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Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell
Justin W Magrath1, Dane A Flinchum1, Alifiani B Hartono1,2
1Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA, USA.
Abstract:
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.
Insights
Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer. Targeting B-lymphocyte kinase (BLK) shows promise for overcoming chemoresistance in DSRCT cancer stem cells.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer driven by the EWSR1-WT1 fusion oncoprotein.
- Current treatments are largely ineffective, with a low 5-year survival rate, highlighting the need for novel therapeutic strategies.
- Cancer stem cell-like (CSC-like) models of DSRCT exhibit increased chemoresistance and stemness marker expression.
Purpose of the Study:
- To investigate the transcriptional and epigenetic alterations in DSRCT cells cultured under CSC-like conditions.
- To identify novel therapeutic targets within chemoresistant DSRCT CSC-like cells.
- To evaluate the therapeutic potential of targeting identified CSC-enriched pathways.
Main Methods:
- RNA sequencing (RNA-seq) to analyze gene expression differences between CSC-like spheres and adherent DSRCT cells.
- ATAC-seq to identify accessible chromatin regions and epigenetic modifications.
- Pathway analysis to elucidate biological processes altered in CSC-like DSRCT.
- Functional assays to assess the impact of targeting B-lymphocyte kinase (BLK).
Main Results:
- RNA-seq revealed significant alterations in chromatin assembly/disassembly and cell junction pathways.
- ATAC-seq identified over 10,000 differentially accessible genomic regions, including near the SOX2 gene.
- B-lymphocyte kinase (BLK) was identified as a druggable, CSC-enriched target regulated by EWSR1-WT1.
- BLK inhibition or knockdown reduced CSC-like properties and chemoresistance in DSRCT cells.
Conclusions:
- Epigenetic dysregulation, particularly in chromatin remodeling, plays a crucial role in the DSRCT CSC-like state.
- BLK is a promising therapeutic target for overcoming chemoresistance in DSRCT.
- Targeting BLK may enhance the efficacy of combination therapies for DSRCT.
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