Introduction: Acute lymphoblastic leukaemia (ALL) is one of the most common malignancies in children. Despite advancements in treatment and survival rates exceeding 90%, relapse remains to be a major cause of mortality. Glucocorticoid (GC)-based chemotherapy is a critical part of standard treatment protocols for paediatric ALL, with patient’s response to this therapy serving as a critical prognostic marker. However, GC resistance remains a significant contributor to relapse and poses a barrier to cure.
Objective: To identify and functionally characterise gene regulatory regions that are differentially regulated in GC-sensitive and GC-resistant ALL and are important for conferring GC sensitivity.
Methods and Results: Candidate gene regulatory regions were identified bioinformatically using the following criteria: (i) open chromatin regions in lymphocytes marked by DNase hypersensitivity signal, (ii) presence of glucocorticoid receptor (GR) binding in GC-treated sensitive ALL, and (iii) active chromatin marked by histone modification H3K27ace in GC-treated sensitive ALL. Through this pipeline, we identified 320 unique regions and selected the top 100 regions to generate a customised dual guide RNA library for an in vivo CRISPR/Cas9 enrichment screen in a clinically relevant GC-sensitive ALL patient-derived xenograft (PDX) model, to identify enriched gRNAs targeting regions mediating GC-sensitivity. Using an improved lentiviral transduction method, Cas9-expressing ALL PDX cells were infected ex vivo with the dgRNA library and then injected into immune-compromised mice. Mice were treated with vehicle control or dexamethasone for 4 weeks and leukaemia level tracked until mice reach experimental end point (>25% human leukaemia cells in the mouse peripheral blood). Relapsed/resistant clones were isolated, DNA extracted, amplified and sequenced to identify the enriched dgRNAs. The most enriched dgRNA deleted an intragenic enhancer region within the JAK1 gene which encodes a tyrosine kinase involved in haematopoiesis, implicating the involvement of JAK/STAT signalling in GC response. This finding will be validated independently in additional PDX models.
Conclusions: This is the first study using in vivo CRISPR/Cas9-mediated gene editing to characterise critical regulatory regions responsible for GC response, providing valuable insights that will inform the development of novel therapeutic strategies to enhance GC sensitivity.