Enhancement of synthetic lethality via combinations of ABT-888, a PARP inhibitor, and carboplatin in vitro and in vivo using BRCA1 and BRCA2 isogenic models

Abstract

Individuals with an inherited BRCA1 or BRCA2 mutation have an elevated risk of developing breast cancer. The resulting tumors typically lack homologous recombination repair as do a subset of sporadic tumors with acquired BRCA deficiency. Clinical responses to monotherapy with platinum drugs or poly PARP inhibitors (PARPi) have been shown for BRCA-associated cancers. However, there are limited data on combination therapy with PARPi and platinum drugs, the mechanism of action of this combination, and the role of BRCA1 or BRCA2 in chemosensitivity. We compared the efficacy of ABT-888 (a PARPi) with that of cisplatin or carboplatin (platinum drugs) alone or in combinations by examining the survival of treated Brca-proficient and -deficient mouse embryonic stem cells. In addition, drug-induced growth inhibition of a BRCA1 and a BRCA2 null cell line were compared with their isogenic BRCA-complemented lines. Although each monotherapy killed or inhibited proliferation of Brca/BRCA-deficient cells, an enhanced effect was observed after treatment with ABT-888 in combination with carboplatin. Moreover, the ABT-888/carboplatin combination delayed tumor growth in Brca2 xenografts. The drugs caused DNA damage and apoptosis. Along with greater PARP activity in Brca/BRCA-deficient cells, these effects correlated with increased chemosensitivity. Our data suggest that ABT-888 and carboplatin combination treatment will be more successful than monotherapy in addressing many BRCA-associated cancers. A randomized phase II trial has recently been initiated to test this hypothesis to assist in the discovery of more effective therapies for patients with BRCA.

FIGURE 1. Clonogenic cell survival and MTT assays of mouse and human BRCA-deficient or -proficient cell lines treated with ABT-888 or carboplatin as single agents or in combination

(A) ABT-888 structure. (B) carboplatin structure. (C–E) WT (AB2.2, Brca^+/+), Brca1 (Brca1^−/−), or Brca2 (Brca2^−/−) mESCs were treated for 48 h with the indicated drugs and concentrations. (C) ABT-888, (D) carboplatin, (E) ABT-888/carboplatin. (F–K) Human BRCA-deficient and BRCA-complemented cell growth was assessed by MTT assay after 72 h of continuous drug treatment. Percent inhibition for HCC1937 (BRCA1) or HCC1937+BRCA1 (BRCA1 Comp.) cells after treatment with (F) ABT-888 (G) carboplatin and (H) ABT-888/carboplatin combination. Percent inhibition for EUFA423F (BRCA2) and EUFA423F+BRCA2 (BRCA2 Comp.) after treatment with (I) ABT-888, (J) carboplatin, (K) ABT-888/carboplatin combination. Data are the average of triplicate measurements; error bars are ± SEM.

FIGURE 2. In vivo efficacy of ABT-888 in combination with carboplatin in Brca2 isogenic CHO xenograft models

(A) Relative tumor volume (RTV) growth in mice (5 per group) bearing VC8 or VC8+Brca2 xenografts after vehicle, ABT-888, carboplatin or ABT-888/carboplatin treatment (relative to the volume on day 1 of treatment). (B) RTV after the 14 day treatment course. Horizontal lines are averages. (C) Tumor mass (mg) after the 14 day treatment course. Horizontal lines are averages. (D) Representative image of one tumor sample for each treatment group endpoint.

FIGURE 3. DNA damage response in BRCA and BRCA-complemented cell lines after ABT-888, carboplatin, or ABT-888/carboplatin combination treatment

All cells were analyzed after 24h of treatment and counterstained with 4′,6-diamidino-2-phenylindole. (A) Immunostained γH2AX foci in HCC1937 or HCC1937+BRCA1 cells treated with vehicle or 200 μM ABT-888/25 μM carboplatin combination. (B) Immunostained γH2AX foci in EUFA423F or EUFA423F+BRCA2 cells treated with vehicle or 12.5 μM ABT-888/12.5 μM carboplatin combination.(C) Quantification of γH2AX foci formation in HCC1937 or HCC1937+BRCA1 cells treated with vehicle, 200 μM ABT-888 (ABT), 25 μM carboplatin (Pt), or 200 μM ABT-888/25 μM carboplatin combination. (D) Quantification of γH2AX foci formation in EUFA423F or EUFA423F+BRCA2 cells treated with vehicle, 12.5 μM ABT-888 (ABT), 12.5 μM carboplatin (Pt), or 12.5μM ABT-888/12.5 μM carboplatin combination. (E) Quantification of RAD51 foci in HCC1937 and HCC1937+BRCA1 cells. (F) Quantification of RAD51 foci in EUFA423F and EUFA423F+BRCA2 cells. RAD51 foci were analyzed after the same treatment conditions as for the γH2AX foci. The RAD51 foci vehicle control was used as the denominator (Foci Untreated) to determine the fold increase with the RAD51 Foci Treated. Raw data examples for RAD51 foci are found in Supplementary Fig S5.

FIGURE 4. Cell death of BRCA and BRCA-complemented cell lines after treatment with ABT-888, carboplatin, or ABT-888/carboplatin combination

All cells were analyzed 72 hours after initial drug treatment. (A) Percentage of apoptotic cells determined from Annexin V staining of HCC1937 and HCC1937+BRCA1 cells incubated in medium containing vehicle or ABT-888 (200μM), carboplatin (12.5μM) or ABT-888/carboplatin (12.5 μM/200μM). (B) Western blots of full and cleaved caspase 3 in HCC1937 and HCC1937+BRCA1 cells. (C) Percentage of apoptotic cells determined from Annexin V staining of EUFA423F or EUFA423F+BRCA1 cells incubated in medium containing vehicle or ABT-888 (12.5μM), carboplatin (12.5μM) or ABT-888/carboplatin (12.5μM/12.5μM). (D) Western blots of full and cleaved caspase 3 in EUFA423F and EUFA423F+BRCA2 cells.

FIGURE 5. PARP activity and NADPH levels in Brca/BRCA and HRR-proficient cells and ERCC1 levels in BRCA and -complemented cell lines

(A) PARP activity in the cellular extracts of three mESC (WT, Brca1, and Brca2) and four human cell lines (HCC1937, HCC1937+BRCA1, EUFA423F, and EUFA423F+BRCA2). (B) As an indirect measure of PARP activity, NADPH levels were determined using reduction of WST-8. (C) Western blot of ERCC1 levels in cell extracts.