei, the TRNs are reconstructed that govern root tip development under osmotic stress. In contrast to commonly used computational integration at cell-type level, 12,968 peak-to-gene linkage is captured at the bona fide single-cell level and construct TRNs at an unprecedented resolution. Furthermore, the unprecedented datasets allow to more accurately reconstruct the coordinated changes of gene expression and chromatin states during cellular state transition. During root tip development, chromatin accessibility of initial cells precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for subsequent differentiation steps. Pseudo-time trajectory analysis reveal that osmotic stress can shift the functional differentiation of trichoblast. Candidate stress-related gene-linked cis-regulatory elements(gl-cCREs) as well as potential target genes are also identified, and uncovered large cellular heterogeneity under osmotic stress.

Unlocking adult T-cell leukemia/lymphoma's epigenetic secrets: delving into the mechanism and impact of EZH1/2 inhibition.

Epigenetic modifications, particularly through methylation of DNA packaging histones, play a pivotal role in controlling gene expression. Aberrant patterns of histone methylation have been associated with the development and progression of hematological malignancies. Unraveling the impact of aberrant histone marks on gene expression and leukemogenesis has spurred a concerted effort to develop clinically effective epigenetic therapies. In malignancies associated with the accumulation of histone H3 lysine trimethylation (H3K27me3), one such intervention involves preventing the deposition of this repressive histone mark by inhibiting the histone-modifying enzymes EZH1 and EZH2. While inhibition of EZH1/2 has demonstrated efficacy in both preclinical studies and clinical trials in various cancers, studies delineating the dynamic effect of EZH1/2 inhibition on H3K27me3 and disease relapse in clinical samples are lacking. In a recent publication, Yamagishi etal. explore how responses of a patient with adult T-cell leukemia/lymphoma to valemetostat, an EZH1/2 inhibitor, are associated with changes in H3K27me3, chromatin accessibility and gene expression, and how these changes can be circumvented in relapsed disease.

Abstract LB234: Identifying cancer vulnerabilities associated with changes in chromatin accessibility by simultaneously profiling hundreds of cancer cell lines with ATAC-seq

Abstract The goal of the Cancer Dependency Map project (DepMap) is to identify the landscape of cancer vulnerabilities by systematically profiling hundreds of cell lines representing the heterogeneity of human cancer. Cancer cells are known to undergo significant alterations in the cancer genome including changes in the accessible chromatin regions (ACRs). These alterations could lead to novel cancer vulnerabilities but chromatin accessibility has not been systematically profiled in DepMap. Profiling hundreds of cell lines individually for ACRs would require large resources. Here we show that we can efficiently profile hundreds of cell lines using two single-cell approaches developed by our labs. SHARE-seq is a single-cell high-throughput combinatorial indexing method to measure both chromatin accessibility (scATAC-seq), and gene expression (scRNA-seq) in the same single cell. MIX-Seq is an approach that leverages the ability to pool hundreds of unique cancer cell lines and then apply single-cell genomics across cell populations from each cell line, resolving its identity using single nucleotide polymorphism (SNP) profiles. Here, we used a combination of MIX-seq (pooling cell lines) and SHARE-seq (scATAC-seq and RNA-seq) to simultaneously profile 500 unique cell lines covering 110 lineages within 22 cancer types at the single-cell level. Encouragingly, in a UMAP representation of the data, cancer cell lines cluster by lineage. Interestingly, we identified hundreds of thousands of peaks within cis-regulatory elements (CREs), 1/3 of which are unique to cancer cell lines and not present in normal cell types. Using published tools, we calculated an ATAC-seq gene activity prediction score per cell line. To identify cancer vulnerabilities associated with ACRs changes, we combined the ATAC-seq gene-activity predictions with RNAseq-based features to construct machine-learning models of cancer dependency scores. We found that the prediction accuracy for multiple dependencies improved with the addition of the ATAC-seq-based scores, indicating that chromatin accessibility provides information beyond gene expression. Additional analyses are underway to create other features. Overall, we have generated a large high-quality dataset to simultaneously map the chromatin accessibility in hundreds of cancer cell lines. Importantly, the chromatin accessibility adds to other omics profiling to identify biomarkers of response and understand how changes in chromatin can lead to new vulnerabilities. Citation Format: Patricia Borck, Kirsty Wienand, Fabiana Duarte, Alvin Qin, Ruochi Zhang, Juliana Babu, Simone Zhang, Samuel Maffa, Max Horlbeck, Rojesh Shrestha, Joshua Dempster, Jennifer Roth, Catarina Campbell, Jason Buenrostro, Francisca Vazquez. Identifying cancer vulnerabilities associated with changes in chromatin accessibility by simultaneously profiling hundreds of cancer cell lines with ATAC-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB234.

Abstract LB008: Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470

Abstract Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric di-methylation of arginine (SDMA) of multiple substrates that regulate biological processes including RNA splicing and cell cycle. The role of PRMT5 in controlling chromatin accessibility has been investigated in some cancer contexts, but its role in non-small cell lung cancer (NSCLC) global chromatin regulation is not known. AstraZeneca has developed an MTA-cooperative PRMT5 inhibitor (AZD3470) that selectively inhibits PRMT5 in MTAP-null tumors and currently is in Phase I clinical trial (NCT06130553, NCT06130553). Here we applied integrative dynamic MultiOMICs analysis upon AZD3470 treatment in six MTAP-null NSCLC cancer cell lines to inform on PRMT5’s function in maintaining tumor cell growth and survival. Methods: Utilizing an integrative approach, our study explored the effects of AZD3470 on human MTAP-deleted NSCLC cells. MultiOMICs analysis including SDMA post-translational modification (PTM) scans, ATAC-seq, RNA-seq, and mass spectrometry proteomics were employed to delineate the immediate effects and downstream phenotypes of AZD3470 PRMT5 inhibition. Results: AZD3470 induced significant SDMA modification of splicing factors consistent with known roles for PRMT5. PRMT5 inhibition by AZD3470 led to changes in global chromatin accessibility in NSCLC cells. Specific changes in genes involved in the cell cycle pathway were observed including CDK6. Integrative analysis of transcriptomics and proteomics data consistently showed alterations in cell cycle, DNA damage repair and splicing related pathways upon treatment, consistent with the established biological roles of PRMT5. Alternative splicing analysis showed that AZD3470 treatment induced a unique retained intron within the ATM gene and resulted in reduced ATM protein levels. Conclusion: Here we identify AZD3470-induced changes to protein post-translational modification, chromatin accessibility, gene expression and RNA splicing in MTAP-deficient NSCLC cancer cell lines. Different area of MultiOMICs data together pointed to changes in cell cycle and DNA repair pathways. Further, we identified alternative splicing of ATM as a consequence of AZD3470 PRMT5i treatment resulting in significant reduction of ATM protein expression. Keywords: AZD3470, NSCLC, PRMT5 Inhibition, Chromatin Accessibility, Alternative Splicing, DNA Repair, ATM Gene. Citation Format: Ted Hong, Steven Criscione, Andrew Jarnuczak, Jelena Urosevic, Stephanie Ashenden, Ghaith Hamza, Praveen Kumar, Nevena Cvetesic, Lauren Bradshaw, Shaun Moore, Daniel Karl, Tianhui Zhang, Abel Sousa, Jonathan Cairns, Anthony Iannetta, Andrew Zhang, Eric Miele, John Reicha, Chris Chambers, Ramy Elgendy, Julia Lindgren, Daniel Jachimowicz, Maryam Clausen, Graham Belfield, Tony Cheung, Michael Grondine, James T. Lynch, Ho Man Chan, Susan Critchlow, Emma Dean. Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB008.

Abstract CT218: Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Abstract Background: Triptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. Preclinical investigations indicate significant activity against super enhancer targets. In the phase I clinical trial of Minnelide™ activity in highly refractory metastatic pancreatic carcinoma was observed. Hence, we undertook this study in patients with chemotherapy refractory metastatic PDAC. Our aims were to determine clinical anticancer effects, and to define effects of the drug on super enhancer expression and downstream effects. Methods: Eligibility criteria included stage IV PDAC progressing on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease by RECIST 1.1. Planned sample size was up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 was administered intravenously over 30 minutes on days 1-21, followed by a 7-day rest period in a 28-day cycle. Exploratory objectives included: analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, proteomics and metabolism. Results: 17 patients were treated. There were no RECIST responses or stable disease. However, evidence of activity was observed in 4 of 15 evaluable patients. This activity included progression after 117 days treatment in 1 patient and reductions in CT tumour size or FDG-PET uptake and/or significant decreases in circulating CA19.9 levels in 3 others. Minnelide was generally well tolerated. Treatment related toxicities > 3 were: neutropenia 4 patients and cerebellar ataxia 1 patient.Exploratory analyses included ATAC-Seq on paired biopsies where gene ontology analyses showed downregulation of genes negatively regulating cell cycle and upregulation of genes controlling cell cycle checkpoints. Other exploratory analyses will be presented. Conclusions: The study identified a tolerable drug dose and evidence of activity in a highly chemotherapy-refractory population which provides encouraging proof for the principle of targeting TFIIH in human PDAC. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Erkut Borazanci, Peter J. O'Dwyer, Daniel Von Hoff, Joseph Hartlebury, Corina E. Antal, Gerard I. Evan, HaiYong Han, Michael Downes, Ronald Evans, Joshua Rabinowitz, Emmanuel Petricoin, Ashok Saluja, Mohana Velagapudi, Hitendra Patel. Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT218.

Genome-Wide Analysis Identifies Nuclear Factor 1C as a Novel Transcription Factor and Potential Therapeutic Target in SCLC

IntroductionRecent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. MethodsTranscriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitivity (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors. ResultsSignificant commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DNase I hypersensitive site sequencing to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment. ConclusionsNFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.

Abstract 1696: ACTL6A deficiency drives cell death through KLF4-involved chromatin accessibility changes in colorectal cancer

Abstract ACTL6A is a subunit of the SWI/SNF and INO80 chromatin remodeling complexes and plays roles in neural development and cancer cell proliferation. Additionally, it is known to be upregulated in various types of cancer and is associated with a decrease in patient survival rates. However, the mechanism of how chromatin remodelers including ACTL6A cause cell death has yet to be defined. Therefore, we would like to investigate how the depletion of ACTL6A promotes cell growth reduction by mediating changes in the epigenetic landscape in colorectal cancer cells. We engineered ACTL6A knockout by CRISPR-Cas9. We performed Assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), and Chromatin Immunoprecipitation sequencing (ChIP-seq) for SMARCB1 and INO80, as representative members of their respective families, and KLF4. Moreover, HCT116 cells stably transduced with doxycycline-inducible shGFP or shACTL6A were implanted into the flank of nude mice. One week after tumor implantation, mice were randomly allocated to 4 groups (n=13-15 per group) and doxycycline treated group was administered with doxycycline (1mg/ml) and 5% sucrose in the drinking water. Tumor volume was measured with calipers. Upon depletion of ACTL6A, the regions of increased chromatin accessibility were correlated with the upregulated genes. After the loss of ACTL6A, there was a significant increase in KLF4 expression and genomic binding largely increased, coinciding with an increase in newly accessible regions containing KLF4 motifs. Interestingly, there is a strong positive correlation between changes in chromatin accessibility and overall changes in the binding of KLF4, SWI/SNF, and INO80 complexes. We find that changes in chromatin accessibility are co-regulated by KLF4, the SWI/SNF and the INO80 complex, while SWI/SNF and INO80 complex have a low correlation in binding between the SWI/SNF and INO80 complexes at newly accessible regions, indicating no physical interactions between these two chromatin complexes. Overall, we demonstrated that the redistribution of ACTL6A-deficient two chromatin remodeling complexes and KLF4 is associated with changes in chromatin accessibility, which are also linked to upregulated genes that promote the p53 pathway. These findings revealed the role of KLF4 along with the SWI/SNF and INO80 complexes, in regulating the chromatin landscape and gene expression in ACTL6A-depleted colorectal cancer cells. Citation Format: Hye-Ju Yang, Eun-Ju Kim, Sang-Hyun Song, Tae-You Kim. ACTL6A deficiency drives cell death through KLF4-involved chromatin accessibility changes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1696.

Abstract 1703: SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility

Abstract Acute Myeloid Leukemia (AML) is characterized by the unfettered proliferation and poor differentiation of myeloid cells causing reduced survival. The mammalian BAF complexes (also known as SWI/SNF) are important regulators of MYC and the cell cycle. There are three BAF complexes (cBAF, ncBAF and pBAF) that share several subunits. SMARCA2 and 4 are mutually exclusive ATPase subunits within the BAF complexes that use ATP hydrolysis and helicase activity to remodel chromatin via nucleosome sliding and removal. PBRM1 is a SMARCA4 associated factor present in pBAF. The association of SMARCA4 with regulatory elements of transcription factors makes it an intriguing target for AML. To evaluate the role of SMARC2/4 in AML, we analyzed RNA transcripts from highly purified primary AML initiating populations and control samples. Sorted leukemia stem cells (Lin-ve, CD34+, CD38-) overexpressed SMARCA2/4 when compared to healthy cells. A novel and highly selective SMARCA2/4 and PBRM1 PROTAC degrading agent AU-15330 was used to investigate the therapeutic effectiveness of targeting BAF complexes as well as the role of SMARCA2/4 in leukemic pathogenesis. Our data demonstrate that AU-15330 effectively degrades SMARCA2/4 and PBRM1 in human leukemia cell lines with varying mutational profiles; MV4-11 was the most sensitive cell line. When evaluating healthy CD34+ HSPCs as well as patient derived AML cells in colony assays, AU-15330 treatment increased differentiation in patient AML samples with minimal effects on healthy CD34+ cells. A xenograft of patient derived AML cells in irradiated mice was performed to determine the requirement of SMARCA2/4 in leukemia stem cell maintenance. Mice treated with AU-15330 had greater myeloid differentiation, fewer human cells in their bone marrows and reduced leukemia stem cell activity, seen in secondary transplants. To understand the underlying mechanism of how SMARCA2/4 and PBRM1 promote leukemic pathogenesis, we used a multi-omic analysis of transcription and chromatin dynamics in MV4-11 cells: ATAC-seq and RNA-seq identified downregulated genes with decreased accessibility, PRO-seq determined early transcriptional changes and HiChIP showed changes in enhancer promoter loops with SMARC2/4 degradation. Several leukemia related transcription factors as well as inflammatory genes were identified from these analyses as possible targets of the BAF complexes in the context of AML. Finally, to determine the effect of SMARCA2/4 degradation on pBAF association with chromatin, single cell imaging was performed of U2OS cells with HALO-tagged PBRM1 treated with the degrader or DMSO. pBAF in the degrader treated cells improved engagement with target chromatin however, a decrease in the stability of the complex binding to the nucleosome when compared to DMSO treated cells was shown. Taken together, these studies will determine the role of SMARCA2/4 in leukemia disease. Citation Format: Sarah Aminov, Kith Pradhan, Srabani Sahu, Shanisha Gordon-Mitchell, Emma Rabinovich, Ariel Fromowitz, Joyeeta Chakraborty, Srinivas Aluri, Rongbao Zhao, Nayem Haque, Milagros Carbajal, Aditi Shastri, Ulrich Steidl, Susanta Samajdarr, Akhil Kumar, Murali Ramachandra, Robert A. Coleman, Kristy Stengel, Amit K. Verma. SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1703.

Abstract 4339: Chromatin landscapes of colorectal cancer development and cfDNA fragmentation

Abstract Introduction: The stepwise progression of colorectal cancer from healthy epithelium, to premalignant adenoma, to cancer is accompanied by genome-wide epigenetic reprogramming. However, current analyses of these processes have been hampered by complex mixtures of cells in normal, adenoma and cancer tissue samples, as well as absence of suitable adenoma model systems. Cultured colorectal organoids, comprised of pure epithelial cells, could enable insights on the epigenetic changes leading to progression from healthy tissue to cancer. Methods: Using 43 patient-derived colorectal organoids derived from healthy, adenoma, and cancer tissues, we examined the chromatin landscape of tumor progression using transposase accessible chromatin analyses with next generation sequencing. We defined a consensus set of nucleosome depleted regions for each disease stage and used principal component analyses to identify peaks that contributed the most variation across the collection of organoids. Consensus regions were linked to genes they overlapped, and intergenic consensus regions were linked to the nearest gene within 1 Mbp. Subsequently, we performed a gene set enrichment analysis to assess pathways affected by differential chromatin accessibility. To identify the transcription factor drivers of regulatory reprogramming, the DNA sequences within the consensus peaks were analyzed for enrichment of characteristic binding motifs. We analyzed regions of consensus peaks for fragmentation characteristics in the circulating cell-free DNA (cfDNA) of 250 healthy individuals and 51 patients with stage IV colorectal cancer. Results: Across all organoid samples, we identified >35 million nucleosome depleted regions that we coalesced into a consensus set of 61,755 regions. We excluded 13,040 regions that had low variation across the organoid tissues, leaving 48,715 regions that could potentially define tissue-specific signatures of chromatin accessibility. As 78% of these regions could be linked to genes, we assessed whether these signatures identify known pathways involved in colorectal cancer. We identified unique signatures of healthy tissues, adenomas, and cancers comprising genes of known and novel pathways, including those involved in WNT, hippo, and RAS signaling. We found that changes in chromatin accessibility can be detected in circulating cfDNA profiles and were associated with independent circulating tumor DNA abundance by droplet digital PCR (R=0.78, p<0.0001). Conclusions: These analyses highlight important targets of chromatin remodeling in colorectal tumorigenesis and provide an avenue for evaluating these through genome-wide analyses of cfDNA fragmentation. Citation Format: Nicholas A. Vulpescu, Zachariah H. Foda, Pieter H. Wisse, Jamie E. Medina, Vilmos Adleff, Remond J. Fijneman, Robert B. Scharpf, Gerrit A. Meijer, Beatriz Carvalho, Victor E. Velculescu. Chromatin landscapes of colorectal cancer development and cfDNA fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4339.

Abstract 3936: Altered endosomal pH regulation: molecular mechanisms of oncogenesis and therapeutic dependencies

Abstract ATP6AP1 and ATP6AP2, which we previously identified to be recurrently altered in granular cell tumors (GCTs), encode for accessory proteins of the vacuolar (V)-ATPase, which controls endosomal acidification. The mechanistic basis of how ATP6AP1/AP2 loss of function (LOF) leads to oncogenesis remains to be elucidated. Here, we sought to unravel the molecular mechanisms downstream of ATP6AP1/AP2 inactivation resulting in oncogenesis and identify associated therapeutic dependencies. Using CRISPR/Cas9 technologies, we generated ATP6AP1-knock out (KO), ATP6AP2-KO and non-target (NT) control immortalized Schwann cells, the likeliest cell of origin of GCTs. Compared to NT controls, ATP6AP1/AP2-KOs displayed increased cellular proliferation, migration, and anchorage independent growth. ATP6AP1/AP2-KOs, but not NT controls, demonstrated in vivo tumorigenesis upon subcutaneous implantation in NSG mice. In addition, ATP6AP1/AP2-KOs, compared to NT controls, displayed abnormal bulk endocytic function, evidenced by reduced endocytic cargo delivery to lysosomes in a DQ-Red BSA assay, and increased LC3BII/LC3BI ratio and expression of the autophagy marker GABARAP, indicating altered autophagic flux. ATAC-seq analysis of our cell modes revealed changes in chromatin accessibility upon ATP6AP1/AP2 inactivation, affecting transcription factors (TFs) associated to the Wnt pathway, including LEF/TCF (canonical Wnt) and NFAT (non-canonical Wnt), and TFs associated to autophagy pathways, among others. RNA-sequencing analysis revealed expression changes in key Wnt pathway components upon ATP6AP1/AP2 LOF, such as biphasic modulators (DKK1, DKK2 and SFRP1), secreted ligands (WNT2, WNT9a and WNT7B), transcriptional regulators (TLE1, TLE2, TLE3 and TLE4) and receptors (LGR5, FZD7 and FZD1). Consistent with these findings, ATP6AP1/AP2-KOs displayed upregulation of downstream canonical (MYC, Cyclin D1) and non-canonical (NFATC1, CALM1) Wnt targets at the mRNA and protein levels, and increased sensitivity to pharmacologic inhibition of the Wnt pathway, with significantly lower IC50s to IWR-1, compared to NT controls. Lastly, ATP6AP1/AP2-KOs displayed increased sensitivity to pharmacologic inhibition or induction of autophagy and/or endocytic processes, with lower IC50s to Chloroquine, N-Ethylmaleimide, Torin-1 and Bafilomycin-A1 compared to NT controls. Taken together, we demonstrate that ATP6AP1/AP2 inactivation leads to the acquisition of oncogenic properties in vitro and in vivo, altered endocytosis and autophagy. Furthermore, our findings suggest that ATP6AP1/AP2 LOF results in chromatin accessibility changes leading to activation of an oncogenic transcriptional program with increased signaling via the Wnt pathway. Notably, inactivation of these genes results in unique and previously unknown vulnerabilities, offering potential avenues for therapeutic exploration. Citation Format: Higinio Dopeso, Yingjie Zhu, Laxmi Gusain, Thais Basili, David Brown, Fatemeh Derakhshan, Edaise M. da Silva, Richard Koche, Pierre-Jacques Hamard, Xinyu Guo, Eneda Toska, Elisa DeStanchina, Hong Zhang, Jorge Reis-Filho, Britta Weigelt, Fresia Pareja. Altered endosomal pH regulation: molecular mechanisms of oncogenesis and therapeutic dependencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3936.