Deciphering mechanisms of neuroblastoma drug resistance using single cell transcriptomics and developing targeted combination therapies for neuroblastoma

Abstract

Peripheral neuroblastic tumors (PNTs) represent a spectrum of tumors derived from the neural crest and account for 10% of paediatric malignancies. PNTs comprise four histological subtypes, including neuroblastoma (NB). Half of high-risk NB patients relapse after chemotherapy, and relapsed patients seldom respond to therapy afterward. This study aimed to identify chemoresistance mechanisms and effective combination therapies against chemoresistant NB. In the first results chapter, intratumoural heterogeneity in PNT subtypes were first investigated using single cell RNA sequencing (scRNA-seq) on 7 primary PNTs spanning all histological subtypes. This revealed extensive transcriptome heterogeneity among PNTs and identified malignant neuroblasts differentiating through a novel “transitional” state. This state resembled known chemoresistant cell phenotypes and predicted poor NB patient prognoses. In the second results chapter, drug resistance mechanisms in NB were explored in vitro using longitudinal IMR-32 cisplatin-resistant cell line models. Following scRNA-seq, resistant cells undergoing cytoskeletal remodelling were identified and then characterised in vitro. Additionally, tumour samples from 5 high-risk NB patients at diagnosis and post-chemotherapy were analysed with scRNA-seq, where an enrichment of oncogenic cell states was observed. In the third results chapter, potential combination therapies were identified using a combinatorial drug screen against the chemoresistant SK-N-BE(2)-C cell line. The highest synergy manifested between two histone methyltransferase inhibitors, GSK343 and SGC0946. Mechanistic studies revealed the induction of ER stress programs by the combination treatment. The administration of this combination therapy in vivo reduced NB tumour growth. In the fourth results chapter, an integrative approach was used to identify mitochondrial drug targets in NB, whereby the mitochondrial translocase SLC25A5 was prioritized. Targeting SLC25A5 with the selective inhibitor, PENAO, reduced cell viability in NB cell lines, however TP53 mutations conferred chemoresistance. The histone deacetylase inhibitor SAHA combined with PENAO overcame mutant TP53 mediated chemoresistance to synergistically reduce NB cell and tumour growth. Collectively, this thesis has identified transcriptional cell states that drive chemoresistance and combination therapies that overcome chemoresistant NB, thereby contributing novel therapeutic targets and treatment avenues for NB patients.