Aug 2026· Molecules and Cells· pp.
100391
· 0 citations
Medicine
TL;DR
Results indicate that targeted reactivation of BFL-1 and SQOR increases cell-death susceptibility in liver cancer cells, providing proof-of-concept for locus-specific epigenetic therapy in liver cancer.
Abstract
Liver cancer treatment with epigenetic drugs remains challenging because demethylating agents such as 5-azacytidine (5-AZA) induce genome-wide toxicity and may activate oncogenes. We hypothesized that a low, nontoxic dose of 5-AZA could prime liver cancer cells by partially relaxing chromatin at selected loci to restore silenced cell-death regulators. HepG2 cells treated with 2 μM 5-AZA underwent ATAC-seq and RNA-seq to identify genes with promoter opening and increased expression. Among ten candidates, BFL-1 and SQOR were prioritized for roles in cell death and redox control. Forced expression of either gene increased sensitivity to TNF-α/cycloheximide (CHX) and sorafenib, both of which elevated mitochondrial reactive oxygen species. To establish causality in a physiological context, we used CRISPR-dCas9-TET1 to demethylate CpG-rich promoter regions of BFL-1 or SQOR. Locus-specific editing sensitized cells to TNF-α/CHX more rapidly than conventional overexpression and reproduced the heightened death response elicited by low-dose 5-AZA without baseline toxicity. Analysis of the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas datasets showed consistent BFL-1 downregulation in liver cancer, variable SQOR expression across cancers, and positive correlations of both genes with tumor-suppression markers and immune-cell infiltration. These results indicate that targeted reactivation of BFL-1 and SQOR increases cell-death susceptibility in liver cancer cells. Integrating low-dose pharmacologic priming with precise epigenetic editing may preserve genome-wide methylation while restoring cell-death competence, providing proof-of-concept for locus-specific epigenetic therapy in liver cancer.
DNA methylation can function as a toxic alkylation reaction exploited by chemotherapeutic agents to induce cancer cell death. However, finely tuned DNA methylation plays a fundamental role in cellular physiology, particularly in the epigenetic regulation of gene expression. Once thought to act solely as a repressor of gene transcription, its functional role has since been elucidated as genomic locus-specific and deeply connected with other epigenetic factors. Following the clinical approval of DNA methyltransferase inhibitors, such as Azacitidine and Decitabine, for the treatment of hematological malignancies, considerable efforts have been devoted to developing pharmacological tools that modulate epigenetic DNA methylation. However, the lack of gene selectivity in these agents limits their therapeutic efficacy and increases off-target toxicity. Moreover, the non-gene-selective nature of current DNA methylation-targeting molecules fails to meet the standards required to discern the nuanced roles of DNA methylation across diverse pathophysiological contexts and genomic loci, particularly in an era where next-generation sequencing and omics technologies enable high-resolution epigenetic analyses. In this review, we examine the mechanisms and roles of DNA methylation in epigenetic regulation, evaluate the current landscape of DNA methylation modulators, from traditional DNMT inhibitors to cutting-edge CRISPR-dCas9 fusion systems and protein-protein interaction disruptors, and discuss their clinical relevance. Finally, we emphasize the need for precise, locus-specific tools to advance both cancer research and therapeutic strategies.
Julie Gilbert, Francesco Calzaferri· Chemical Research in Toxicol...· 0 citations
The anthracycline family, with its prime member doxorubicin, is one of the cornerstones in cancer chemotherapy and acts by poisoning topoisomerase II, resulting in DNA double-stranded breaks. One of its members, aclarubicin, is considered a distinct member of this family due to its inability to inflict DNA double-strand breaks. Despite this, aclarubicin is an effective anti-cancer drug by evicting histones, resulting in chromatin damage. How aclarubicin-induced chromatin damage induces cell death remains largely unknown. Here, we performed a genome-wide CRISPR screen to identify factors regulating sensitivity of cells to aclarubicin and identified p53 as a critical factor for cellular sensitivity. Even though aclarubicin does not induce DNA breaks, treatment resulted in swift activation of ATM independent of the MRN complex, which stabilized and activated p53, resulting in apoptosis and cell cycle arrest. Furthermore, response to aclarubicin treatment could reasonably be predicted by the p53-status of cell lines. These data suggest that ATM and p53 can be activated for apoptosis induction by at least two different pathways, DNA- and chromatin damage. Together, these data suggest p53 could be an important factor to stratify patients for treatment with Aclarubicin.
Sabina Y. van der Zanden, Koen Schipper, Merle A. van Gelder et al.· Cell Death & Disease· 0 citations
Pancreatic cancer remains at a stagnant 5-year survival of <13%, attributed to the high heterogeneity and plasticity of these tumors. To circumvent this, we focus on the abundant nuclear protein high mobility group-box protein 2 (HMGB2). HMGB2 depletion is key for establishing replicative senescence in normal cells, but it is significantly overexpressed across multiple cancer types. Here, we combine single-cell and spatial genomics with patient-derived organoids and tumor samples to show how increased HMGB2 availability represents a transcriptional addiction that fuels cell-cycle progression and growth. We repurpose a small molecule inhibitor targeting HMGB2 to interfere with its binding to DNA, restrict chromatin accessibility at promoters, and constrain tumor growth both in vitro and in vivo. 3D chromatin interactions involving HMGB2-bound enhancers and promoters also collapse upon drug treatment. Thus, pharmacological HMGB2 targeting represents a universal strategy for managing cancer progression irrespective of its genetic or molecular characteristics.
Adi Danieli-Mackay, Ioanna Papadionysiou, Markos Tsitsianopoulos et al.· Cell Reports· 0 citations
Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.
O. Yedier-Bayram, Elif Ayca Guvener, T. Bagci-Onder· Journal of Visualized Experi...· 0 citations
The effectiveness of T cell-based immunotherapy is limited due to T cell exhaustion, a state of impaired cytotoxicity, diminished cytokine production, and restricted proliferative capacity during chronic antigen exposure. Epigenetic modifications, such as the acquisition of repressive DNA methylation, sustain T cell exhaustion. Here, we investigate the role of hypomethylating agents in reversing T cell dysfunction via DNA demethylation. We hypothesize that using a selective inhibitor of DNA methyltransferase 1 (DNMT1i), GSK-3685032, can overcome the fate restrictions unique to terminally differentiated T-cells. Our preliminary data show that the fate and subset specificity of human and mouse T-cells can be epigenetically altered to mediate the reversal of repressive epigenetic programs.
Methylation profiling revealed that DNMT1i treatment results in robust loss of DNA methylation, notably at critical memory and stem-associated gene loci including TCF7 and LEF1.
Notably, DNMT1i-treated T cells that underwent division had decreased methylation coupled to heightened multipotency and plasticity. Single-cell transcriptomics post DNMT1i treatment reinforced our findings by revealing a unique subset of memory T cells that have homing potential and a long-lived memory gene signature.
Collectively, we describe an epigenetic approach that facilitates the reversal of repressive DNA methylation and are now using this approach to alter the fate of T cell subsets for immunotherapy.
R01CA237311
Lymphocyte Differentiation and Peripheral Maintenance (LYM)
A. Norman, Ben Youngblood, Caitlin C. Zebley· Journal of Immunology· 0 citations
Cisplatin (DDP), a core chemotherapeutic agent for osteosarcoma (OS), induces DNA cross‐linking to cause damage, yet the underlying regulatory mechanisms remain elusive. Our study has revealed that in DDP‐resistant osteosarcoma (OS) cells, both the expression level of the lysine acetyltransferase KAT8 and its mediated site‐specific acetylation of histone H4K16ac are significantly decreased. Using CUT&Tag technology and proteomic analysis, we demonstrated that the KAT8–H4K16ac axis modulates dynamic histone acetylation to epigenetically regulate DNA repair pathways. The biological inactivation of KAT8 results in a targeted decrease in the epigenetic mark H4K16ac at the promoter regions of genes associated with DNA repair, leading to diminished chromatin accessibility and inhibition of the p53 signaling pathway. Consequently, the DNA damage response is compromised, promoting cisplatin resistance in OS. In contrast, targeted restoration of H4K16ac reverses chemotherapy resistance through the reinstatement of chromatin dynamics and transcriptional activation of DNA repair programs. This hypothesis emphasizes the need to clarify the molecular mechanisms and signaling pathways associated with KAT8–H4K16ac, especially in relation to apoptotic pathways, which could offer new insights and justifications for treatment strategies in cisplatin‐resistant osteosarcoma.
Zhen-Qun Zhao, Guohui Yu, Shikui Wu et al.· Med Research· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
