YU238259 Is a Novel Inhibitor of Homology-Dependent DNA Repair That Exhibits Synthetic Lethality and Radiosensitization in Repair-Deficient Tumors

Abstract

Radiotherapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair, but not non-homologous end-joining, in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation, etoposide, and PARP inhibition, but this synergism is heightened by BRCA2 deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU238259 treatment even in the absence of concomitant DNA-damaging therapy. The cytotoxicity of these small molecules in repair-deficient cells results from an accumulation of unresolved DNA double-strand breaks. These findings suggest that YU238259 or related small molecules may have clinical benefit to patients with advanced BRCA2-negative tumors, either as a monotherapy or as an adjuvant to radiotherapy and certain chemotherapies.

Implications: We have identified a novel series of compounds that demonstrate synthetic lethality in DNA repair-deficient cell and animal models and have strong potential for clinical translation.

Fig. 1. High-throughput screening reveals a novel class of compounds with synthetic lethality in the context of HDR defects

(A) Methodology of synthetic lethality screen. (B) LD50 values and maximal percent effect values for given sulfonamide compounds were calculated in cells proficient or deficient in either BRCA2 or FANCD2 (C) Structures of sulfonamide compounds identified in screen of ChemBridge DIVERSet small molecule library or through structural homology screening. (D) Structure of lead agent, YU238259.

Fig. 2. Sulfonamide compounds do not inhibit PARP activity or intercalate into DNA

(A) PARP activity in the presence of YU128440, YU238259, or vehicle was assessed by in vitro enzymatic assay. 3-aminobenzamide, a known PARP inhibitor, was a positive control. (B) Plasmid DNA was incubated with YU128440, caffeine (a negative control), or a known intercalating agent, doxorubicin. Disruption of the duplex DNA helical structure was analyzed by circular dichroism spectroscopy.

Fig. 3. YU238259 specifically inhibits HDR activity

(A) Schematic of DR-GFP and EJ5-GFP cell-based reporter assays for HDR and NHEJ, respectively. (B) Effect of YU238259 on the frequency of HDR and NHEJ, as assessed by FACS analysis of GFP+ cells. (C) Relative percent of cells positive for DNA repair foci at 8 h post-irradiation with 10 Gy IR, following pretreatment with 25 uM YU238259 or vehicle. (D) Neutral comet assay of DLD-1 or DLD-1 BRCA2-KO cells pretreated with YU238259 and irradiated with 5 Gy IR. DSB levels, as assessed by comet tail moment, were measured at 0 h and 24 h post-irradiation. (E) Cell cycle analysis of DLD-1 and DLD-1 BRCA2-KO cells treated with YU238259 for 6 h and stained with propidium iodide.

Fig. 4. YU238259 demonstrates synthetic lethality with loss of HDR proteins

Paired, isogenic human cell lines deficient and proficient in (A) BRCA2, (B) ATM, and (C) PTEN were treated with increasing doses of YU238259 for 48 h and toxicity assessed by clonogenic survival assay. (D) U2OS cells were transfected with siRNAs against ATRIP, NBS1, or non-targeting control, or mock-transfected. 72 h later, cells were treated with YU238259 as above and survival assessed by clonogenic assay. Differences between repair-deficient and proficient lines were analyzed statistically via the Wilcoxon signed-rank test and were significant at the p < 0.01 level.

Fig. 5. YU238259 exhibits synergism with radiotherapy and DNA-damaging chemotherapy that is potentiated by BRCA2 loss

Clonogenic assay of DLD-1 and DLD-1 BRCA2-KO cells pretreated with YU238259 for 24 h and then co-treated with (A, B) ionizing radiation, (C, D) etoposide, or (E, F) olaparib for an additional 24 h in the presence of YU238259. Analysis using the Chou-Talalay model yielded CI values < 0.9 for all dose combinations in the DLD-1 BRCA2-KO cell line, mathematically proving synergism. Higher doses of YU238259 were also synergistic with IR in the DLD-1 cell line.

Fig. 6. YU238259 inhibits growth of BRCA2-deficient tumor xenografts in nude mice

Growth curves of subcutaneous DLD-1 or DLD-1 BRCA2-KO tumor xenografts in athymic mice treated with (A) four doses of 5 mg/kg YU128440 or (B) twelve doses of 3 mg/kg Yu238259. Survival rate of mice, as measured by number of days required for tumors to reach 4x their volume at time of treatment, for (C) YU128440 and (D) YU238259.