A high-throughput pharmaceutical screen identifies compounds with specific toxicity against BRCA2-deficient tumors

Abstract

Purpose: Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. Although patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA deficiency and to test new combination therapies to establish optimal treatment strategies.

Experimental design: We did a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors.

Results: Three alkylators-chlorambucil, melphalan, and nimustine-displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine doing better than cisplatin and the poly-(ADP-ribose)-polymerase inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 of 5 animals tested.

Conclusions: We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators, especially melphalan and nimustine, alone or in combination with the poly-(ADP-ribose)-polymerase inhibitors, for the treatment of breast cancers with a defective BRCA pathway.

Figure 1. Functional reconstitution of BRCA2-deficient mammary tumor cells

BRCA2-Deficient mouse mammary tumor cells (KB2P1.21 and KB2P3.4) were reconstituted with transduction of an iBAC carrying the complete Brca2 gene to result in KB2P1.21R2 and KB2P3.4R3 lines. A. RT-PCR Experiments show that while the KB2P1.21 and KB2P3.4 cell lines lack expression of exon 11 due to cre-mediated recombination, full-length Brca2 expression is restored upon stable introduction of a Brca2 containing iBAC. B. Co-localization of γH2A.X (green) with RAD51 (red) in the nucleus (blue) of cells exposed to 20 Gray γ-irradiation is restored in KB2P1.21R2 and KB2P3.4R3 versus KB2P1.21 and KB2P3.4 cell lines. BRCA2-proficient KP cells were shown as controls. C. Cytotoxicity profiles of olaparib (upper graphs) and cisplatin (lower graphs) clearly show sensitivity associated with BRCA2 deficiency.

Figure 2. A pharmacological screen identifies three alkylators with specific toxicity to BRCA2-deficient cells

A. Scatter-plot depicting differential toxicities of 97 compounds for which IC50s could be determined. On the x-axis, differential toxicity between BRCA2-proficient (KP) and BRCA2-deficient cells is depicted. The y-axis indicates differential toxicity between BRCA2-reconstituted (KB2P) and BRCA2-deficient cells. B. Cytotoxicity profiles of chlorambucil, nimustine and melphalan performed on the complete cell-line panel validate the alkylators picked up from the screen. C. Immunofluorescence of γH2A.X (green) and RAD51 (red) in the nucleus (blue) of cells exposed for 24 hours to the alkylators at indicated concentrations. Co-localization of γH2A.X and RAD51 indicates induction of recombinogenic lesions.

Figure 3. In vivo validation of drug activity on orthotopically transplanted cell-lines

FVB × 129/Ola F1 Hybrid animals were injected with 500,000 KB2P3.4 cells in the 4^th fat pad. When tumor volume exceeded 200mm³, daily treatment for 28-days with olaparib was started, or single dose injections with cisplatin, chlorambucil, melphalan or nimustine were given. For each treatment and for untreated controls, transplantations in three mice were performed.

Figure 4. In vivo validation of drug activity on allografted BRCA2-deficient tumors

A. Small pieces of 5 independent mammary tumors from K14cre;Brca2^F11/F11;p53^F2-10;F2-10 mice were transplanted orthotopically into multiple wild-type recipient mice. When tumor volumes exceeded 200mm³, control animals were left untreated and either 28 consecutive daily olaparib treatments were given, or single dose injections of cisplatin, chlorambucil, melphalan or nimustine. B. Average survival time after treatment is indicated for the complete cohort of 5 tumors with error bars indicating s.e.m. Log-rank p-values between olaparib and cisplatin vs. chlorambucil, melphalan and nimustine are indicated when <0.05. *One animal in the melphalan treatment group was still alive 290 days after the start of treatment. ^†Two animals in this group were sacrificed at day 143 and 204 due to other health problems (see Discussion).