In vitro and in vivo genome wide CRISPR screening under drug treatment in T-ALL

The basis of UK T-ALL induction therapy is VXLD (Vincristine, Dexamethasone, L-Asparaginase and Daunorubicin). Treatment resistance and relapse are pivotal problems in T-ALL, and identifying involved genes and pathways is key to improving current therapy. We have combined a genome wide CRISPR screening approach (using the GeCKOv2 library) with VXLD chemotherapy, both in vitro and in vivo to identify drug resistance mechanisms, and new drug targets. T-ALL cell lines were examined for transducability, karyotype, engraftment potential, and in vitro response to VXLD (individually and in drug matrix assays). Cell line engraftment in RAG2-/-gc-/- (RG) immunocompromised mice was assessed and monitored by flow cytometry and IVIS imaging. HPB-ALL cells (pseudodiploid with TLX3 translocation) engrafts consistently in RG mouse bone marrow and has the required characteristics for screening. A VXLD dosing regimen was optimised so that it was both tolerable and efficacious at reducing leukaemic burden in RG mice injected intra-femorally with HPB-ALL cells. Screening is underway in HPB-ALL cells, and we look to commence validation studies in the near future.