High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs

Abstract

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.

Conflict of interest statement

Conflict of interest statement: A.O.H.N. is an employee of MRC-Holland BV, which markets the MLPA test used in this article. A.L., R.B., A.C., M.J.O., and N.M.B.M. are employees of KuDOS Pharmaceuticals, which developed AZD2281.

Figures

Fig. 1.

Overview of tumor transplantations and drug treatments in this study. Small tumor fragments of 9 spontaneous mammary tumors (T1–T9), which developed in K14cre;Brca1F/F;p53 F/F mice (Liu et al., ref. 12) were transplanted orthotopically into syngeneic wild-type female mice. After a mean latency of ≈4 weeks, when tumors reached a size of 150–250 mm3 (V = length × width2 × 0.5), the indicated drug treatments were carried out. Dosing was as follows: 50 mg of AZD2281 per kg i.p. daily for 28 days (28 days) or daily for 100 days (100 days), 6 mg of cisplatin per kg i.v. on day 0 (30 min after the first AZD2281 injection), 100 mg of carboplatin per kg i.v. on day 0 (30 min after the first AZD2281 injection), 2 mg of tariquidar per kg every other day (if combined with AZD2281, tariquidar was given 30 min in advance). Treatment of tumors was resumed once the tumor relapsed to its original size (100%).

Fig. 2.

Treatment of mice carrying orthotopically transplanted Brca1−/−;p53−/− tumors with 50 mg of AZD2281 per kg i.p. (A) Intratumoral concentration of AZD2281 and PARP1 activity over time. Error bars indicate SEM. (B–D) Animals carrying 9 individually transplanted tumors (T1–T9) were either left untreated, or received AZD2281 daily for 28 or 100 days. Graphs show relative tumor volume (RTV, ratio of tumor volume to initial size at start of treatment) as a function of time. T8 and T9 showed stable disease and received continuous dosing beyond the 28 days (see Fig. S1). Once the tumors relapsed, treatment was resumed when the tumor size reached 100% of the original volume. Days on which AZD2281 was given are indicated by circles, triangles, or squares.

Fig. 3.

AZD2281 treatment induces DNA damage-associated foci and caspase 3-mediated apoptosis. (A) Example of the IHC analysis of T4 using anti-activated caspase 3 and anti-γH2AX-specific antibodies. Sections of the sensitive tumor (before or 7 days after daily injection of 50 mg of AZD2281 per kg i.p.) and resistant tumor (day 73 after unsuccessful daily treatment of the relapsing tumor, see Fig. 2C) are shown. Bar = 50 μm. (B) Quantification of γH2AX or cleaved caspase 3-positive cells of 3 individual Brca1−/−;p53−/− tumors (T3, T4, T6) and Ecad−/−;p53−/− tumor 1 before, 7 days after daily AZD2281 treatment or of the outgrown AZD2281-resistant tumor (see Fig. 2C and Fig. S2). For P values see Table S1.

Fig. 4.

Increased expression of Abcb1a and Abcb1b is associated with AZD2281 resistance in vivo. (A) RT-MLPA analysis of the ratios of Abcb1a, Abcb1b, Abcc1, Abcg2, Parp1, and Hprt1 expression in AZD2281-resistant tumors and samples from the corresponding untreated tumors. Actin-β expression was used as internal reference. The values presented are the mean ratio of 3 independent reactions. The suffix 28 indicates the 28-day schedule of AZD2281 and 100 the 100-day schedule. Error bars indicate standard deviation. For the complete dataset see Table S2. (B) Three Brca1−/−;p53−/− doxorubicin-resistant tumors (2 with up-regulation of Abcb1a/b and 1 without; ref. 13) were tested for their response to AZD2281. Days on which 50 mg of AZD2281 per kg were given have open squares. (C) T1–T4 and T6 were treated with a daily injection of 50 mg of AZD2281 per kg for 28 days. When tumors relapsed to 100% of their original volume, they were retreated by i.p. injection of 2 mg of tariquidar per kg every other day (light blue line) or 50 mg of AZD2281 per kg daily (red line) or both (dark blue line). Days on which animals were treated are indicated by rhombi, triangles, or squares. Graphs in B and C show relative tumor volume (RTV, ratio of tumor volume to initial size at start of treatment) as a function of time.

Fig. 5.

Combination of AZD2281 with carboplatin and cisplatin prolongs recurrence-free survival and overall survival. (A) Box plots indicate the time after therapy start before tumors relapse back to the size when treatment was initiated. Dosing was as follows: 50 mg of AZD2281 per kg i.p. daily for 28 days (28d) or daily for 100 days (100d), 6 mg of cisplatin per kg i.v. on day 0 (30 min after the first AZD2281 injection), 100 mg of carboplatin per kg i.v. on day 0 (30 min after the first AZD2281 injection). (B) Kaplan–Meyer curves showing the overall survival after 400 days. Wilcoxon signed rank tests: control vs. AZD2281–28d: P < 0.009 (n = 7); control vs. AZD2281–100d: P < 0.009 (n = 7); AZD2281–28d vs. AZD2281–100d: P < 0.009 (n = 7); cisplatin vs. cisplatin+AZD2281–28d: P < 0.019 (n = 9); cisplatin vs. cisplatin+AZD2281–100d: P < 0.014 (n = 7).