Seclidemstat blocks the transcriptional function of multiple FET-fusion oncoproteins

Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in several rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting pediatric, adolescent, and young and middle-aged adults, with prognosis depending on the fusion and whether the disease is localized, metastatic, or relapsed. New therapies are needed for these patients. These translocations fuse the 5' portion of the FET family gene with a 3' partner gene encoding a transcription factor. The resulting fusion proteins have the intrinsically disordered low complexity domain (LCD) of the FET protein paired with a DNA binding domain and these chimeras function as oncogenic transcription factors. FET fusion proteins have proven stubbornly difficult to target directly and promising new therapeutic strategies target the critical regulators and co-regulators of these proteins. One such protein is the histone lysine specific demethylase 1 (LSD1). LSD1 physically interacts with and is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 gene regulation and prior studies showed that treatment with the noncompetitive inhibitor SP-2509 blocked the transcriptional activity of the fusion. A similar molecule, seclidemstat (SP-2577), was identified as a promising lead for clinical development and is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against EWSR1::FLI1 or any other FET fusion protein has yet to be demonstrated. In this study, we sought to evaluate the in vitro potency of seclidemstat against multiple cell lines derived from different FET-rearranged sarcomas, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also defined the transcriptomic effect of seclidemstat treatment in these diseases and evaluated the activity of seclidemstat against FET fusion transcriptional regulation in multiple cell lines. Seclidemstat showed potent activity in cell viability assays in all four FET-rearranged sarcomas and disrupted the transcriptional function of all the tested fusion proteins. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas Overall design: To evaluate the pharmacologic effects of seclidemstat treatment on the transcriptomes of FET-rearranged sarcoma cell lines, we treated 9 cell lines expressing a variety of FET fusion oncogenes with either DMSO or the IC90 dose of seclidemstat for 48 hours, collected RNA, and performed RNA-seq. Additionally, to compare with another sarcoma without a FET-rearrangement, we treated 3 fusion positive rhabdomyosarcoma cell lines with 2 uM SP-2509. RNA from these samples was collected at 48 hours and analyzed by RNA-seq.

编码RNA结合蛋白FUS、EWSR1和TAF15（FET蛋白）的基因，参与多种罕见肉瘤的染色体易位过程。FET重排肉瘤多为侵袭性恶性肿瘤，好发于儿童、青少年及中青年群体，其预后取决于融合基因类型，以及疾病处于局限性、转移性还是复发状态。此类患者亟需全新的治疗方案。此类染色体易位会将FET家族基因的5'端区域，与编码转录因子的3'伴侣基因相融合。所形成的融合蛋白携带着FET蛋白固有无序的低复杂度结构域（LCD），并与DNA结合结构域配对，这类嵌合蛋白可作为致癌转录因子发挥功能。FET融合蛋白始终难以直接靶向，而颇具前景的新型治疗策略聚焦于这类蛋白的关键调控因子与共调控因子。其中一类靶标为组蛋白赖氨酸特异性去甲基化酶1（LSD1）。LSD1可与包括EWSR1::FLI1在内的多种FET融合蛋白发生物理相互作用，并被其招募至靶点。LSD1能够促进EWSR1::FLI1的基因调控活动，既往研究证实，使用非竞争性抑制剂SP-2509处理可阻断该融合蛋白的转录活性。同类分子seclidemstat（SP-2577）被鉴定为极具临床开发潜力的先导化合物，目前正针对FET重排肉瘤开展临床试验（NCT03600649）。然而，seclidemstat是否对EWSR1::FLI1或其他FET融合蛋白具有药理学活性，仍有待验证。本研究旨在评估seclidemstat对多种FET重排肉瘤来源细胞系的体外效力，涵盖尤文肉瘤、促结缔组织增生性小圆细胞肿瘤、透明细胞肉瘤及黏液样脂肪肉瘤。同时，我们明确了seclidemstat治疗上述疾病后的转录组学效应，并评估了seclidemstat对多种细胞系中FET融合转录调控的活性。Seclidemstat在四种FET重排肉瘤的细胞活力实验中均展现出强效活性，并可破坏所有被测融合蛋白的转录功能。尽管表观遗传抑制剂与靶向抑制剂单药治疗在临床中大概率难以取得理想效果，但本研究数据表明，seclidemstat仍是FET重排肉瘤患者颇具前景的新型治疗策略。整体实验设计：为评估seclidemstat处理对FET重排肉瘤细胞系转录组的药理学影响，我们将9株携带多种FET融合致癌基因的细胞系分别以二甲基亚砜（DMSO）或seclidemstat的IC90剂量处理48小时，收集RNA并进行RNA-seq。此外，为与无FET重排的其他肉瘤进行对照，我们将3株融合阳性横纹肌肉瘤细胞系以2 μM的SP-2509处理。上述样本的RNA均在处理48小时后收集，并通过RNA-seq完成分析。