ABSTRACT
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Subject(s)
Antineoplastic Agents , Drug Discovery , Enzyme Inhibitors , Microsatellite Instability , Neoplasms , Synthetic Lethal Mutations , Werner Syndrome Helicase , Animals , Female , Humans , Mice , Administration, Oral , Allosteric Regulation/drug effects , Antineoplastic Agents/adverse effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Clinical Trials as Topic , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Damage/drug effects , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Mice, Nude , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Protein Domains , Reproducibility of Results , Suppression, Genetic , Synthetic Lethal Mutations/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Werner Syndrome Helicase/antagonists & inhibitors , Werner Syndrome Helicase/genetics , Werner Syndrome Helicase/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.
