Exome-scale CRISPR screening reveals master controllers of cell state maintenance in high-risk neuroblastoma

A major limitation in cancer treatment is chemoresistance, particularly at relapse. Previously, relapse was thought to be driven by chemoresistance arising from new acquired genetic mutations; however, emerging evidence indicates that cancer cells exploit non-genetically-driven processes such as epigenetically regulated transcriptional plasticity to drive chemoresistance, invasion and proliferation. High-risk neuroblastoma (NB) demonstrates transcriptional plasticity with defined cell states and low mutational burden. NB cells exist primarily in two distinct states: a chemosensitive adrenergic state (ADRN) and a less common drug-tolerant persister cell state, the mesenchymal (MES) state. MES cells are enriched at relapse, suggesting that cells switch to this state under therapeutic pressure. Despite a myriad of approaches to treating high risk NB, relapsed patients have exceptionally poor survival. Unfortunately, mechanisms maintaining cell state and permitting state switching in NB are poorly understood. We hypothesize that dissection of the pathways promoting NB cell state plasticity will reveal new approaches to drive cell state interconversions that facilitate chemosensitivity. Thus, here, we took advantage of a recently developed novel fluorescent reporter system of the NB MES cell state developed by the Durbin Lab, to perform whole exome-CRISPR-cas12 knockout screening and identify master controllers of cell state maintenance in ADRN and MES-dominant cell lines. Integrated pathway analysis demonstrated conserved pathway modules involved in maintenance of cells in distinct cell states. To validate these findings, we performed targeted CRISPR knockouts in reporter carrying cells and identified changes in chemoresistance by chemosensitivity assays. To profile the effects of chemotherapy directly on cell state maintenance, we performed fluorescence reporter assays, followed by RNAseq to confirm changes in target loss nominated from CRISPR screens. Cell state switching is an intriguing paradigm by which NB cells may evade conventional therapies. We have identified pathways playing a role in maintaining either the ADRN or MES cell state in NB. Continued interrogation of these fundamental mechanisms represents a new approach that may potentially be leveraged for therapeutic gain.