Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15–25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy.

促纤维增生性小圆细胞肿瘤 (DSRCT) 是一种由 EWSR1-WT1 融合癌蛋白引起的侵袭性儿科癌症。该肿瘤难以治疗，5 年生存率仅为 15-25%，因此需要开发新的治疗方法，特别是能够针对化疗耐药亚群的治疗方法。新型体外癌症干细胞样（CSC样）培养条件可增加干细胞标记物（SOX2、NANOG）的表达，降低DSRCT细胞系对化疗的敏感性，同时保持DSRCT细胞形成异种移植物的能力。为了深入了解这种化疗耐药模型，我们进行了 RNA-seq 来阐明在 CSC 样球体中生长的 DSRCT 细胞与正常二维贴壁状态之间的转录变化。在通路分析中鉴定并利用了常见上调和下调的基因，揭示了与染色质组装和分解相关的通路的上调以及包括细胞连接组装和细胞外基质组织的通路的下调。染色质组装的改变表明表观遗传学在 DSRCT CSC 样状态中发挥作用，并使用 ATAC-seq 对其进行了进一步研究，识别了超过 10,000 个差异可及峰，包括 4444 个球体可及峰和 6,120 个粘附可及峰。可接近区域与较高的基因表达相关，包括在类 CSC 培养条件下 CSC 标记 SOX2 的可接近性增加。这些分析进一步用于确定潜在的 CSC 治疗靶点，从而确定 B 淋巴细胞激酶 (BLK) 作为富含 CSC 的、EWSR1-WT1 调节的可药物靶点。BLK 抑制和敲低降低了 CSC 样特性，包括消除肿瘤球形成和干性标记表达。重要的是，BLK 敲低降低了 DSRCT CSC 样细胞的化疗耐药性，使其抑制成为未来联合治疗的有希望的目标。