BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

BACKGROUND: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. RESULTS: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. CONCLUSIONS: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

Post-surgical ctDNA-based molecular residual disease detection in patients with stage I uterine malignancies.

INTRODUCTION: Among uterine malignancies, endometrial cancer (EC) is the most common cancer of the female reproductive tract. Traditionally, risk stratification in EC is determined by standard clinicopathological risk factors. Although circulating tumor DNA (ctDNA) has emerged as a prognostic biomarker in various malignancies, its clinical validity in EC remains to be established. METHODS: In this analysis of real-world data, 267 plasma samples from 101 patients with stage I EC were analyzed using a tumor-informed ctDNA assay (Signatera™ bespoke mPCR-NGS). Patients were followed post-surgically and monitored with ctDNA testing for a median of 6.8 months (range: 0.37-19.1). RESULTS: Patients who tested ctDNA-positive at both their first time point and longitudinally experienced inferior recurrence-free survival (RFS) (HR = 6.2; p = 0.0006 and HR = 15.5; p < 0.0001, respectively), and showed a recurrence rate of 58% and 52%, vs. 6% and 0%, respectively for the ctDNA-negative patients. Most ctDNA-positive patients had high-risk histologies or sarcoma, versus low-risk and high-intermediate risk (H-IR) EC. Furthermore, patients with high-risk histologies who were ctDNA-positive showed shorter RFS compared to those who tested negative (HR = 9.5; p = 0.007), and those who tested positive in the low/H-IR cohort (HR = 0.25; p = 0.04). Post-surgically, detectable ctDNA was highly prognostic of clinical outcome and remained the only significant risk factor for recurrence when adjusted for clinicopathological risk factors, such as histologic risk group, mismatch repair (MMR), and p53 status. CONCLUSION: Incorporating ctDNA monitoring along with traditional known risk factors may aid in identifying patients with stage I EC who are at highest risk of recurrence, and possibly aid in treatment stratification.

A Therapeutic Vaccine Targeting Rat BORIS (CTCFL) for the Treatment of Rat Breast Cancer Tumors.

Cancer testis antigens are ideal for tumor immunotherapy due to their testis-restricted expression. We previously showed that an immunotherapeutic vaccine targeting the germ cell-specific transcription factor BORIS (CTCFL) was highly effective in treating aggressive breast cancer in the 4T1 mouse model. Here, we further tested the therapeutic efficacy of BORIS in a rat 13762 breast cancer model. We generated a recombinant VEE-VRP (Venezuelan Equine Encephalitis-derived replicon particle) vector-expressing modified rat BORIS lacking a DNA-binding domain (VRP-mBORIS). Rats were inoculated with the 13762 cells, immunized with VRP-mBORIS 48 h later, and then, subsequently, boosted at 10-day intervals. The Kaplan-Meier method was used for survival analysis. Cured rats were re-challenged with the same 13762 cells. We demonstrated that BORIS was expressed in a small population of the 13762 cells, called cancer stem cells. Treatment of rats with VRP-BORIS suppressed tumor growth leading to its complete disappearance in up to 50% of the rats and significantly improved their survival. This improvement was associated with the induction of BORIS-specific cellular immune responses measured by T-helper cell proliferation and INFγ secretion. The re-challenging of cured rats with the same 13762 cells indicated that the immune response prevented tumor growth. Thus, a therapeutic vaccine against rat BORIS showed high efficacy in treating the rat 13762 carcinoma. These data suggest that targeting BORIS can lead to the elimination of mammary tumors and cure animals even though BORIS expression is detected only in cancer stem cells.

OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma.

PURPOSE: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. EXPERIMENTAL DESIGN: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. RESULTS: We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. CONCLUSIONS: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

Proteogenomic discovery of neoantigens facilitates personalized multi-antigen targeted T cell immunotherapy for brain tumors.

Neoantigen discovery in pediatric brain tumors is hampered by their low mutational burden and scant tissue availability. Here we develop a proteogenomic approach combining tumor DNA/RNA sequencing and mass spectrometry proteomics to identify tumor-restricted (neoantigen) peptides arising from multiple genomic aberrations to generate a highly target-specific, autologous, personalized T cell immunotherapy. Our data indicate that aberrant splice junctions are the primary source of neoantigens in medulloblastoma, a common pediatric brain tumor. Proteogenomically identified tumor-specific peptides are immunogenic and generate MHC II-based T cell responses. Moreover, polyclonal and polyfunctional T cells specific for tumor-specific peptides effectively eliminate tumor cells in vitro. Targeting tumor-specific antigens obviates the issue of central immune tolerance while potentially providing a safety margin favoring combination with other immune-activating therapies. These findings demonstrate the proteogenomic discovery of immunogenic tumor-specific peptides and lay the groundwork for personalized targeted T cell therapies for children with brain tumors.

The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis.

CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf+/+Boris+/+, Ctcf+/-Boris+/+, and Ctcf+/+Boris-/- males are fertile, Ctcf+/-Boris-/- (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are "toxic" to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.

Frondoside A Inhibits an MYC-Driven Medulloblastoma Model Derived from Human-Induced Pluripotent Stem Cells.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the MYC oncogene combined with p53 loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non-small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.

Germline microsatellite genotypes differentiate children with medulloblastoma.

BACKGROUND: The germline genetic events underpinning medulloblastoma (MB) initiation, and therefore the ability to determine who is at risk, are still unknown for the majority of cases. Microsatellites are short repeated sequences that make up ~3% of the genome. Repeat lengths vary among individuals and are often nonrandomly associated with disease, including several cancers such as breast, glioma, lung, and ovarian. Due to their effects on gene function, they have been called the "tuning knobs of the genome." METHODS: We have developed a novel approach for identifying a microsatellite-based signature to differentiate MB patients from controls using germline DNA. RESULTS: Analyzing germline whole exome sequencing data from a training set of 120 MB subjects and 425 controls, we identified 139 individual microsatellite loci whose genotypes differ significantly between the groups. Using a genetic algorithm, we identified a subset of 43 microsatellites that distinguish MB subjects from controls with a sensitivity and specificity of 92% and 88%, respectively. This microsatellite signature was validated in an independent dataset consisting of 102 subjects and 428 controls, with comparable sensitivity and specificity of 95% and 90%, respectively. Analysis of the allele genotypes of those 139 informative loci demonstrates that their association with MB is a consequence of individual microsatellites' genotypes rather than their hypermutability. Finally, an analysis of the genes harboring these microsatellite loci reveals cellular functions important for tumorigenesis. CONCLUSION: This study demonstrates that MB-specific germline microsatellite variations mark those at risk for MB development and suggests mechanisms of predisposition.

MYC Drives Group 3 Medulloblastoma through Transformation of Sox2+ Astrocyte Progenitor Cells.

A subset of group 3 medulloblastoma frequently harbors amplification or overexpression of MYC lacking additional focal aberrations, yet it remains unclear whether MYC overexpression alone can induce tumorigenesis and which cells give rise to these tumors. Here, we showed that astrocyte progenitors in the early postnatal cerebellum were susceptible to transformation by MYC. The resulting tumors specifically resembled human group 3 medulloblastoma based on histology and gene-expression profiling. Gene-expression analysis of MYC-driven medulloblastoma cells revealed altered glucose metabolic pathways with marked overexpression of lactate dehydrogenase A (LDHA). LDHA abundance correlated positively with MYC expression and was associated with poor prognosis in human group 3 medulloblastoma. Inhibition of LDHA significantly reduced growth of both mouse and human MYC-driven tumors but had little effect on normal cerebellar cells or SHH-associated medulloblastoma. By generating a new mouse model, we demonstrated for the first time that astrocyte progenitors can be transformed by MYC and serve as the cells of origin for group 3 medulloblastoma. Moreover, we identified LDHA as a novel, specific therapeutic target for this devastating disease. SIGNIFICANCE: Insights from a new model identified LDHA as a novel target for group 3 medulloblastoma, paving the way for the development of effective therapies against this disease.

Proteomic analysis of Medulloblastoma reveals functional biology with translational potential.

Genomic characterization has begun to redefine diagnostic classifications of cancers. However, it remains a challenge to infer disease phenotypes from genomic alterations alone. To help realize the promise of genomics, we have performed a quantitative proteomics investigation using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and 41 tissue samples spanning the 4 genomically based subgroups of medulloblastoma and control cerebellum. We have identified and quantitated thousands of proteins across these groups and find that we are able to recapitulate the genomic subgroups based upon subgroup restricted and differentially abundant proteins while also identifying subgroup specific protein isoforms. Integrating our proteomic measurements with genomic data, we calculate a poor correlation between mRNA and protein abundance. Using EPIC 850 k methylation array data on the same tissues, we also investigate the influence of copy number alterations and DNA methylation on the proteome in an attempt to characterize the impact of these genetic features on the proteome. Reciprocally, we are able to use the proteome to identify which genomic alterations result in altered protein abundance and thus are most likely to impact biology. Finally, we are able to assemble protein-based pathways yielding potential avenues for clinical intervention. From these, we validate the EIF4F cap-dependent translation pathway as a novel druggable pathway in medulloblastoma. Thus, quantitative proteomics complements genomic platforms to yield a more complete understanding of functional tumor biology and identify novel therapeutic targets for medulloblastoma.

Corrigendum: Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells.

This corrects the article DOI: 10.1038/srep41279.

Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells.

Despite sharing the same sequence specificity in vitro and in vivo, CCCTC-binding factor (CTCF) and its paralog brother of the regulator of imprinted sites (BORIS) are simultaneously expressed in germ cells. Recently, ChIP-seq analysis revealed two classes of CTCF/BORIS-bound regions: single CTCF target sites (1xCTSes) that are bound by CTCF alone (CTCF-only) or double CTCF target sites (2xCTSes) simultaneously bound by CTCF and BORIS (CTCF&BORIS) or BORIS alone (BORIS-only) in germ cells and in BORIS-positive somatic cancer cells. BORIS-bound regions (CTCF&BORIS and BORIS-only sites) are, on average, enriched for RNA polymerase II (RNAPII) binding and histone retention in mature spermatozoa relative to CTCF-only sites, but little else is known about them. We show that subsets of CTCF&BORIS and BORIS-only sites are occupied by several testis-specific transcriptional regulators (TSTRs) and associated with highly expressed germ cell-specific genes and histone retention in mature spermatozoa. We also demonstrate a physical interaction between BORIS and one of the analyzed TSTRs, TATA-binding protein (TBP)-associated factor 7-like (TAF7L). Our data suggest that CTCF and BORIS cooperate with additional TSTRs to regulate gene expression in developing male gametes and histone retention in mature spermatozoa, potentially priming certain regions of the genome for rapid activation following fertilization.

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions.

BACKGROUND: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation. RESULTS: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. CONCLUSIONS: We discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells.