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

癌症/睾丸特异性 CTCF 样 BORIS 因子对 CTCF 功能和靶位点的调节

• 批准号: 10272128
• 负责人: Victor Lobanenkov
• 金额: $ 85.59万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272128
• 关键词: 3-Dimensional; 4T1; ATAC-seq; Adenoviruses; Affect; Alphavirus; Animals; Antigens; Attention; Autologous; Binding; Binding Sites; Breast Cancer Model; Brothers; CRISPR/Cas technology; Cancer Patient; Cancer cell line; Cell Maintenance; Cells; Cessation of life; ChIP-seq; Chromatin; Chromatin Loop; Chromatin Remodeling Factor; Clinic; Clinical trial protocol document; Control Animal; Coupled; DNA; DNA Binding; DNA Binding Domain; Data; Defect; Dendritic Cells; Dependence; Disease; Distant; ERBB2 gene; Elements; Embryo; Endogenous Retroviruses; Epigenetic Process; FDA approved; Family; Gametogenesis; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genome; Genomic Segment; Germ Cells; Goals; Housekeeping; Human; Immune; Immunize; Immunological Diagnosis; Immunotherapeutic agent; Immunotherapy; In Vitro; Journals; K-562; K562 Cells; Knock-out; Knockout Mice; Lead; Light; Link; MCF7 cell; MYCN gene; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of testis; Maps; Mediating; Meiosis; Methods; Mitotic; Modeling; Molecular; Mus; Mutate; NIH 3T3 Cells; NIH Mouse; Names; Nature; Neoplasm Metastasis; Neuroblastoma; Oncogenic; Organ; Outcome; Pathologic; Patients; Peripheral Blood Mononuclear Cell; Phenotype; Play; Population; Primates; Protein Isoforms; Proteins; Protocols documentation; Rattus; Recombinants; Recurrence; Regulation; Regulatory Element; Reporting; Research; Resistance; Retrotransposon; Role; Side; Site; Somatic Cell; Specificity; Spermatids; Spermatogenesis; Structure; Tandem Repeat Sequences; Testing; Testis; Tissues; Transcript; Transcription Initiation Site; Transcriptional Activation; Translating; Translational Research; Tumor Suppressor Genes; Ukraine; Undifferentiated; Up-Regulation; Ursidae Family; Vaccines; base; cancer cell; cancer immunotherapy; cancer site; cancer stem cell; cancer survival; cancer therapy; cell type; chemotherapy; chromatin immunoprecipitation; chromatin remodeling; clinical application; design; disease diagnosis; driving force; experimental study; gene product; genome-wide; human model; humanized mouse; immunogenic; imprint; improved; in vivo; inhibitor/antagonist; knock-down; knockout animal; male; malignant breast neoplasm; mouse model; next generation sequencing; novel; overexpression; paralogous gene; pre-clinical; programs; promoter; recruit; senescence; stem-like cell; stemness; subfertility; therapy resistant; tissue culture; tool; transcriptome sequencing; treatment site; tumor; tumor growth; virtual; working group; zinc finger nuclease

BORIS is a paralog of CTCF, the global three-dimensional genome organizer. While CTCF is ubiquitously expressed, BORIS expression is restricted to germ and cancer cells, making BORIS as a cancer-testis gene. Our research on CTCF and BORIS is focused on developing appropriate tools to study the functions of both factors. First, we generated knockout animals for the two factors and found that CTCF is essential for survival of the embryo, while BORIS is involved in normal spermatogenesis. BORIS-/- mice show a sub-fertility phenotype and multiple defects in spermatogenesis. Gene expression profiling of BORIS-/- mice uncovered a role for BORIS in transcriptional activation of many testis-specific genes during spermatogenesis. In particular, expression of Gal3st1 and FerT testes-specific isoforms is induced by BORIS binding to intronic CTCF sites. Interestingly, both transcripts are products of genes that are male germ cell-specific homologs of genes expressed in somatic cells and both transcripts are aberrantly activated in cancer cells, coinciding with BORIS expression. To extend these two examples to a genome-wide scale, we recently performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) to map both CTCF and BORIS vinding sites in several cancer cell lines. Combining our ChIP-Seq, RNA-seq, and deep-CAGE-seq data with ENCODE data, we found that an aberrant expression of BORIS in cancer cells results in activation of multiple testis-specific genes from intronic CTCF binding sites. The interspecies conservation analysis of BORIS sites revealed the high conservation of over 80% of all sites between human and mice, allowing us to confirm a similar BORIS genome-wide occupancy in both human cancer cell lines and mouse round spermatids. Moreover, our data suggest that although single CTCF sites are always bound by CTCF-only (in all cells types), normal CTCF function gets lost upon recruitment of BORIS to CTA-genes as well as to many house-keeping tumor suppressor genes and anti-death pro-oncogenic gene' promoters through de novo formation of BORIS and CTCF heterodimers driven by spatial proximity at 2xCTS-containing reg. DNA elements bound simultaneously by two adjacent paralogous 11 ZF DNA-bindig domains. Remarkably, this and our other major conclusions on aberrant chromatin regulatory interactions in BORIS-positive cancer cell lines have been convincingly confirmed in actual human neuroblastoma tumors with ALK-mutated & MYCN-amplified cells that gained resistance against chemotherapy with TAE684 ALK inhibitor through the initial loss of MYCN expression followed by subsequent activation and increasing over-expression of BORIS leading to the concomitant switch in proliferation dependence from MYCN to BORIS as described recently in a prestigious journal (Nature, Aug 2019) available at https://doi.org/10.1038/s41586-019-1472-0 online. Notably, aberrantly activated BORIS in the treatment-resistant cancer cells of real human neuroblastoma patients has been found to be enriched at 3D DNA-looping anchoring-points mapped with BORIS- vs. CTCF- specific ChIP_Seq and Hi-C methods within de-regulated genomic regions defined, as expected, by the same dual 2xCTS rather that single 1xCTS elements that we have previously mapped in a number of human cancer lines with high BORIS levels, including undifferentiated K562 and OvCa cells. We also recently confirmed that intronic CTCF sites could be reprogrammed into alternative transcription start sites via BORIS occupancy through epigenetic remodeling of chromatin. To identify the molecular mechanisms by which BORIS activates alternative transcription from intronic promoters, we plan to analyze what protein-partners are recruited by BORIS to initiate transcription and what kind of chromatin remodeling factors are involved in the activation of intronic class of Transcription Start Sites (TSSs) and whether any CTCF-assisted chromatin looping could be also involved by such TSS as well. RNA-seq following BORIS knockdown revealed a widespread upregulation of SVA repeat expression, suggesting that BORIS acts as a repressor of SVA transcription. Given that SVA repeats are primate-specific, these observations suggest that germline-restricted BORIS continued to evolve after the divergence of the primate lineage, acquiring a specific function in germline defense. A deeper analysis of CTCF and BORIS binding to a vast majority of currently known mouse and human repetitive DNA elements also revealed a striking distinction between repeat-contained CTCF-only, CTCF & BORIS, and BORIS-only sites. CTCF-only sites were enriched in evolutionarily ancient and inactive types of repeats, while CTCF & BORIS sites were mainly located in many other tandem repeats. In contrast, BORIS-only sites were found primarily within the evolutionarily young SVA class of repeats. SVA elements are primate specific, active retrotransposons, and so their uncontrolled activity presents a threat to the stability of the germline. Generation of BORIS humanized mice would provide an ideal model to test direct link between normal BORIS expression transposition-prone repeats including human-specific HERV and SVA families. Furthermore, CTCFL/BORIS has recently received additional attention as a very attractive immunotherapy target because it was found expressed in cancer stem cells (CSC) in vivo and in CSC-like side populations of cancer cell lines in tissue culture. Since SCS are believed to drive tumor growth recurrence, metastasis, and treatment resistance, while CTCFL/BORIS silencing lead to senescence and death of CSC, it appears therefore that our immunotherapeutic strategy that targets CTCFL/BORIS may lead to the selective destruction of CSC and potential eradication of metastatic disease. Indeed, the high immunotherapeutic potential of CTCFL/BORIS was shown in the same FDA-approved stringent 4T1 mouse model of human breast cancer that was used for pre-clinical experiments designed to develop the HER2-based immunotherapy that has been eventually approved by the FDA. Using these highly metastatic, poorly immunogenic carcinoma cells inoculated into Th2 prone mice we showed that Dendritic Cells (DC) fed with recombinant CTCFL/BORIS as immune antigen inhibited tumor growth and reduced metastases numbers in distant organs. While about 20% of CTCFL/BORIS immunized animals become tumor-free, approximately 50% (i.e., every second one) of the BORIS-immunized animals remained metastasis-free. Hence, at least the same widely used rat model of breast cancer showed that alphavirus-based CTCFL/BORIS immunotherapy (reviewed recently by D. Loukinov) was capable of cancer elimination as we were able literally cure 50% of animals compared to a 100% lethal outcome with untreated control animals. Based on the above data we believe that our most recently advanced and currently ongoing attempt of translating CTCFL/BORIS targeting immunotherapeutic strategies to the clinic will succeed to provide new avenues for improving survival of cancer patients with advanced metastatic disease driven by a therapy-resultant cell population of BORIS-expressing CSCs also known as Cancer Stem Cells. The above data suggest that BORIS is likely to be not only a marker, but also a driving force for the cancer stemness phenotype. Notably, BORIS has been included in the list of high priority TAAs generated by the NCI Translational Working Group (NCI TWG) 91. As a proof of principle two animal breast cancer models have been successfully tested. On this basis, we developed UVAX-002, an autologous cellular vaccine comprising peripheral blood mononuclear cell-derived DC transfected with adenovirus-containing BORIS. The clinical trial protocol was already approved in the Ukraine by a Coordinating Center for extra-corporal cell maintenance and returning DCs to real patients

BORIS 是全球三维基因组组织者 CTCF 的旁系同源。 CTCF 普遍表达，而 BORIS 表达仅限于生殖细胞和癌细胞，这使得 BORIS 成为一种癌症睾丸基因。我们对 CTCF 和 BORIS 的研究重点是开发适当的工具来研究这两个因素的功能。首先，我们培育了这两个因子的基因敲除动物，发现 CTCF 对于胚胎的存活至关重要，而 BORIS 则参与正常的精子发生。 BORIS-/- 小鼠表现出生育力低下的表型和精子发生的多种缺陷。 BORIS-/- 小鼠的基因表达谱揭示了 BORIS 在精子发生过程中许多睾丸特异性基因转录激活中的作用。特别是，Gal3st1 和 FerT 睾丸特异性亚型的表达是由 BORIS 与内含子 CTCF 位点结合诱导的。有趣的是，这两个转录本都是与体细胞中表达的基因雄性生殖细胞特异性同源的基因的产物，并且这两个转录本在癌细胞中异常激活，与 BORIS 表达一致。为了将这两个例子扩展到全基因组范围，我们最近进行了染色质免疫沉淀与下一代测序 (ChIP-seq) 相结合，以绘制几种癌细胞系中 CTCF 和 BORIS 结合位点的图谱。将我们的 ChIP-Seq、RNA-seq 和 deep-CAGE-seq 数据与 ENCODE 数据相结合，我们发现癌细胞中 BORIS 的异常表达会导致内含子 CTCF 结合位点的多个睾丸特异性基因激活。 BORIS位点的种间保守性分析揭示了人类和小鼠之间超过80%的所有位点的高度保守性，这使我们能够确认人类癌细胞系和小鼠圆形精子细胞中相似的BORIS全基因组占有率。此外，我们的数据表明，虽然单个 CTCF 位点总是仅与 CTCF 结合（在所有细胞类型中），但在将 BORIS 招募到 CTA 基因以及许多管家肿瘤抑制基因和抗肿瘤基因后，正常的 CTCF 功能就会丧失。 -通过由包含 2xCTS 的 reg 的空间邻近性驱动的 BORIS 和 CTCF 异二聚体的从头形成来启动死亡原癌基因的启动子。 DNA 元件同时与两个相邻的旁系同源 11 ZF DNA 结合结构域结合。 值得注意的是，这一结论以及我们关于 BORIS 阳性癌细胞系中染色质调控相互作用异常的其他主要结论，已在具有 ALK 突变和 MYCN 扩增细胞的实际人类神经母细胞瘤肿瘤中得到令人信服的证实，这些细胞通过最初的 TAE684 ALK 抑制剂获得了对化疗的耐药性。 MYCN 表达缺失，随后 BORIS 激活和过度表达增加，导致增殖依赖性从 MYCN 转变为 BORIS，正如最近在著名的一篇著名杂志中所描述的那样期刊（《自然》杂志，2019 年 8 月）可在线访问 https://doi.org/10.1038/s41586-019-1472-0。值得注意的是，在真正的人类神经母细胞瘤患者的治疗耐药性癌细胞中，异常激活的 BORIS 被发现在 3D DNA 环锚定点处富集，这些锚定点是在解除管制的情况下使用 BORIS 与 CTCF 特异性 ChIP_Seq 和 Hi-C 方法绘制的正如预期的那样，基因组区域由相同的双 2xCTS 而不是单 1xCTS 元件定义，我们之前在许多具有高 BORIS 水平的人类癌症系（包括未分化的癌症系）中绘制了图谱K562 和 OvCa 细胞。 我们最近还证实，通过染色质的表观遗传重塑，内含子 CTCF 位点可以通过 BORIS 占据重新编程为替代转录起始位点。为了确定 BORIS 从内含子启动子激活替代转录的分子机制，我们计划分析 BORIS 招募哪些蛋白质伙伴来启动转录，以及哪些染色质重塑因子参与内含子类转录起始位点的激活。 TSS）以及此类 TSS 是否也涉及任何 CTCF 辅助的染色质环化。 BORIS 敲低后的 RNA-seq 揭示了 SVA 重复表达的广泛上调，表明 BORIS 充当 SVA 转录的抑制因子。鉴于SVA重复是灵长类动物特异性的，这些观察结果表明，种系限制的BORIS在灵长类谱系分化后继续进化，获得种系防御中的特定功能。 对 CTCF 和 BORIS 与目前已知的绝大多数小鼠和人类重复 DNA 元件结合的更深入分析也揭示了仅重复包含 CTCF、CTCF & BORIS 和仅 BORIS 位点之间的显着区别。仅 CTCF 位点在进化上古老且不活跃的重复类型中富集，而 CTCF 和 BORIS 位点主要位于许多其他串联重复中。相比之下，只有 BORIS 的位点主要发现于进化上年轻的 SVA 重复序列中。 SVA 元件是灵长类动物特异性的、活跃的逆转录转座子，因此它们不受控制的活性对种系的稳定性构成威胁。 BORIS 人源化小鼠的产生将提供一个理想的模型来测试正常 BORIS 表达易转座重复序列（包括人类特异性 HERV 和 SVA 家族）之间的直接联系。 此外，CTCFL/BORIS 最近作为一个非常有吸引力的免疫治疗靶点受到了额外的关注，因为它被发现在体内的癌症干细胞 (CSC) 和组织培养中的癌细胞系的 CSC 样侧群中表达。由于SCS被认为会导致肿瘤生长、复发、转移和治疗耐药，而CTCFL/BORIS沉默会导致CSC衰老和死亡，因此，我们针对CTCFL/BORIS的免疫治疗策略可能会导致CSC和CSC的选择性破坏。潜在根除转移性疾病。事实上，CTCFL/BORIS 的高免疫治疗潜力在 FDA 批准的严格 4T1 人类乳腺癌小鼠模型中得到了体现，该模型用于临床前实验，旨在开发基于 HER2 的免疫疗法，该疗法最终已获得 FDA 批准。将这些高转移性、低免疫原性的癌细胞接种到 Th2 易感小鼠中，我们发现，用重组 CTCFL/BORIS 作为免疫抗原喂养的树突状细胞 (DC) 可抑制肿瘤生长并减少远处器官的转移数量。虽然大约 20% 的 CTCFL/BORIS 免疫动物没有肿瘤，但大约 50%（即每隔一个）的 BORIS 免疫动物仍然没有转移。因此，至少同样广泛使用的乳腺癌大鼠模型表明，基于甲病毒的 CTCFL/BORIS 免疫疗法（最近由 D. Loukinov 审查）能够消除癌症，因为我们实际上能够治愈 50% 的动物，而 100% 的动物则被治愈。未经治疗的对照动物的致死结果。基于上述数据，我们相信，我们最近和目前正在进行的将 CTCFL/BORIS 靶向免疫治疗策略转化为临床的尝试将成功地为改善由治疗产生的细胞驱动的晚期转移性疾病癌症患者的生存提供新途径表达 BORIS 的 CSC 群体也称为癌症干细胞。 上述数据表明，BORIS很可能不仅是一个标志物，而且是癌症干性表型的驱动力。值得注意的是，BORIS 已被纳入 NCI 转化工作组 (NCI TWG) 91 生成的高优先级 TAA 列表中。作为原理证明，两种动物乳腺癌模型已成功进行测试。在此基础上，我们开发了UVAX-002，一种自体细胞疫苗，包含转染含有腺病毒的BORIS的外周血单核细胞来源的DC。该临床试验方案已在乌克兰获得体外细胞维护和将树突状细胞返还给真实患者协调中心的批准