Identifying new driver genes in well differentiated retroperitoneal liposarcoma

Tyler, Robert William ORCID: 0000-0002-8771-158X (2022). Identifying new driver genes in well differentiated retroperitoneal liposarcoma. University of Birmingham. Ph.D.

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Abstract

Introduction: Well differentiated retroperitoneal liposarcoma (WDLS) has a high local recurrence rate, a poor response to neoadjuvant therapy and has the potential to become a high-grade dedifferentiated tumour (DDLS). Efforts to target the hallmark MDM2 and CDK4 amplification have been met with limited successes and led to efforts to target outside of this region. Preliminary work identified a possible somatic mutation (chr9: 68,303, 273A>T) in FOXD4L3. Secondly, a recurrent fusion within the TCA cycle gene SDHA was found. Both of these genes required further exploration, as well as a broader approach to identify new genes of interest.

Aims: Validate the presence of the somatic mutation (chr9: 68,303, 273A>T) in FOXD4L3 in a larger patient cohort. Demonstrate SDHA fusion in vitro and then explore SDHA functionality in vivo, using metabolic tracing. Identify novel driver genes in WDLS through the combination of CRISPR and single-cell RNA sequencing.

Methods: Nanopore sequencing of WDLS tissue samples was performed. Sanger sequencing was performed on both WDLS samples, as well as healthy controls. SDHA expression was examined using an SDHA fusion assay and Western blotting. To explore the in vivo function of SDHA, \(^{13}\)C\(_6\) metabolic tracing experiments were undertaken in four patients undergoing surgery for WDLS. To identify new targets in WDLS, a Genome wide CRISPR knockout (GeCKO) screen was performed on a WDLS cell line (93T449). To complement this, total RNA sequencing on paired tumour/normal samples was performed as well as single-cell RNA sequencing (scRNA-seq).

Results: The previously assumed somatic mutation in FOXD4L3 (chr9: 68,303, 273A>T)) was identified as a single nucleotide variant – rs7034645. In WDLS, rs7034645 was more highly prevalent with an odds ratio of 5.3, classifying this as a high-risk germline variant. Elevated expression levels of SDHA were observed in normal tissue compared to tumour. Metabolic tracing experiments and RNA sequencing suggested an impairment of SDHA function in WDLS, although with no convincing evidence of a complete loss of function attributable to gene fusions. Significant levels of aspartate and succinate were found in tumours shedding new light on their metabolic phenotype. Both the Wnt and mTOR signalling pathways were consistently implicated in the GeCKO screen, total RNA sequencing and scRNA-seq, which merit further analysis.

Conclusion: Rs7034645 is the first risk variant described in WDLS. Fusions within SDHA do not appear responsible for tumourigenesis, but there is evidence for a downregulation of SDHA both in the transcriptome and metabolome. Furthermore, tracing experiments offer the first insights into the metabolome of WDLS with reliance on aspartate worthy of future investigation. The Wnt and mTOR signalling pathways are possible drivers in WDLS, which demand further inquiry.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):