Background: T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy with dismal survival rates after relapse. Aldo-keto reductase family 1 member C3 (AKR1C3) is an NADPH-dependent enzyme that is highly expressed in T-ALL, which can be exploited therapeutically. ACHM-025, a novel prodrug activated by AKR1C3 into a nitrogen mustard-based DNA alkylating agent, was previously demonstrated by our group to exhibit profound in vivo activity against T-ALL patient-derived xenografts (PDXs), suggesting its potential for T-ALL targeted therapy. The aim of this project was to utilise a genome-wide CRISPR/Cas9 knockout screen to identify genes and pathways critical for T-ALL cell sensitivity or resistance to ACHM-025.
Methods: The T-ALL cell line RPMI-8402, which had previously been engineered to express the Cas9 protein with confirmed expression and functionality, was lentivirally transduced with the Cellecta CRISPR Human Genome 80K Knockout Library consisting of almost 80,000 single guide RNAs (sgRNAs) targeting approximately 19,000 genes at an abundance of 4 sgRNAs per gene. Transduction was performed at a multiplicity of infection of 0.4 to ensure that cells received a single sgRNA. Following a period of equilibration and selection for puromycin resistance, transduced cells were equally divided into ACHM-025-treated and vehicle-treated groups and harvested at multiple time points up to 21 days post treatment initiation. Genomic DNA was extracted from the harvested cells, libraries prepared, and next-generation sequencing was performed to identify enriched or depleted sgRNAs.
Results: SgRNAs targeting the AKR1C3 gene, as well as an epigenetic regulator involved in histone acetylation, along with several genes whose protein products are involved in NADPH production, all of which play a role in ACHM-025 activation, were highly enriched suggesting that they mediate ACHM-025 sensitivity. In contrast, sgRNAs targeting genes whose protein products repress AKR1C3 expression, as well as those involved in DNA repair, antioxidant pathways, and an epigenetic regulator involved in histone demethylase, were all depleted indicating their role in mediating resistance to ACHM-025.
Conclusion: This study has identified novel genes and pathways that regulate sensitivity and resistance to ACHM-025, providing insights for designing rational combination therapies to improve T-ALL treatment.