SIRT6, a member of the NAD+-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9–Rad1–Hus1 (9–1–1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes. We show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9–1–1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres. Although SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells.

Cancer stem cells (CSCs) are cells within tumors that maintain the ability to self-renew, drive tumor growth, and contribute to therapeutic resistance and cancer recurrence. In this study, we investigate the role of Zinc finger and SCAN domain containing 4 (ZSCAN4) in human head and neck squamous cell carcinoma (HNSCC). The murine Zscan4 is involved in telomere maintenance and genomic stability of mouse embryonic stem cells. Our data indicate that the human ZSCAN4 is enriched for, marks and is co-expressed with CSC markers in HNSCC. We show that transient ZSCAN4 induction for just 2 days increases CSC frequency both in vitro and in vivo and leads to upregulation of pluripotency and CSC factors. Importantly, we define for the first time the role of ZSCAN4 in altering the epigenetic profile and regulating the chromatin state. Our data show that ZSCAN4 leads to a functional histone 3 hyperacetylation at the promoters of OCT3/4 and NANOG, leading to an upregulation of CSC factors. Consistently, ZSCAN4 depletion leads to downregulation of CSC markers, decreased ability to form tumorspheres and severely affects tumor growth. Our study suggests that ZSCAN4 plays an important role in the maintenance of the CSC phenotype, indicating it is a potential therapeutic target in HNSCC.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between ‘stemness’ and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

Head and neck squamous cell carcinoma (HNSCC) originates in the epithelial lining of the oral cavity, pharynx, and larynx, with over 830,000 new cases diagnosed globally in 2020, making it the seventh most prevalent cancer. Despite treatment advances, high-grade HNSCCs remain associated with poor outcomes and a high risk of recurrence. Although Cancer Stem Cells (CSCs) are rare in HNSCC tumors, they are key drivers of tumor relapses, as they evade apoptosis and survive current therapies through enhanced DNA repair and quiescence. This review integrates recent discoveries into a unified framework for understanding CSC mechanisms. It examines the role of pluripotency factors, biomarkers, replicative immortalization, metabolic reprogramming, redox regulation, and immune evasion in shaping CSC behavior and survival under treatment stress. Non-coding RNAs are also discussed as modulators of gene expression via epigenetic regulation in CSCs. Importantly, it highlights how these adaptive mechanisms intersect as potential vulnerabilities that could be exploited to eliminate CSCs through targeting multiple pathways. Finally, it provides recent and emerging precision therapeutics, including CAR-T cells, immune checkpoint inhibitors, metabolic and redox-targeting agents, and epigenetic modulators currently in preclinical and clinical trials applications designed to eliminate CSCs and improve patient outcomes in HNSCC.

Telomeres are a unique structure of DNA repeats covered by proteins at the ends of the chromosomes that protect the coding regions of the genome and function as a biological clock. They require a tight regulation of the factors covering and protecting their structure, as they are shortened with each cell division to limit the ability of cells to replicate uncontrollably. Additionally, they protect the chromosome ends from DNA damage responses and thereby, prevent genomic instability. Telomere dysfunction can lead to chromosomal abnormalities and cancer. Therefore, dysregulation of any of the factors that regulate the integrity of the telomeres will have implications to chromosomal stability, replicative lifespan and may lead to cell transformation. This review will cover the main factors participating in the normal function of the telomeres and how these are regulated by the ubiquitin and SUMO systems. Accumulating evidence indicate that the ubiquitin and SUMO pathways are significant regulators of the shelterin complex and other chromatin modifiers, which are important for telomere structure integrity. Furthermore, the crosstalk between these two pathways has been reported in telomeric DNA repair. A better understanding of the factors contributing to telomere biology, and how they are regulated, is important for the design of new strategies for cancer therapies and regenerative medicine.