Active Chromatin State Research Articles

Tissue‐specific methylomic responses to a lifestyle intervention in older adults associate with metabolic and physiological health improvements

AbstractAcross the lifespan, diet and physical activity profiles substantially influence immunometabolic health. DNA methylation, as a tissue‐specific marker sensitive to behavioral change, may mediate these effects through modulation of transcription factor binding and subsequent gene expression. Despite this, few human studies have profiled DNA methylation and gene expression simultaneously in multiple tissues or examined how molecular levels react and interact in response to lifestyle changes. The Growing Old Together (GOTO) study is a 13‐week lifestyle intervention in older adults, which imparted health benefits to participants. Here, we characterize the DNA methylation response to this intervention at over 750 thousand CpGs in muscle, adipose, and blood. Differentially methylated sites are enriched for active chromatin states, located close to relevant transcription factor binding sites, and associated with changing expression of insulin sensitivity genes and health parameters. In addition, measures of biological age are consistently reduced, with decreases in grimAge associated with observed health improvements. Taken together, our results identify responsive molecular markers and demonstrate their potential to measure progression and finetune treatment of age‐related risks and diseases.

PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR–Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.

Epigenetic control of metabolic identity across cell types.

Constraint-based network modelling is a powerful tool for analysing cellular metabolism at genomic scale. Here, we conducted an integrative analysis of metabolic networks reconstructed from RNA-seq data with paired epigenomic data from the EpiATLAS resource of the International Human Epigenome Consortium (IHEC). Applying a state-of-the-art contextualisation algorithm, we reconstructed metabolic networks across 1,555 samples corresponding to 58 tissues and cell types. Analysis of these networks revealed the distribution of metabolic functionalities across human cell types and provides a compendium of human metabolic activity. This integrative approach allowed us to define, across tissues and cell types, i) reactions that fulfil the basic metabolic processes (core metabolism), and ii) cell type-specific functions (unique metabolism), that shape the metabolic identity of a cell or a tissue. Integration with EpiATLAS-derived cell type-specific gene-level chromatin states and enhancer-gene interactions identified enhancers, transcription factors, and key nodes controlling core and unique metabolism. Transport and first reactions of pathways were enriched for high expression, active chromatin state, and Polycomb-mediated repression in cell types where pathways are inactive, suggesting that key nodes are targets of repression. This integrative analysis forms the basis for identifying regulation points that control metabolic identity in human cells.

Systematic Multi-Omics Investigation of Androgen Receptor Driven Gene Expression and Epigenetics changes in Prostate Cancer.

Prostate cancer, a common malignancy, is driven by androgen receptor (AR) signaling. Understanding the function of AR signaling is critical for prostate cancer research. We performed multi-omics data analysis for the AR+, androgen-sensitive LNCaP cell line, focusing on gene expression (RNAseq), chromatin accessibility (ATACseq), and transcription factor binding (ChIPseq). High-quality datasets were curated from public repositories and processed using state-of-the-art bioinformatics tools. Our analysis identified 1004 up-regulated and 707 down-regulated genes in response to androgen deprivation therapy (ADT) which diminished AR signaling activity. Gene-set enrichment analysis revealed that AR signaling influences pathways related to neuron differentiation, cell adhesion, P53 signaling, and inflammation. ATACseq and ChIPseq data demonstrated that as a transcription factor, AR primarily binds to distal enhancers, influencing chromatin modifications without affecting proximal promoter regions. In addition, the AR-induced genes maintained higher active chromatin states than AR-inhibited genes, even under ADT conditions. Furthermore, ADT did not directly induce neuroendocrine differentiation in LNCaP cells, suggesting a complex mechanism behind neuroendocrine prostate cancer development. In addition, a publicly available online application LNCaP-ADT (https://pcatools.shinyapps.io/shinyADT/) was launched for users to visualize and browse data generated by this study. This study provides a comprehensive multi-omics dataset, elucidating the role of AR signaling in prostate cancer at the transcriptomic and epigenomic levels. The reprocessed data is publicly available, offering a valuable resource for future prostate cancer research.

SMCHD1 maintains heterochromatin and genome compartments in human myoblasts.

Mammalian genomes are subdivided into euchromatic A compartments that contain mostly active chromatin, and inactive, heterochromatic B compartments. However, it is unknown how A and B genome compartments are established and maintained. Here we studied SMCHD1, an SMC-like protein in human male myoblasts. SMCHD1 colocalizes with Lamin B1 and the heterochromatin mark H3K9me3. Loss of SMCHD1 leads to extensive heterochromatin depletion at the nuclear lamina and acquisition of active chromatin states along all chromosomes. In absence of SMCHD1, long range intra-chromosomal and inter-chromosomal contacts between B compartments are lost while many new TADs and loops are formed. Inactivation of SMCHD1 promotes numerous B to A compartment transitions accompanied by activation of silenced genes. SMCHD1 functions as an anchor for heterochromatin domains ensuring that these domains are inaccessible to epigenome modification enzymes that typically operate in active chromatin. Therefore, A compartments are formed by default when not prevented by SMCHD1.

Abstract LB028: Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies

Abstract The paralogous histone acetyltransferases EP300 and CREBBP are two of the master regulators of transcription by controlling both enhancer and promoter activity through histone and non-histone acetylation. EP300 and CREBBP activity is primarily driven by their acetylation of K27 and K18 on histone H3 leading to open and active chromatin state. Targeting the EP300/CREBBP axis of chromatin regulation may be a way to disrupt the signaling pathways downstream of key oncogenic signaling networks like MYC/IRF4 in hematopoietic cancers. Successful development of dual EP300/CREBBP BRDi and HATi has led to promising pre-clinical efficacy, but due to observed tolerability issues may potentially limit their utility in the clinic. We hypothesized, based upon published mouse KO phenotypes, that a selective EP300 compound may improve tolerability. Due to the high sequence homology between EP300 and CREBBP in both the BRD and HAT domains, developing selective inhibitors has proven to be challenging. To combat this, we have utilized heterobifunctional protein degrader technology (ProDegs, also known as PROTACS) to develop EP300 selective ProDegs with greater than 100-fold selectivity over CREBBP. We observe rapid targeted degradation of EP300 with maximal degradation occurring within the first 2-4 hours after treatment. Concomitant with the loss of EP300, we see modulation of the associated H3K27ac and H3K18ac biomarkers as well as alterations in key oncogenic transcription networks. In both in vitro and in vivo assays looking at impact to platelet maturation, we observed that selective loss of EP300 largely abrogated observed thrombocytopenia compared to dual EP300/CREBBP inhibitors. Upon treatment, MM and NHL models show strong downregulation of MYC/IRF4 oncogenic signaling pathways, both of which culminate in a loss of cellular viability and cell death. Additionally, we observe in vivo dose responsive tumor growth inhibition in OPM2 and Pfeiffer xenograft models. Together, the data demonstrate the potential utility of EP300 selective ProDegs for the treatment of hematopoietic malignancies. Citation Format: Frederick A. Derheimer, Ninvita Givarkes, Natalie Miller, Rita Grantner, Casey Quinlan, Sonja Brun, Thomas Paul, Astrid Ruefli-Brasse, Stefan Steyn, Derek Bartlett, Matthew Hayward, Matthew Brown, Daniel Uccello, Benjamin J. Burke, Kris Borzilleri, Adam Gilbert, Michelle Hemkens. Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies [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 LB028.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Temporally-coordinated bivalent histone modifications of BCG1 enable fungal invasion and immune evasion

Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.

Abstract A050: A Phase 1 dose escalation study of TACH101, a first-in-class KDM4 inhibitor for advanced solid tumors

Abstract The KDM4 family of histone demethylases (KDM4A, B, C, D) have been identified as key oncogenic drivers in various cancers. KDM4 proteins control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histones H3K9 and H3K36 and have been shown to play an important role in epigenetic dysregulation during tumorigenesis. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform is not effective. TACH101 is a novel, first-in-class small molecule KDM4 inhibitor that targets isoforms A-D. In vitro and in vivo studies have shown potent anti-proliferative activity and strong tumor growth inhibition across multiple xenograft models. Preclinical studies demonstrated favorable pharmacokinetics (PK) and safety profiles. Initial clinical development of TACH101 will target advanced or metastatic solid tumors. TACH101-CS-0001 is an open-label, single-arm, phase 1 dose escalation study evaluating the safety and tolerability of orally administered TACH101 in subjects with solid tumors. Patients receive TACH101 in a single-dose lead-in period followed by repeated dosing on a weekly schedule. Using a Bayesian optimal interval (BOIN) design, the trial will evaluate up to 8 dose levels following a 3-days on/4-days off dosing schedule. Escalation across 2 dose cohorts (5 and 10mg) has been completed and enrollment into cohort 3 (25 mg) has begun. The planned enrollment is up to 33 patients including dose escalation and dose expansion (highest safe dose level) cohorts. Inclusion criteria include advanced or metastatic solid tumor that has progressed or was non-responsive or intolerant to available therapies and for which no standard or available curative therapy exists. Patients must have disease that is measurable according to RECIST (v1.1) and an ECOG performance status score of 0 to 1. Exclusion criteria include severe hematologic, hepatic, or renal insufficiency. Primary endpoints will assess safety and tolerability to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). Secondary endpoints will evaluate PK and preliminary anti-tumor activity as assessed by objective response rate (ORR), duration of response (DOR), and clinical benefit rate (CBR). Exploratory endpoints include evaluation of circulating tumor DNA (ctDNA)-based genetic markers as well as molecular characterization of optional tissue biopsies. This trial will be the first to evaluate the full potential of KDM4 inhibition in solid tumors with the novel epigenetic-targeting drug candidate TACH101 in subjects with advanced or metastatic cancers. (NCT05076552). Citation Format: Apostolia Tsimberidou, Andrae L Vandross, David Sommerhalder, Charmaine Joseph, Chandtip Chandhasin, Frank Perabo, Michael F Clarke. A Phase 1 dose escalation study of TACH101, a first-in-class KDM4 inhibitor for advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A050.

Chromatin state transitions in the Drosophila intestinal lineage identify principles of cell-type specification

Tissue homeostasis relies on rewiring of stem cell transcriptional programs into those of differentiated cells. Here, we investigate changes in chromatin occurring in a bipotent adult stem cells. Combining mapping of chromatin-associated factors with statistical modeling, we identify genome-wide transitions during differentiation in the adult Drosophila intestinal stem cell (ISC) lineage. Active, stem-cell-enriched genes transition to a repressive heterochromatin protein-1-enriched state more prominently in enteroendocrine cells (EEs) than in enterocytes (ECs), in which the histone H1-enriched Black state is preeminent. In contrast, terminal differentiation genes associated with metabolic functions follow a common path from a repressive, primed, histone H1-enriched Black state in ISCs to active chromatin states in EE and EC cells. Furthermore, we find that lineage priming has an important function in adult ISCs, and we identify histone H1 as a mediator of this process. These data define underlying principles of chromatin changes during adult multipotent stem cell differentiation.

Pre-Existing Chromatin States Regulate KLF4 Binding to Eliminate Leukemia Stem Cells

Transcription factors collaborate with chromatin's epigenetic states to regulate cell fate decisions. While recent in vitro work using cryo-EM (Sinha et al., Nature, 2023) suggested that histone modifications could regulate the activity of pioneer transcription factors, it was not clear whether these findings applied in vivo, or if chromatin states could influence transcription factor binding to affect cell fate. Our previous work (Liu et al., Leukemia,2014; Wang et al., Nat Commun,2019) showed that reprogramming factors (Oct4, Sox2, Klf4, and cMyc, collectively known as OSKM) selectively eliminated leukemia cells in vivo, with minimal impact on normal Hematopoietic Stem and Progenitor Cells (HSPCs). This suggested that cell fates induced by OSKM could be influenced by pre-existing chromatin states. Building on this hypothesis, we profiled the pre-existing chromatin states of leukemia cells and HSPCs using MNase-ChIP-seq, ATAC-seq, and WGBS-seq. In our Tet-on-induced models, Klf4 activation resulted in the targeting of distinct gene sets and exhibited similar dynamic changes in chromatin accessibility, despite these regions sharing the same active chromatin state. Using machine learning algorithms, we identified H3K18ac –a feature of the active chromatin states preferred by Klf4– as a dominant factor influencing Klf4's genomic binding. We then altered H3K18ac through an enzymatically deficient SIRT7, which is an H3K18ac deacetylase, and examined where Klf4 binds. We observed an increase in Klf4 binding at sites where H3K18ac levels were elevated. This result suggested that Klf4 binding was influenced by changes in H3K18ac. We exposed human CD34+ cells, several leukemia cell lines, and samples from leukemia patients to ATPO-253. This small molecule, currently in phase 1 clinical trials, has been found to enhance the levels of the KLF4 protein. This treatment led to an increase in KLF4 protein levels and we observed activation of apoptosis genes in the tested leukemia cell lines that possessed high pre-existing H3K18ac levels. In addition, we observed a decline across all tested leukemia samples, while the population of CD34+ cells essentially remained stable. In conclusion, our work demonstrated that pre-existing chromatin states, such as H3K18ac, regulate Klf4's genomic binding, thereby influencing distinct cell fates in leukemia cells and HSPCs. This opens potential avenues for clinical applications in targeted leukemia cell clearance using KLF4 or ATPO-253.

TMIC-70. A TRANSCRIPTIONALLY ACTIVE EPIGENETIC IMPRINTING OF THE JAK/STAT PATHWAY MEDIATED BY H3.3-G34 MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG) INDUCES AN IMMUNOREACTIVE TME

Abstract Pediatric high-grade gliomas (pHGGs) stand as the primary cause of cancer-associated fatalities among children. Sixteen percent of pHGGs found in children and young adults present Gly34Arg/Val substitutions within the histone H3.3 (H3.3-G34R/V). Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we showed that this mutation leads to downregulation of the DNA repair pathways and enhanced susceptibility to DNA damage. DNA damage response (DDR) inhibitors in vivo, further increased genetic instability, and activation of the cGAS-STING pathway, which induces the release of immune-stimulatory cytokines. We carried out an epigenetic analysis using ChIP-seq in our GEMM, and our findings indicate that this mutation triggers transcriptionally active chromatin states within genes linked to immune reactivity, particularly JAK-STAT signaling pathway genes. Correspondingly, G34-mutant pHGG cells release immune-activating cytokines that reprogram the immune cells within the tumor microenvironment (TME). Myeloid derived suppressor cells from G34-mutant pHGG are phenotypically less immunosuppressive than the MDCS form histone wild type pHGG. Moreover, G34-mutant pHGG TME exhibit higher frequency of CD8 T cells. Based on these findings, we assessed the efficacy of an adenoviral-mediated immunostimulatory gene therapy (GT). This therapy mediates expression of Flt3L within pHGG TME, which aids dendritic cell recruitment. Thymidine kinase (TK), converts ganciclovir (GSV) into a cytotoxic compound, leading to pHGG cells’ death. GT resulted in 60% of long-term survival in mice harboring G34R pHGG. The animals that survived due to the GT did not develop tumors after a rechallenge with G34R pHGG cells, demonstrating that the treatment induces antitumoral immunological memory. In conclusion, our findings suggest that G34-mutant pHGG patients could benefit from combined therapies that target genetic instability and stimulate the immune system. Funded by NIH/NINDS.

Non-small Cell Lung Cancer Epigenomes Exhibit Altered DNA Methylation in Smokers and Never-smokers

Epigenetic alterations are widespread in cancer and can complement genetic alterations to influence cancer progression and treatment outcome. To determine the potential contribution of DNAmethylation alterations to tumor phenotype in non-small cell lung cancer (NSCLC) in both smoker and never-smoker patients, we performed genome-wide profiling of DNA methylation in 17 primary NSCLC tumors and 10 matched normal lung samples using the complementary assays, methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation sensitive restriction enzyme sequencing (MRE-seq). We reported recurrent methylation changes in the promoters of several genes, many previously implicated in cancer, including FAM83A and SEPT9 (hypomethylation), as well as PCDH7, NKX2-1, and SOX17 (hypermethylation). Although many methylation changes between tumors and their paired normal samples were shared across patients, several were specific to a particular smoking status. For example, never-smokers displayed a greater proportion of hypomethylated differentially methylated regions (hypoDMRs) and a greater number of recurrently hypomethylated promoters, including those of ASPSCR1, TOP2A, DPP9, and USP39, all previously linked to cancer. Changes outside of promoters were also widespread and often recurrent, particularly methylation loss over repetitive elements, highly enriched for ERV1 subfamilies. Recurrent hypoDMRs were enriched for several transcription factor binding motifs, often for genes involved in signaling and cell proliferation. For example, 71% of recurrent promoter hypoDMRs contained a motif for NKX2-1. Finally, the majority of DMRs were located within an active chromatin state in tissues profiled using the Roadmap Epigenomics data, suggesting that methylation changes may contribute to altered regulatory programs through the adaptation of cell type-specific expression programs.

KDM6A epigenetically regulates subtype plasticity in small cell lung cancer.

Small cell lung cancer (SCLC) exists broadly in four molecular subtypes: ASCL1, NEUROD1, POU2F3 and Inflammatory. Initially, SCLC subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoural subtype heterogeneity and plasticity between subtypes. Here, using a CRISPR-based autochthonous SCLC genetically engineered mouse model to study the consequences of KDM6A/UTX inactivation, we show that KDM6A inactivation induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumours that express both ASCL1 and NEUROD1. Mechanistically, KDM6A normally maintains an active chromatin state that favours the ASCL1 subtype with its loss decreasing H3K4me1 and increasing H3K27me3 at enhancers of neuroendocrine genes leading to a cell state that is primed for ASCL1-to-NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC genetically engineered mouse model to model ASCL1 and NEUROD1 subtype heterogeneity and plasticity, which is found in 35-40% of human SCLCs.

Abstract A006: Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss

Abstract KRAS-targeted inhibition yielded promising, yet far from ideal, clinical responses, revealing that cancer cells easily bypass KRAS loss. Therefore, we aim to understand how mutant KRAS cancer cells tolerate so well the loss of a key oncogene to which they were addicted to. KRAS expression was silenced through siRNAs in colorectal cancer (CRC) cell lines that carry KRAS mutations. The proteome characterization was obtained by mass spectrometry and the expression of significantly altered proteins was validated by western blotting. Chromatin states were investigated using electron microscopy. Upon KRAS inhibition, there was a significant reduction in the cell number, accompanied by changes in cell cycle, thus supporting KRAS-dependency. Proteomics analysis revealed that KRAS inhibition-tolerant cells upregulated several proteins associated with the extracellular exosome or nuclear compartments. Molecular function and biological process gene ontology terms revealed an up-regulation of proteins mainly associated with binding activities (RNA, protein, nucleosome, and core promoter binding) as well as gene expression regulation, mRNA splicing and processing, and nucleosome assembly and repositioning. In addition, the proteomics data also revealed an upregulation of proteins associated with active chromatin states. Moreover, KRAS-silenced persister cells also presented alterations in some histone post-translational modifications and in chromatin packing, both suggesting that transcription is impacted. Overall, our results suggest an epigenetic mechanism underlying tolerance to KRAS inhibition that involves chromatin structural changes and transcription alterations, which we are currently pursuing. Citation Format: Flavia Martins, Ana L. Machado, Patricia D. Carvalho, Joana Carvalho, Rune Matthiesen, Vadim Backman, Sergia Velho. Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A006.

Abstract 5774: Small cell lung cancer subtype plasticity is regulated by KDM6A

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that accounts for ~15% of lung cancers. While nearly all SCLCs are genetically driven by near universal loss of function (LOF) mutations in RB1 and TP53; several recent studies show that there are different phenotypic SCLC molecular subtypes characterized by expression of lineage transcription factors. These include the neuroendocrine ASCL1 and NEUROD1 subtypes which together comprise ~70-80% of SCLCs. Initially subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoral subtype heterogeneity and plasticity between subtypes. A recent study found that 35-40% of human SCLCs express both ASCL1 and NEUROD1, but the mechanisms driving ASCL1 and NEUROD1 intra-tumoral heterogeneity are not well understood. My laboratory previously developed an autochthonous CRISPR-based SCLC genetically-engineered mouse model (GEMM) generated by intratracheally injecting adenoviruses encoding Cre recombinase and sgRNAs targeting Rb1, Trp53, and Rbl2. Cre turns on Cas9 expression and allows for CRISPR/Cas9 editing of Rb1, Trp53, and Rbl2 in somatic cells in the lungs. The unique advantage of this model is that it allows the inclusion of sgRNAs targeting additional genes of interest in the same adenovirus. Using this CRISPR-based autochthonous SCLC GEMM approach, we studied the consequences of inactivating the epigenetic modifier KDM6A during SCLC tumorigenesis. KDM6A functions as an H3K27 histone demethylase and also exists in the COMPASS complex with KMT2C/D to promote H3K4 mono-/di-methylation at enhancers. KDM6A along with its protein binding partner KMT2D are mutated in SCLC and KDM6A has been implicated in controlling differentiation in other lineages. Strikingly, we found that KDM6A inactivation in SCLC GEMMs induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumors that expressed both ASCL1 and NEUROD1. ATAC-sequencing showed open chromatin at the promoters of NEUROD1 and NEUROD1 target genes in KDM6A inactivated tumors. Interestingly, KDM6A inactivated tumors showed a spectrum of ASCL1 to NEUROD1 heterogeneity where some KDM6A inactivated tumors completely lost ASCL1 and solely expressed NEUROD1, some tumors expressed ASCL1 and NEUROD1 in a mutually exclusive manner, while others primarily expressed ASCL1 with very few NEUROD1 positive cells. Mechanistically, KDM6A binds and maintains ASCL1 target genes in an active chromatin state with its loss increasing H3K27me3 near both promoters and enhancers, and decreasing H3K4me1/2 at enhancers together leading to a cell state primed for ASCL1 to NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC GEMM to model ASCL1 and NEUROD1 subtype heterogeneity, which is found in 35-40% of human SCLCs. Citation Format: Leslie Duplaquet, Yixiang Li, Matthew A. Booker, Yingtian Xie, Radhika A. Patel, Deli Hong, Thomas Denize, Emily Walton, Yasmin N. Laimon, Roderick Bronson, Jackson Southard, Shuqiang Li, Sabina Signoretti, Michael Y. Tolstorukov, Paloma Cejas, Henry W. Long, Michael C. Haffner, Matthew G. Oser. Small cell lung cancer subtype plasticity is regulated by KDM6A. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5774.

TACH101, a first-in-class pan-inhibitor of KDM4 histone demethylase.

Histone lysine demethylase 4 (KDM4) is an epigenetic regulator that facilitates the transition between transcriptionally silent and active chromatin states by catalyzing the removal of methyl groups on histones H3K9, H3K36, and H1.4K26. KDM4 overamplification or dysregulation has been reported in various cancers and has been shown to drive key processes linked to tumorigenesis, such as replicative immortality, evasion of apoptosis, metastasis, DNA repair deficiency, and genomic instability. KDM4 also plays a role in epigenetic regulation of cancer stem cell renewal and has been linked to more aggressive disease and poorer clinical outcomes. The KDM4 family is composed of four main isoforms (KDM4A-D) that demonstrate functional redundancy and cross-activity; thus, selective inhibition of one isoform appears to be ineffective and pan-inhibition targeting multiple KDM4 isoforms is required. Here, we describe TACH101, a novel, small-molecule pan-inhibitor of KDM4 that selectively targets KDM4A-D with no effect on other KDM families. TACH101 demonstrated potent antiproliferative activity in cancer cell lines and organoid models derived from various histologies, including colorectal, esophageal, gastric, breast, pancreatic, and hematological malignancies. In vivo , potent inhibition of KDM4 led to efficient tumor growth inhibition and regression in several xenograft models. A reduction in the population of tumor-initiating cells was observed following TACH101 treatment. Overall, these observations demonstrate the broad applicability of TACH101 as a potential anticancer agent and support its advancement into clinical trials.

A pair of readers of bivalent chromatin mediate formation of Polycomb-based “memory of cold” in plants

The Polycomb-group chromatin modifiers play important roles to repress or switch off gene expression in plants and animals. How the active chromatin state is switched to a Polycomb-repressed state is unclear. In Arabidopsis, prolonged cold induces the switching of the highly active chromatin state at the potent floral repressor FLC to a Polycomb-repressed state, which is epigenetically maintained when temperature rises to confer "cold memory," enabling plants to flower in spring. We report that the cis-acting cold memory element (CME) region at FLC bears bivalent marks of active histone H3K4me3 and repressive H3K27me3 that are read and interpreted by an assembly of bivalent chromatin readers to drive cold-induced switching of the FLC chromatin state. In response to cold, the 47-bp CME and its associated bivalent chromatin feature drive the switching of active chromatin state at a recombinant gene to a Polycomb-repressed domain, conferring cold memory. We reveal a paradigm for environment-induced chromatin-state switching at bivalent loci in plants.

The dynamics of chromatin states mediated by epigenetic modifications during somatic cell reprogramming.

Somatic cell reprogramming (SCR) is the conversion of differentiated somatic cells into totipotent or pluripotent cells through a variety of methods. Somatic cell reprogramming also provides a platform to investigate the role of chromatin-based factors in establishing and maintaining totipotency or pluripotency, since high expression of totipotency- or pluripotency-related genes usually require an active chromatin state. Several studies in plants or mammals have recently shed light on the molecular mechanisms by which epigenetic modifications regulate the expression of totipotency or pluripotency genes by altering their chromatin states. In this review, we present a comprehensive overview of the dynamic changes in epigenetic modifications and chromatin states during reprogramming from somatic cells to totipotent or pluripotent cells. In addition, we illustrate the potential role of DNA methylation, histone modifications, histone variants, and chromatin remodeling during somatic cell reprogramming, which will pave the way to developing reliable strategies for efficient cellular reprogramming.

Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages.