Chromatin-associated Proteins Research Articles

Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Disrupting the interaction between a p53 gain-of-function mutant and the transcriptional co-activator PC4 reverses drug resistance in cancer cells.

PC4 is a chromatin-associated protein and transcriptional coactivator whose role in gene regulation by wild-type p53 is now well known. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. We show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. Here, we introduced a peptide that disrupts the PC4-R273Hp53 interaction to tumor cells bearing the R273HTP53 gene, which led to a lowering of MDR1 expression and abrogation of drug resistance in a mutant-specific manner. The results suggest that the PC4-R273Hp53 interaction may be a promising target for reducing proliferation and drug resistance in tumors.

Prediction of cell-type-specific cohesin-mediated chromatin loops based on chromatin state

Chromatin loop is of crucial importance for the regulation of gene transcription. Cohesin is a type of chromatin-associated protein that mediates the interaction of chromatin through the loop extrusion. Cohesin-mediated chromatin interactions have strong cell-type specificity, posing a challenge for predicting chromatin loops. Existing computational methods perform poorly in predicting cell-type-specific chromatin loops. To address this issue, we propose a random forest model to predict cell-type-specific cohesin-mediated chromatin loops based on chromatin states identified by ChromHMM and the occupancy of related factors. Our results show that chromatin state is responsible for cell-type-specificity of loops. Using only chromatin states as features, the model achieved high accuracy in predicting cell-type-specific loops between two cell types and can be applied to different cell types. Furthermore, when chromatin states are combined with the occurrence frequency of CTCF, RAD21, YY1, and H3K27ac ChIP-seq peaks, more accurate prediction can be achieved. Our feature extraction method provides novel insights into predicting cell-type-specific chromatin loops and reveals the relationship between chromatin state and chromatin loop formation.

Abstract 7026: Multi-omic genomic mapping with long read sequencing

Abstract Gene transcription is regulated by the complex interplay between histone post-translational modifications (PTMs), chromatin associated proteins (CAPs), and DNA methylation (DNAme). Mapping their genomic locations and examining the relationships between these chromatin elements is a powerful approach to decipher mechanisms of disease, thereby enabling discovery of novel biomarkers and therapeutics. Leading epigenomic mapping technologies (e.g., ChIP-seq, CUT&RUN) rely upon DNA fragmentation to isolate regions of interest for sequencing on short read platforms (e.g., Illumina). This strategy leads to substantial loss of contextual information regarding the surrounding DNA, precluding the identification of multiple co-occurring epigenomic features on a single DNA molecule. By contrast, long-read sequencing (LRS) platforms are capable of sequencing very long reads from a single molecule (typically >10kb), allowing relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. Here we report a robust multi-omic method that leverages LRS to simultaneously profile histone PTMs (or CAPs), DNAme, and parental haplotype in a single assay. This nondestructive, epigenomic mapping approach leverages a novel DNA methyltransferase fusion protein (pAG-M.EcoGII) to label DNA underneath antibody-targeted chromatin features, thereby marking sites of interest while preserving DNA molecules intact for LRS. Inspired by our work with state-of-the-art immunotethering-based approaches (CUT&RUN/CUT&Tag), nuclei are bound to magnetic beads to streamline and automate sample processing. Next, adenosines nearby antibody-targeted chromatin features are methylated with pAG-M.EcoGII, which are then directly read from genomic DNA using Oxford Nanopore Technologies or Pacific Biosciences LRS platforms. To determine the capabilities and limitations of this assay, we tested multiple chromatin targets in various cell lines. Importantly, this method is highly reproducible across biological replicates, and highly concordant with orthogonal SRS assays (e.g., CUT&RUN). Further, we showed that this method is a true multi-omic approach by simultaneously profiling histone PTMs, native DNAme (5mC), and parental single-nucleotide variants from single DNA molecules within a single reaction. Finally, this workflow preserves chromatin integrity for LRS, revealing heterogeneity (e.g., haplotype or paternal origin) within/between data types and providing access to previously unmappable genomic regions (e.g., centromeres). Citation Format: Bryan J. Venters, Paul W. Hook, Vishnu S. Kumary, Alli R. Hickman, James T. Anderson, Anup Vaidya, Ryan J. Ezell, Jonathan M. Burg, Zu-Wen Sun, Martis W. Cowles, Winston Timp, Michael-Christopher Keogh. Multi-omic genomic mapping with long read sequencing [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 7026.

Abstract 7027: Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq

Abstract DNA methylation (DNAme) is an epigenetic mark that includes the modification of cytosine resides (5mC) within CpG islands. In addition to well characterized roles regulating gene expression, imprinting and silencing parasitic DNA elements, the misregulation of DNAme is implicated in multiple diseases. Evidence is emerging that DNAme is not an independent epigenetic mark but rather closely linked to the post translational modification (PTM) of histone proteins. However, examining the relationship between 5mC and PTMs are hampered by the usual approach of independent analyses that cannot establish a direct linkage. Furthermore, the traditional approach to measure 5mC relies upon harsh bisulfite chemical conversation of DNA, which introduces damage and systemic biases. To address these limitations, we developed Targeted Enzymatic Methylation-sequencing (TEM-seq): an ultra-sensitive multi-omic genomic mapping technology that delivers high resolution DNAme profiles at epitope-defined chromatin features. Importantly this assay is capable of directly examining the link between 5mC and histone PTMs and/or chromatin associated proteins (ChAPs). The TEM-seq workflow integrates CUT&RUN for genomic mapping with NEB’s enzymatic methyl-seq (EM-seq) for unbiased DNAme analysis. CUT&RUN is a highly sensitive, standards-driven assay that uses antibodies to locally tether protein A/G-micrococcal nuclease (pAG-MNase) to chromatin within intact cells or nuclei, followed by controlled activation of the MNase to cleave nearby DNA. EM-seq then enzymatically converts [5mC/5hmC] to generate single-base resolution, unbiased DNAme profiles from sub-ng samples. To determine the capabilities and limitations of this novel approach, we tested multiple chromatin targets in various cell lines. We demonstrate that TEM-seq is highly reproducible, specific, and efficient (<10% off-target binding, >90% enzymatic conversion of DNAme, <0.5% conversion of unmethylated DNA). TEM-seq is also highly-sensitive, requiring only five million short-read sequences per assay (10-50x less than whole-genome 5mC-sequencing), demonstrating the disruptive potential of the assay to enable cost-effective approach for targeted DNAme analysis. Finally, we leveraged TEM-seq to gain mechanistic insights to Rett syndrome a neurodegenerative disorder that results from mutations in the 5mC-binding domain of the MECP2 gene. Citation Format: Keith E. Maier, Vishnu U. Sunitha Kumary, Bryan J. Venters, Allison Hickman, Anup Vaidya, James Anderson, Ryan Ezell, Jonathan M. Burg, Louise Williams, Chaithanya Ponnaluri, Pierre Esteve, Isaac Meek, Zu-wen Sun, Martis W. Cowles, Sriharsa Pradhan, Michael-Christopher Keogh. Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq [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 7027.

Abstract 5116: Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. We previously reported that ALL cells are vulnerable to metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and we and others have reported it interacts with chromatin-associated proteins to regulate gene expression in response to metabolic stress. To investigate AMPK’s mechanistic role in epigenetic gene regulation under metabolic stress, and identify new AMPK partners, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1. Following treatment with the allosteric AMPK activator PF-06409577 (PF064) for 1h, to mimic the cell’s rapid response to energy/metabolic stress, the samples underwent mass spectrometry-based proteomics. Proteomics data analysis identified novel altered (enhanced or reduced) AMPK protein-protein interactions (PPI) following PF064 treatment. The GO analysis of proteins interacting with AMPK showed that metabolic stress enhances PPIs involved in regulating gene expression, cellular metabolic processes, DNA repair, chromatin organization, histone modifications, etc. More importantly, we identified that AMPK interacted with members of the non-canonical Polycomb repressive complex 1 (PRC1.1) such as PCGF1, RING1, KDM2B, BCORL1, USP7. PRC1 is a known epigenetic regulator that monoubiquitinylates histone H2A at Lys119 (H2AK119ub) to control transcription and regulate gene expression. Using Co-IP, we confirmed these interactions in KASUMI-2 (Bp-ALL) and KE-37 (T-ALL) cells. When cells were treated with PF064 or 991 (another AMPK activator), we found that the interactions of AMPK with the catalytic core of PCR1.1 (PCGF1, RING1) were increased compared to untreated cells. To examine the effect of activated AMPK on PCR1.1 activity, we determined the level of H2AK119ub in ALL cells treated with PF064 and 991 for 1h. We found that the level of H2AK119ub protein expression was increased in ALL-treated cells indicating the PRC1.1 was activated. Together, these data suggest that stress-induced AMPK activation increased the catalytic activity of the PRC1.1 via PPI in ALL. Our findings are consistent with our previous published RNA-Seq and ChIP-Seq data indicating an epigenetic role for AMPK in altering gene expression examined under metabolic stress (two thirds exhibited downregulation), and provide insights into the AMPK-driven mechanism(s) involved. Further elucidation of the uncovered PPIs and its mechanistic role(s) may lead to potential epigenetic-based strategies to overcome resistance/refractoriness in ALL and other malignancies. Citation Format: Anna Shvab, Guy J. Leclerc, Julio C. Barredo. Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling in acute lymphoblastic leukemia [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 5116.

Increased genomic instability and reshaping of tissue microenvironment underlie oncogenic properties of Arid1a mutations.

Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur invivo. Using denaturing purification of invivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Nucleosome conformation dictates the histone code.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.

Chromatin accessibility and cell cycle progression are controlled by the HDAC-associated Sin3B protein in murine hematopoietic stem cells

BackgroundBlood homeostasis requires the daily production of millions of terminally differentiated effector cells that all originate from hematopoietic stem cells (HSCs). HSCs are rare and exhibit unique self-renewal and multipotent properties, which depend on their ability to maintain quiescence through ill-defined processes. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignancy. In particular, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Previous studies have identified chromatin coordination as a key regulator of differentiation in embryonic stem cells.ResultsHere, we utilized genetic inactivation of the chromatin-associated Sin3B protein to manipulate cell cycle control and found dysregulated chromatin accessibility and cell cycle progression in HSCs. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant progression through the G1 phase of the cell cycle, which correlates with the engagement of specific signaling pathways, including aberrant expression of cell adhesion molecules and the interferon signaling program in LT-HSCs. In addition, we uncover the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, which points to cell cycle progression possibly dictating the priming of HSCs for differentiation.ConclusionsOur findings provide new insights into controlled cell cycle progression as a potential regulator of HSC lineage commitment through the modulation of chromatin features.

Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response.

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.

Abstract C076: New insights into the role of FOXA1- HNF4 axis in pancreatic cancer

Abstract Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and have limited treatment options. Therefore, there is an immediate need for the generation of new and innovative therapeutic targets. Little is known about the role of pioneer factors such as FOXA1 and their interaction partners in a PDAC context. Inspired by literature reports implicating FOXA1 and GATA5/GATA6 in regulating pancreatic cancer resistance and metastasis, our study hypothesised the possibility of an undiscovered nuclear receptor that works with FOXA1 (similar to Estrogen receptor in breast cancer). Using innovative ‘omic’ based approaches (RIME) developed in our laboratory 1 we discovered a nuclear receptor (NR) complex involving HNF4A and HNF4G in the classical sub-type of pancreatic cancer. Subsequently the interaction was independently validated in Whipple surgical biopsies from PDAC patients using ChIP-sequencing studies. Across multiple patient tumors (n=7) a binding overlap specifically between FOXA1 and HNF4G (3461 sites) was established. To investigate the therapeutic potential of HNF4G in the classical subtype of PDAC, we generated CRISPR deletions (KO) of HNF4G in the HPAF-II cells. HNF4G-KO cells were orthotopically implanted into the pancreas of NSG mice. A significant survival advantage of 12 days (<0.001) and reduced growth (<0.02) was observed in these mice compared to the controls. To better understand the impact of HNF4G-KO on gene expression, the orthotopic tumors were subjected to RNA-seq analyses. Gene set enrichment and pathway analysis revealed significantly down-regulated pathways to include EMT transition. TCGA data highlighted HNF4G amplification in 9% of PDAC patients with concomitant decreased progression free survival. These data point towards HNF4G being a therapeutically viable target for further exploration. Protein Arginine Methyl Transferase 1 (PRMT1) is a common interactor of both FOXA1 and HNF4G. Our study reveals a unique dependency of PRMT1 on the HNF4G-FOXA1 complex in PDAC biopsies. HNF4G-KO drastically reduces the chromatin binding of PRMT1, implicating them as functionally dependent. Treatment of HNF4G-KO cells with GSK3368715 (PRMT1 inhibitor) further sensitizes these cells and results in a significant survival advantage (10 days <0.02). We propose a model of HNF4G inhibition in combination with PRMT1 as a novel therapeutic opportunity to treat the classical sub-type of PDAC. Further, our study reveals a unique reliance of primary classical tumors on HNF4G. Although HNF4G appears to dominate FOXA1 functionality in the primary disease, FOXA1 remains instrumental in priming metastasis and enhancer reprograming. 1. Papachristou EK, Kishore K, Holding AN, Harvey K, Roumeliotis TI, Chilamakuri CSR, et al. A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes. Nature Communications. 2018;9(1):2311. Citation Format: Shalini V. Rao, Lisa Young, Danya Cheeseman, Stephanie Mack, Jill Temple, Chandra Shekar Reddy Chilamakuri, Evangelia Papachristou, Catherina Pelicano, Amy Smith, Dominique-Laurent Couturier, Michael Gill, Duncan Jodrell Jodrell, Alasdair Russell, Igor Chernukhin, Jason Carroll. New insights into the role of FOXA1- HNF4 axis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C076.

Abstract B017: VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks

Abstract Valosin-containing protein (VCP) is an evolutionarily conserved AAA+ ATPase which plays pleiotropic roles in global proteostasis by extracting polyubiquitinated proteins from various cellular organelles and structures and facilitating their turnover. Our recent work demonstrated that nuclear VCP activity during DNA damage response specifically depends on ATM/ATR/DNA-PK-mediated Ser784 phosphorylation. Ser784phosphorylation increases VCP ability to facilitate chromatin-associated protein degradation and is required for DNA repair, checkpoint signaling, and cell survival. Clinically, high nuclear pSer784-VCP levels are significantly associated with poor survival outcome among chemotherapy-treated breast and pancreatic cancer patients. Thus, pSer784-VCP is a broadly important and therapeutically relevant genome stabilizer. Here, we focused specifically on the role of pSer784-VCP in DNA replication fork stability during stress, a known source of DNA damage if not properly regulated. By performing single-molecule DNA fiber analysis using human cell lines with genetic gain and loss-of-function of VCP, we observed that VCP protects nascent DNA from hydroxyurea (HU)-induced over-resection and this activity requires Ser784 phosphorylation. Using proximity ligation assay (PLA), we detected HU-induced pSer784-VCP on nascent DNA, suggesting that it may protect stressed forks by extracting functionally important regulators. Consistent with this theory, we detected an interaction of pSer784-VCP with SNF2H, a nucleosome-sliding enzyme which we recently found essential for nascent DNA over-resection at unprotected forks. In support of the hypothesis that SNF2H is a novel substrate of pSer784-VCP at stressed forks, we observed SNF2H retention on nascent DNA upon VCP knockdown in HU-treated cells, and the functional rescue of SNF2H retention by VCP re-expression requires Ser784phosphorylation. In support of their epistatic relationship, SNF2H depletion fully rescued fork over-resection caused by VCP loss. Similarly, blocking fork reversal by depleting SMARCAL1 or FBH1 also rescued fork over-resection caused by VCP loss. Importantly, mutating the ATPase activity of SNF2H abolished its ability to promote fork over-resection, suggesting that nucleosome remodeling by SNF2H underlies its effect on fork stability and may be a key molecular event enabling fork reversal upstream of fork degradation. Collectively, our data suggest that pSer784-VCP is a previously unrecognized stabilizer of stressed DNA replication forks, and it does so at least in part via its physical extraction of SNF2H from nascent DNA and the consequent attenuation of nucleosome remodeling needed for fork reversal and over-resection. More experiments are currently underway to further dissect the mechanistic aspects of this working model as well as to evaluate the potential of developing pSer784-VCP into a clinically relevant chemo-predictive biomarker and chemo-sensitizing target for different types of cancer. Citation Format: Jieya Shao, Mari Iwase, Rong Xu, Shuyang Lin. VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B017.

Therapeutic Targeting of the Menin–KMT2A Interaction

The direct targeting of chromatin-associated proteins is increasingly recognized as a potential therapeutic strategy for the treatment of cancer. In this review, we discuss a prominent example, namely, small-molecule inhibitors that target the menin–KMT2A interaction. These molecules are currently being investigated in clinical trials and showing significant promise. We describe the unique specificity of menin–KMT2A protein complexes for the transcriptional regulation of a small subset of genes that drive developmental and leukemic gene expression. We review the chromatin-associated KMT2A complex and the protein–protein interaction between menin and KMT2A that is essential for the maintenance of different types of cancer cells, but most notably acute myeloid leukemia (AML). We also summarize the development of menin inhibitors and their effects on chromatin. Finally, we discuss the promising early results from clinical trials in patients with AML and the recent discovery of therapy-resistant menin mutants that validate menin as a therapeutic target but also may present therapeutic challenges. Expected final online publication date for the Annual Review of Cancer Biology, Volume 8 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors.

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydroisoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physicochemical properties. We identified the 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physicochemical properties compared to our best-in-class dihydroisoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics.

Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.

Automated Cut&run Brings Scalable Epigenomic Profiling to Hematology

Genome-wide association studies (GWAS) and corresponding transcriptomics research have been leveraged to identify disease risk variants in hematological disorders with the hopes of developing more personalized treatment regimens. Interestingly, >90% of GWAS-identified variants are found in non-coding regions of the genome, indicating they likely affect the regulatory machinery that governs chromatin structure. Non-coding elements, such as enhancers, play important roles in supporting chromatin mechanisms, including cell-type specific gene expression, cell fates and disease states. However, these variants are difficult to study compared to protein-coding mutations and have been challenging to directly associate with phenotypic readouts. To date, efforts to understand the effects of non-coding variants on chromatin dynamics have been focused on chromatin accessibility (ATAC-seq) and DNA methylation profiling. Notably, these assays can only provide a binary, “open-or-closed,” view of chromatin and often fail to provide mechanistic insight into disease etiology. Epigenomic features - such as histone post-translational modifications (PTMs) and chromatin-associated proteins - mark distinct genomic compartments (e.g., promoters, enhancers) and regulate chromatin structure, gene expression, and cell function. Mapping these features provides a rich context to study cell fate and has great potential for discovering new biomarkers and drug targets. Despite this, efforts to integrate epigenomics into large scale lymphatic and myeloid research have been hampered by the poor sensitivity, high background, and low throughput of traditional chromatin mapping technology (i.e., ChIP-seq). Here, we present autoCUT&RUN, a high throughput assay for rapid, ultra-sensitive profiling of epigenomic features from FACS-isolated primary cells or tissues. This workflow generates reliable profiles from <10,000 cells per reaction, and is supported by a rigorous optimization strategy, high-quality antibodies, and quantitative spike-in controls. As part of a multi-site collaboration with the Immunological Genome Consortium, we used our autoCUT&RUN platform to build a comprehensive epigenomic database of mouse immune cells - composed of >1,500 epigenomic profiles from >100 different FACS-isolated primary immune cell types. These studies set the stage to leverage high-throughput epigenomics for multi-site clinical studies to identify and/or characterize novel biomarkers for precision medicine applications. Further, we will discuss our integration of CUT&RUN and Enzymatic Methylation-sequencing (CUT&RUN-EM) to deliver a true multiomic view of the co-occurrence of epigenomic features and DNA methylation, which can reveal multivalent cellular mechanisms and deconvolute sample heterogeneity.

Stress-Induced AMPK Interactome Analysis Reveals Novel Protein-Protein Interactions Associated with Chromatin Remodeling and Transcription Machinery in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. Survival rates for relapsed/refractory ALL remain dismal. We have previously reported that ALL cells are vulnerable to energy/metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and our lab and others have reported it interacts with chromatin-associated proteins to epigenetically regulate gene expression in response to energy/metabolic stress. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we previously used ChIP-seq and RNA-seq in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well-known AMPK activator, and found that (i) AMPK interacts with components of the transcription machinery including the TATA-Box Binding Protein Associated Factor (TAF) and RNA polymerase II, (ii) AMPKα2 was enriched for genes involved in histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization, (iii) AMPKa2 was recruited to promoter regions of the epigenetic regulators KMT2A and SETD1A, and (iv) the mRNA expression of KMT2A and SETD1A genes was upregulated in KASUMI-2 cells treated with AICAR. To further investigate AMPK's involvement in epigenetic mechanisms in response to energy/metabolic stress, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. AMPK is a highly conserved heterotrimeric kinase complex composed of catalytic α-subunit (α1 or α2) and regulatory β (β1 or β2) and γ (γ1, γ2, or γ3) subunits. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1 (which are the most abundant AMPK subunits expressed in ALL cells), and these stable cell lines were treated with the potent allosteric AMPK activator PF-06409577 for 1 hr, to mimic the cell's rapid response to energy/metabolic stress. Following characterization of these stable cell lines and small-scale optimization experiments, treated samples and controls were submitted to mass spectrometry analysis. Proteomics data analysis identified novel AMPK's protein-protein interactions (PPI) which were altered (enhanced or reduced) following PF-06409577 treatment. Among PPI altered by PF-06409577 treatment, 148 were enhanced and 144 reduced for AMPKα1, 169 enhanced and 172 reduced for AMPKα2, 250 enhanced and 188 reduced for AMPKβ1 and 289 enhanced and 153 reduced for AMPKγ1. For proteomics data visualization we used Cytoscape software. The GO analysis of proteins interacting with AMPKα1β1γ1 and/or AMPKα2β1γ1 heterotrimeric complexes in cells treated with PF-06409577 showed that energy/metabolic stress enhances their interaction with proteins involved in gene expression, cellular metabolic processes, chromatin organization, DNA repair, histone modifications, etc.. Using the STRING database, we performed MCL clustering analysis and uncovered a major cluster of known epigenetic regulators (e.g., histone modifiers such as KDM6A, HDAC2, SETD2, KMT2A and SETD1A etc.) and components of the transcription machinery (such as NELFE, POLR2A etc.) that interacted with AMPK under stress-induced conditions. These data support our previous findings uncovered by ChIP-seq and RNA-seq analysis. Consequently, our findings further validate an epigenetic role for AMPK via interactions with putative members of chromatin-associated and transcription machinery complexes and chromatin modifying enzymes that allow ALL cells to respond to conditions of energy/metabolic stress. To our knowledge, this is the first report describing the AMPK interactome in pediatric ALL. Further elucidation of the uncovered protein-protein interactions may lead to potential epigenetic-based targeted therapies to overcome therapeutic resistance in relapse/refractory ALL and other hematological malignancies.

Role of HMGB1 and its associated signaling pathways in human malignancies

The High-Mobility Group Box-1 (HMGB1), a non-histone chromatin-associated protein, plays a crucial role in cancer growth and response to therapy as it retains a pivotal role in promoting both cell death and survival. HMGB1 has been reported to regulate several signaling pathways engaged in inflammation, genome stability, immune function, cell proliferation, cell autophagy, metabolism, and apoptosis. However, the association between HMGB1 and cancer is complex and its mechanism in tumorigenesis needs to be further elucidated. This review aims to understand the role of HMGB1 in human malignancies and discuss the signaling pathways linked to this process to provide a comprehensive understanding on the association of HMGB1 with carcinogenesis. Further, we will review the role of HMGB1 as a target/biomarker for cancer therapy, the therapeutic strategies used to target this protein, and its potential role in preventing or treating cancers. In light of the recent growing evidence linking HMGB1 to cancer progression, we think that it may be suggested as a novel and emergent therapeutic target for cancer therapy. Hence, HMGB1 warrants paramount investigation to comprehensively map its role in tumorigenesis.