Background: Paediatric acute lymphoblastic leukaemia (ALL) remains one of the leading causes of cancer-related death in Australian children. The enzyme Aldo-Keto Reductase Family 1 Member C3 (AKR1C3) is highly expressed in T-cell ALL (T-ALL)1. ACHM-025, a new prodrug selectively activated by AKR1C3 to form a DNA crosslinker, exhibited profound in vivo efficacy in T-ALL Patient-Derived Xenografts (PDXs). B-cell ALL (B-ALL), accounting for 85% of ALL cases, mostly express low AKR1C3 expression and therefore are resistant to ACHM-025. Understanding the genetic determinants regulating AKR1C3 expression in both B-ALL and T-ALL is crucial to expanding the therapeutic potential of ACHM-025.
Methods and Results: Using a combination of epigenetic techniques in a panel of B- and T-ALL PDX samples, we identified cis-regulatory elements (CREs) associated with AKR1C3 expression. Specifically, the AKR1C3 promoter (H3K4me3 ChIP-Seq) was more accessible (ATAC-Seq) in T-ALL with high AKR1C3. Two long-range downstream enhancers were identified by ATAC-Seq and H3K4me1 ChIP-Seq. Both enhancers were active in T-ALL with high AKR1C3 expression, while only one was active in B-ALL and T-ALL with low AKR1C3 expression (H3K27ac ChIP-Seq). H3K27ac HiChIP showed promoter-enhancers looping in T-ALL indicating their role in regulating AKR1C3 expression. We next used CRISPRactivation (CRISPRa) system to functionally test promoter and enhancer activation in a B-ALL cell line with low AKR1C3 expression. Targeting the promoter resulted in increased AKR1C3 expression and concurrent increased sensitivity to ACHM-025. More excitingly, CRISPRa targeting the enhancers showed the same trend. Conversely, using CRISPR/Cas9 to knockout the AKR1C3 gene or delete the enhancers in a T-ALL cell line with high AKR1C3 resulted in decreased AKR1C3 expression and reduced sensitivity to ACHM-025. Having established the functional role of the downstream CREs, we have now performed an unbiased locus-specific proteomic analysis using dCas9-CAPTURE2 targeting AKR1C3 CREs, identifying transcription factors and co-factors that can be targeted for rational drug combinations with ACHM-025.
Conclusion: This study is the first to identify long-range CREs that control AKR1C3 expression, which are both B- and T-ALL specific. The identification of AKR1C3 master regulators provides an avenue for the development of rational drug combinations, extending the use of ACHM-025 to all leukemia patients.