Specific activation of tumor suppressors repressed by oncogenes to treat cancer

肝细胞癌（HCC）是最常见的恶性肝癌类型，是全球癌症相关死亡的第二大常见原因。目前，HCC患者的治疗选择非常有限，驱动HCC形成的潜在机制也很少阐明。因此，迫切需要开发一种副作用很小或没有副作用的专门治疗方法。转录因子sallike protein 4 （SALL4）是一个很有前景的基因靶点，在包括HCC在内的大量实体癌中表达，而在健康成人组织中不表达。SALL4与核小体重塑和去乙酰化酶（NuRD）复合体的视网膜母细胞瘤结合蛋白4 （RBBp4）结合，从而抑制包括肿瘤抑制因子在内的各种基因，最终导致癌症。最近，研究表明，合成肽（FFW）破坏SALL4-RBBp4/NuRD相互作用可导致小鼠异种移植物中HCC生长下降，这表明它是HCC患者非常有希望的靶点。然而，由于RBBp4还存在于其他核复合物中，阻断RBBp4是一种非选择性的治疗方法。为了克服这一障碍，拟议的研究项目旨在特异性靶向SALL4-RBBp4/NuRD复合物特异性抑制肿瘤抑制因子的启动子。在该项目的第一部分，将验证受SALL4-RBBp4/NuRD复合物影响的最有希望的肿瘤抑制基因。因此，它们对HCC细胞的致瘤性的影响，如增殖和迁移，将在培养中进行测试。在第二部分中，SALL4-RBBp4/NuRD相互作用将被新的CRISPR/dCas9技术破坏，该技术位于第一部分中显示出强抗肿瘤作用的经过验证的肿瘤抑制因子的特定启动子处。通过设计特定的引导RNA并将dCas9蛋白与FFW肽融合，FFW将特异性地、专一地破坏预期启动子处的SALL4-RBBp4/NuRD复合物，而不是非特异性地破坏其他基因组位点。因此，这种特殊的肿瘤抑制因子将被重新激活。第三部分将分析激活抑瘤因子在HCC细胞中的抗肿瘤作用。最后，dCas9-FFW构建体治疗肝脏肿瘤的治疗潜力将在异种移植小鼠模型中进行体内研究。综上所述，本研究项目将针对sall4阳性HCC中下调的相关抑癌基因，为肝癌患者特异性激活被抑制的抑癌基因提供一种非常新颖和创新的技术。这代表了一种非常有前途的个性化癌症治疗策略。由于sall4阳性癌症的高丰度，这种策略还可以转移到各种其他类型的癌症。

Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer representing the second most common cause of cancer-related deaths in the world. Currently, the therapeutic options for HCC patients are highly limited and the underlying mechanisms driving HCC formation are only poorly elucidated. Therefore, there is an urgent need to develop a specialized treatment with little or no side effects. The transcription factor Sal-like protein 4 (SALL4) is a promising gene target that is expressed in a significant number of solid cancers, including HCC, and not in healthy adult tissues. SALL4 binds to the retinoblastoma binding protein 4 (RBBp4) of the nucleosome remodeling and deacetylase (NuRD) complex and thereby represses various genes, including tumor suppressors, eventually leading to cancer. Recently, it was demonstrated that disruption of the SALL4-RBBp4/NuRD interaction by a synthesized peptide (FFW) results in decreased HCC growth in murine xenografts, indicating that it represents a very promising target for HCC patients. However, since RBBp4 is additionally present in other nuclear complexes, blocking RBBp4 is a nonselective therapeutic approach. To overcome this obstacle, the proposed research project aims to specifically target the SALL4-RBBp4/NuRD complex at promoters of specific repressed tumor suppressors. In the first part of the project, the most promising tumor suppressor genes that are affected by the SALL4-RBBp4/NuRD complex will be validated. Therefore, their effect on the tumorigenic properties of HCC cells, such as proliferation as well as migration, will be tested in culture. In the second part, the SALL4-RBBp4/NuRD interaction will be disrupted with the novel CRISPR/dCas9 technology at the specific promoter of a validated tumor suppressor demonstrating strong antitumor effects in part one. By designing a specific guide RNA and fusing the dCas9 protein with the FFW peptide, FFW will specifically and exclusively disrupt the SALL4-RBBp4/NuRD complex at the intended promoter and not nonspecifically at other genomic sites. Consequently, this particular tumor suppressor will become reactivated. In the third part, the antitumor effect of the activated tumor suppressor will be analyzed in HCC cells. Finally, the therapeutic potential of the dCas9-FFW construct to treat liver tumors will be investigated in vivo in a xenograft mouse model. In summary, the proposed research project will target relevant tumor suppressors downregulated in SALL4-positive HCC, providing a very novel and innovative technique to specifically reactivate repressed tumor suppressors in HCC patients. This represents a very promising strategy for personalized cancer therapy. Due to the high abundance of SALL4-positive cancers, this strategy can additionally be transferred to a variety of other cancer types.