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. 2012 Nov;2(11):1048-63.
doi: 10.1158/2159-8290.CD-11-0336. Epub 2012 Aug 22.

Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1-related breast cancer

Ashish Juvekar  1 , Laura N Burga, Hai Hu, Elaine P Lunsford, Yasir H Ibrahim, Judith Balmañà, Anbazhagan Rajendran, Antonella Papa, Katherine Spencer, Costas A Lyssiotis, Caterina Nardella, Pier Paolo Pandolfi, José Baselga, Ralph Scully, John M Asara, Lewis C Cantley, Gerburg M Wulf
Affiliations

Combining a PI3K inhibitor with a PARP inhibitor provides an effective therapy for BRCA1-related breast cancer

Ashish Juvekar et al. Cancer Discov. 2012 Nov.

Abstract

There is a need to improve treatments for metastatic breast cancer. Here, we show the activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in a MMTV-CreBrca1(f/f)Trp53(+/-) mouse model of breast cancer. When treated with the pan-class IA PI3K inhibitor NVP-BKM120, tumor doubling was delayed from 5 to 26 days. NVP-BKM120 reduced AKT phosphorylation, tumor cell proliferation, and angiogenesis. Resistant tumors maintained suppression of AKT phosphorylation but exhibited activation of the MAPK pathway at the "pushing margin." Surprisingly, PI3K inhibition increased indicators of DNA damage, poly-ADP-ribosylation (PAR), and γ-H2AX, but decreased Rad51 focus formation, suggesting a critical role of PI3K activity for Rad51 recruitment. The PARP inhibitor olaparib alone attenuated tumor growth modestly; however, the combination of NVP-BKM120 and olaparib delayed tumor doubling to more than 70 days in the mouse model and more than 50 days in xenotransplants from human BRCA1-related tumors, suggesting that combined PI3K and PARP inhibition might be an effective treatment of BRCA1-related tumors.

Significance: Current treatment options for triple-negative breast cancer are limited to chemotherapeutic regimens that have considerable toxicity and are not curative. We report here that the combination of a PI3K inhibitor with a PARP inhibitor provides in vivo synergy for treatment of an endogenous mouse model for BRCA1-related breast cancers, making this a candidate combination to be tested in human clinical trials.

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Conflict of interest statement

Conflicts of interest:

LCC and JB have consulted for Novartis Pharmaceuticals, which is developing NVP-BKM120 for cancer treatment; JB has consulted for Astra Zeneca, which is developing Olaparib for cancer treatment.

Figures

Fig. 1
Fig. 1
PI3K pathway activation in BRCA1-related breast cancer in MMTV-CreBRCA1f/fp53+/−. Tumor-bearing females were euthanized, tissues harvested and processed for immunohistochemistry. Displayed are representative images of immunohistochemistry for phospho-AKT (S473), pospho-(Thr202/Tyr204)-ERK, and the tumor-suppressor phosphatases INPP4B and PTEN. Adjacent normal mammary gland tissue is on the left, tumor tissue on the right. 400 x magnification.
Fig. 2
Fig. 2
Pharmacodynamic effects of PI3K inhibitor NVP-BKM120 on breast carcinomas in MMTV-CreBRCA1f/fp53+/− mice. Female virgin mice developed spontaneous breast cancers at ages 8–12 months. A. Representative 18FDG PET-CT scan images of a tumor-bearing mouse at baseline (image on the left) and within 48 hours of after start of treatments with the PI3K-inhibitor NVP-BKM120 (50 mg/kg/day by gavage, image on the right). This mouse had developed 4 simultaneous tumors. Arrows in red, green, yellow and white are used to identify different tumors upon baseline (left) and post-treatment (right) imaging. The color palette for uptake ranges from dark blue to bright yellow with increasing count intensity. The changes in 18FDG-uptake were determined as described in Materials and Methods. They were a decrease by 45% (tumor with red arrow), 64 % (tumor green arrow), 64% (tumor yellow arrow), 56% (tumor white arrow). B. Suppression of AKT-phosphorylation on S473 as a result of treatments with NVP-BKM120 in vivo. Tumor tissue was obtained via core needle biopsy before and after two weeks of treatments with NVP-BKM120, fixed and processed for IHC with anti-pAKT (S473) antibodies. For additional IHC images see Fig. S1. C. Decrease in FDG-uptake in 6 mammary carcinomas. Relative decrease in FDG-uptake was determined by the ratio of uptake at 48 hours (blue bars) or 2 weeks (red bars) to baseline. Tumor-specific FDG-uptake was determined as described in Materials and Methods. For additional PET-CT images see Fig. S2. D. Concordance of decrease in FDG-uptake and tumor shrinkage during a 2-week treatment with PI3K inhibitor NVP-BKM120. The tumor-bearing animal was imaged with FDG-PET (upper panel, tumor indicated with a yellow arrow before and after treatment, decrease in uptake 93%) and concomitant CT scan (lower panel) before (left) and while on treatment (right). The tumor is again marked in the CT scan with a yellow arrow in the axial and sagittal plane. The red outline indicates the tumor circumference before treatment to visualize treatment effect on tumor size.
Fig. 3
Fig. 3
Anti-angiogenic effects of PI3K inhibitor NVP-BKM120. A. Gross pathologic images of an untreated tumor (left), a tumor treated for 2 weeks (middle) and 6 weeks (right) with NVP-BKM120 at 50 mg/kg/day via gavage. B, C, D Immunohistochemistry to detect CD31 in an untreated tumor (B), the center of a tumor treated for 6 weeks (C) and the tumor capsule of a mammary tumor treated for 6 weeks (D). E: Determination of the Chalkley score to quantify CD31 staining. IHCs with anti-CD31 antibodies were performed in pre-treatment biopsies and in tumor specimen from mice at the time of tumor progression.
Fig. 4
Fig. 4
PI3K-inhibition increases poly-ADP-ribosylation and H2AX phosphorylation. A. Compensatory pathway activation induced by treatments with NVP-BKM120. HCC1937 or SUM149 cells were treated with NVP-BKM120, Olaparib or its combination as indicated for 72 hours, lysed and subjected to immunoblotting with antibodies against total AKT, EGFR, ERK and their phospho-specific epitopes. B. In vivo increase of γH2AX-positive cells after treatment with NVP-BKM120 and proliferative activity at the “pushing margin”. Tumor-bearing mice were subjected to a pre-treatment biopsy and then treated with NVP-BKM120 at 50 mg/kg/day. IHCs of pre-treatment biopsies and post-treatment tumor tissues were performed with antibodies as indicated. C. Effects of combined PI3K- and PARP-inhibition on BRCA1-mutant cells. Cells were treated with NVP-BKM120 at 1 µM and Olaparib 10 µM or their combination for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, pAKT, total AKT and γH2AX and Actin as indicated. D. BRCA1 mutant human HCC1937 or SUM149 cells were treated with vehicle control or NVP-BKM120 at the indicated concentrations for 24 hours, lysed and subjected to immunoblotting with antibodies against PAR, p-AKT (S473), γH2AX, Cleaved Caspase 3(CC3) as an apoptosis marker and Actin as a loading control.
Fig. 5
Fig. 5
Effects of NVP-BKM120, KU-55933 and their combination on the DNA damage response. A. HCC1937 were treated for 18 hours with NVP-BKM120 at 2.5 µM, KU55933 at 10 µM or their combination, subjected to ionizing irradiation with 10 Gy or mock, lysed 6 hours later and subjected to immunoblotting with antibodies as indicated. B-E Loss of Rad51 focus formation in response to ionizing radiation in the presence of NVP-BKM120. Breast cancer cells were isolated from primary tumors from MMTV-Cre MMTV-CreBRCA1f/fp53+/− mice and either treated with vehicle control (B, C) or NVP-BKM120 (D, E) for 18 hours, followed by irradiation with 10 Gy. 6 hours later cells were fixed and processed for immunofluorescence with antibodies against Rad51 and counterstained with DAPI. F. Induction of DNA-PK and H2AX phosphorylation and loss of RAD51 occur in response to PI3Kα, not PI3Kβ-inhibition. SUM149 cells were transfected with siRNA pools depleting PI3Kα (left panel) or PI3Kβ (right panel). Cells were lysed after 48 hrs and subjected to immunoblotting with antibodies as indicated.
Fig. 6
Fig. 6
Anti-tumor efficacy of PI3K inhibitor NVP-BKM120 alone and in combination with Olaparib. A–D Tumor-bearing MMTV-CreBRCA1 p53+/− were treated with either vehicle control (A), NVP-BKM120 (B, 50 mg/kg/day (n=11) or 30 mg/kg/day (n=10)), Olaparib (C, 50 mg/kg/day (n=8)) or the combination of NVP-BKM120 and Olaparib (D, NVP-BKM 50 mg/kg/day+Olaparib 50 mg/kg/day (n=8) or NVP-BKM 30 mg/kg/day+Olaparib 50 mg/kg/day (n=7)) and tumor volumes were measured every 2–3 days using calipers. Trendlines for vehicle control (red curve) and NVP-BKM120 treatments (green curve) were calculated using all data points to determine best fit. The functions of the best-fit curves were used to determine tumor doubling times for all three treatment modalities and controls. E, Stable body mass with PI3K-inhibitor and PARP-inhibitor treatments Mice were weighed before and after treatments. F, G. Target inhibition and pharmacokinetics in vivo. Tumor tissues harvested from animals treated with NVP-BKM120 (30 mg/kg/day) alone or in combination with Olaparib (50 mg/kg/day) as indicated were harvested 3 hours after the last treatment and subjected to immunoblotting with antibodies against Actin, p-AKT and γH2AX (F) or lysed and subjected to Mass Spectrometry (G). For standards used see Materials and Methods and Fig. S5. H, I. Responses of human BRCA1 -related breast cancers implanted as xenotransplants into nude mice to NVP-BKM120, Olaparib or their combination. Breast cancer tissues from two patients, one with a 185delAG germline mutation (H) and the other one with a 2080delA germline mutation (I) were propagated as subcutaneous implants in nude mice. Tumors were allowed to grow to a size of 5 mm when mice were randomized to treatments with either vehicle control (black, —), NVP-BKM120 (red, formula image), Olaparib (green, formula image)or their combination (blue, formula image) (n=6 for each cohort, same dosing as in F). Tumor assessment with electronic calipers was done as described in Materials and Methods.
Fig 7
Fig 7
Signal transduction and proliferative activity of responsive and resistant tumors after treatments with NVP-BKM120 and Olaparib. A. Tumor-bearing MMTV-CreBRCA1f/fp53+/− mice were treated as indicated, and tumor tissues obtained as pre-treatment biopsies and at the time of progression. For tumor-bearing mice that were treated with the combination of NVP-BKM120 and Olaparib we also obtained a day 10-of-treatment biopsy (middle panel), at which point all tumors were responsive. Immunohistochemistry was performed with antibodies against antigens as indicated. B. Ki67 and γ-H2AX were scored by counting and averaging the number of positive nuclei per high-power field (HPF) in pre- and on-treatment biopsies and at the time of progression.
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