Epigenetic and Genetic Vulnerabilities in Synovial Sarcoma

摘要 RP-4 专注于滑膜肉瘤，这是一种由 SS18-SSX 驱动的侵袭性儿童/年轻成人肉瘤 融合癌基因。 SS18-SSX 已成为表观遗传控制的多方面破坏者，可介导 全基因组转录失调，导致增殖和异常或停滞 差异化。我们的总体目标是探讨滑膜肉瘤的基本病理学，从而提名 潜在的治疗靶点。我们提出假设驱动的筛选方法，即功能性筛选方法 基因组筛选以揭示染色质/转录调节因子之间的脆弱性 激酶，以及针对致癌融合本身。基于 SS18-SSX 导致的假设 为了使滑膜肉瘤细胞具有特殊的“表观遗传”依赖性，我们将在目标 1 中，（a）执行广泛的 使用针对表观遗传调节剂的新 CRISPR 敲除混合文库进行功能基因组筛选； (b) 寻求进一步验证和理解 KDM2B，这是在 初步研究，作为人类滑膜肉瘤细胞的脆弱性。基于以下假设 SS18-SSX 的另一个影响是生长信号的转录失调，目标 2 将采用三个 正交策略可以更好地定义这种肉瘤中的靶向激酶漏洞。具体来说，我们将 (a) 对人滑膜肉瘤细胞中的激酶进行基于 CRISPR 的功能基因组筛选 线路； (b) 通过患者来源的磷酸化蛋白分析来鉴定滑膜肉瘤中的活化激酶 异种移植物； (c) 通过两种互补方法确定帕唑帕尼治疗滑膜肉瘤的靶点， 亲和蛋白质组学和 PLATO（翻译 ORF 的并行分析）。这两个分析应该可以澄清 这种多靶点激酶抑制剂的关键激酶靶标在滑膜肉瘤的子集中具有活性 患者。该目标产生的结果将被整合起来，为更合理地瞄准目标奠定基础。 该肉瘤中的关键激酶。由于 SS18-SSX 是滑膜肉瘤的主要驱动癌基因， 融合转录本的连接点代表了基于序列的合理且高度特异性的目标 使用新的反义寡核苷酸（ASO）方法进行治疗。以我们有希望的成果为基础 使用gapmer ASO在体外和体内直接靶向其他肉瘤融合，我们将优化和 对照 SS18-SSX 评估 gapmer ASO。在目标 4 中，我们将通过以下方式验证目标 1-3 中确定的目标： 确认在人类肿瘤中的靶向作用和表达，我们将进行临床前评估 它们作为体外和体内治疗靶点的潜力。最有希望的目标将得到验证并 使用多重、正交、相关的体外和体内进行广泛的临床前评估 系统。这个根本性转化项目的最终总体目标是开始临床评估 至少一种基于我们在患有以下疾病的患者中工作中确定的新目标和策略的药物 这种肉瘤通常会在五年内致命。

ABSTRACT RP-4 focuses on synovial sarcoma, an aggressive pediatric/young adult sarcoma driven by the SS18-SSX fusion oncogene. SS18-SSX has emerged as a multi-faceted disruptor of epigenetic control that mediates genome-wide transcriptional deregulation, resulting in proliferation and aberrant or arrested differentiation. Our overall goal is to probe the basic pathobiology of synovial sarcoma so as to nominate potential therapeutic targets. We propose hypothesis-driven screening approaches, namely functional genomic screens to uncover vulnerabilities among chromatin/transcriptional regulators and among kinases, as well as targeting the oncogenic fusion itself. Based on the hypothesis that SS18-SSX causes synovial sarcoma cells to possess special “epigenetic” dependencies, we will, in Aim 1, (a) perform a broad functional genomic screen using a new CRISPR knock-out pooled library against epigenetic modulators; and (b) seek to further validate and understand mechanistically KDM2B, a dependency identified in preliminary studies, as a vulnerability in human synovial sarcoma cells. Based on the hypothesis that another effect of SS18-SSX is transcriptional deregulation of growth signaling, Aim 2 will employ three orthogonal strategies to better define targetable kinase vulnerabilities in this sarcoma. Specifically, we will (a) perform a CRISPR-based functional genomic screen against kinases in human synovial sarcoma cell lines; (b) identify activated kinases in synovial sarcoma by phospho-protein profiling of patient-derived xenografts; and (c) define the targets of pazopanib in synovial sarcoma by two complementary methods, affinity proteomics and PLATO (parallel analysis of translated ORFs). These two analyses should clarify the critical kinase targets of this multi-targeted kinase inhibitor that is active in a subset of synovial sarcoma patients. The results generated in this Aim will be integrated to form the basis for more rational targeting of critical kinases in this sarcoma. As SS18-SSX is the primary driver oncogene in synovial sarcoma, the junction point of the fusion transcript represents an rational and highly specific target for sequence-based therapeutics using new antisense oligonucleotide (ASO) approaches. Building on our promising results using gapmer ASOs to directly target other sarcoma fusions in vitro and in vivo, we will optimize and evaluate gapmer ASOs against SS18-SSX. In Aim 4, we will validate the targets identified in Aims 1-3 by confirming on-target effects and expression in human tumors, and we will perform preclinical evaluation of their potential as therapeutic targets in vitro and in vivo. The most promising targets will be validated and subjected to extensive preclinical evaluation using multiple, orthogonal, relevant in vitro and in vivo systems. The ultimate overall goal of this fundamentally translational project is to begin clinical evaluation of at least one agent based on the novel targets and strategies identified through our work in patients with this often lethal sarcoma within five years.