Functional Genomic Screens to Elucidate Alternative Splicing and Circular RNA Biogenesis Regulators Driving Neuroendocrine Cancer Progression

Abstract

Cancer progression remains a major clinical challenge, with treatment resistance and recurrence serving as continuing hurdles to overcome. Neuroendocrine (NE) cancers, such as small cell lung carcinomas (SCLC), represent a highly lethal subset of tumors, in which progression can result in rapid proliferation and metastasis, through the expression of NE signalling hormones. Different genetic regulatory networks are implicated in this progression, although the full complexity remains poorly understood. Regulated RNA processing via alternative splicing (AS) and circRNA biogenesis are widely acting levels of regulation, despite being historically overlooked. Recent studies have identified the role of AS and circRNA regulators, such as RNA binding proteins (RBPs), in driving NE cancer progression. To systematically identify novel regulators and directly link them with target RNA events, functional genomic screens such as “Systematic Parallel Analysis of endogenous RNA regulation coupled to barcode Sequencing" (SPAR-Seq) can be employed. This study sought to develop and optimize a new circRNA event panel, coupled to siRNA knockdown treatments for extending SPAR-Seq screen applications. Pilot screens in an SCLC cell line confirmed successful amplification and barcoding through gel electrophoresis and identified changes in circRNA regulation upon siSOX2 knockdown. Further focused experiments also identified SFPQ and PTBP2 as regulators of key NE-associated AS events, establishing them as new RBPs of interest for future SPAR-seq screens. Single-cell profiling was also used as a tool for SPAR-seq lead prioritization. scPipeline was used on published and in-house generated single-cell datasets, exploring candidate RBPs in a cell-specific manner, associating them with NE cancer markers. Together, these provide novel integrated approaches for the discovery of master regulators of AS and circRNA in NE cancer.