Regulation of CTCF Functions and Target Sites by Cancer/Testis-specific CTCF Like BORIS Factor

National Institute Of Allergy And Infectious Diseases

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Abstract

BORIS (CTCFL) is a paralog of CTCF, the global organizer of three-dimensional genome architecture. While CTCF is ubiquitously expressed, BORIS expression is restricted to germ cells and cancer cells, making it a cancer-testis gene. Our research focuses on developing tools to study the functions of both factors. Knockout studies revealed that CTCF is essential for embryonic survival, whereas BORIS plays a critical role in spermatogenesis, with Boris-/- mice showing sub-fertility and multiple spermatogenic defects. Gene expression profiling demonstrated that BORIS activates testis-specific genes such as Gal3st1 and FerT by binding to intronic CTCF sites, both of which are also aberrantly activated in cancers expressing BORIS. Genome-wide mapping with ChIP-seq, combined with RNA-seq, deep-CAGE-seq, and ENCODE data, showed that aberrant BORIS expression in cancer cells reprograms intronic CTCF sites to activate testis-specific genes. Conservation analysis confirmed that more than 80% of BORIS sites are shared between humans and mice, supporting similar genome-wide occupancy in both human cancer cell lines and mouse spermatids. Mechanistically, while single CTCF sites remain CTCF-only, dual closely spaced motifs (2xCTS) allow BORIS recruitment, forming CTCF–BORIS heterodimers that disrupt normal CTCF functions at cancer-testis genes, tumor suppressors, and oncogene promoters. These findings were validated in human neuroblastoma tumors resistant to ALK inhibitor therapy (Nature, 2019), where cells shifted proliferation dependence from MYCN to BORIS, accompanied by BORIS enrichment at 3D DNA-loop anchoring points. We further demonstrated that intronic CTCF sites can be reprogrammed into alternative transcription start sites through BORIS-mediated chromatin remodeling, and ongoing studies aim to identify BORIS protein partners and chromatin remodelers. RNA-seq after BORIS knockdown revealed widespread activation of primate-specific SVA retrotransposons, suggesting BORIS acts as a germline defense factor. Comparative analysis of repeat elements showed CTCF-only sites enriched in ancient inactive repeats, CTCF & BORIS sites in tandem repeats, and BORIS-only sites in young active SVA repeats. This indicates an evolutionary adaptation of BORIS to repress transposition-prone elements. BORIS has also emerged as a powerful immunotherapy target since it is expressed in cancer stem cells (CSCs), which drive recurrence, metastasis, and therapy resistance. Silencing BORIS induces CSC death, and in preclinical breast cancer models, BORIS-based dendritic cell immunotherapy reduced tumor growth and metastasis, with up to 20% of animals becoming tumor-free and ~50% metastasis-free, compared to 100% lethality in controls. An alphavirus-based BORIS vaccine also eradicated tumors in 50% of animals. Building on this, we developed UVAX-002, an autologous dendritic cell vaccine transfected with adenovirus-BORIS, with clinical trial protocols already approved for patient treatment. Collectively, these findings demonstrate that BORIS is not only a biomarker but also a driving force of cancer stemness and given its inclusion on the NCI Translational Working Group’s list of high-priority tumor-associated antigens, it represents both a fundamental player in genome regulation and a highly promising therapeutic target.