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. 2017 Sep;16(9):2022-2034.
doi: 10.1158/1535-7163.MCT-17-0098. Epub 2017 Jun 15.

Modeling Therapy Resistance in BRCA1/2-Mutant Cancers

Amy Dréan  1 , Chris T Williamson  1 , Rachel Brough  1 , Inger Brandsma  1 , Malini Menon  1 , Asha Konde  1 , Isaac Garcia-Murillas  2 , Helen N Pemberton  1 , Jessica Frankum  1 , Rumana Rafiq  1 , Nicholas Badham  1 , James Campbell  1 , Aditi Gulati  1 , Nicholas C Turner  2 , Stephen J Pettitt  1 , Alan Ashworth  3 , Christopher J Lord  3
Affiliations

Modeling Therapy Resistance in BRCA1/2-Mutant Cancers

Amy Dréan et al. Mol Cancer Ther. 2017 Sep.

Abstract

Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or "revertant" mutations in BRCA1 or BRCA2 Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor-sensitive and PARP inhibitor-resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in "revertant" tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022-34. ©2017 AACR.

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Conflict of interest statement

Conflict of interest statement: CJL and AA are named inventors on patents describing the use of PARP inhibitors and stand to gain as part of the ICR “Rewards to Inventors Scheme”.

Figures

Figure 1
Figure 1. Characterization of BRCA1 and BRCA2 secondary mutant, PARP-inhibitor resistant clones.
A. Schematic showing experimental design. SUM149 and CAPAN1 cells were transfected with Cas9 and CRISPR gRNA expression constructs targeting BRCA1 or BRCA2, respectively, to induce DSB and subsequently create a secondary BRCA1 or BRCA2 mutation reinstating the open reading frame. B. DNA sequence for CAPAN1.B2.S* showing 5 bp deletion in BRCA2. PAM sequence is underlined in blue. C. Predicted BRCA2 protein structure for CAPAN1.B2.S*. The predicted amino acid length is shown. D. Dose-response survival curves for CAPAN1.B2.S* (red) and CAPAN1 parental cell lines exposed to olaparib (P <0.0001, ANOVA). Error bars represent SEM (standard error of the mean) from triplicate experiments. E. Representative images for nuclear RAD51 foci formation in CAPAN1 and CAPAN1.B2.S* cells following IR exposure. Scale bar = 10 μm. F. Bar chart illustrating quantitation of nuclear RAD51 foci. Cells containing more than five foci were counted as positive. Mean ± SEM for three independent experiments are shown. p values were calculated using Student’s t test. G. DNA sequence for SUM149.B1.S* showing 80 bp deletion in BRCA1. PAM sequence is underlined in blue. H. Predicted BRCA1 protein structure for SUM149.B1.S*. The predicted amino acid length is shown. I. Dose-response survival curves for SUM149.B1.S* (green) and SUM149 parental cells exposed to olaparib (P <0.0001, ANOVA). Error bars represent SEM from triplicate experiments. J. Bar chart illustrating quantitation of nuclear RAD51 foci. Cells containing more than five foci were counted as positive. Mean ± SEM (standard error of the mean) for three independent experiments are shown. p values were calculated using Student’s t test.
Figure 2
Figure 2. Olaparib exposure induces Darwinian selection favouring secondary BRCA mutants in vitro.
A. Experimental schematic. Secondary mutant and parental cells were mixed at a 1:20 ratio and then exposed to DMSO, olaparib, or cisplatin. After drug exposure, populations were analysed for surviving fraction and secondary mutant:parental proportions using ddPCR. B. Bar graph illustrating the effect of drug exposure on population surviving fraction in either CAPAN1.B2.S*:CAPAN1 or SUM149.B1.S*:SUM149 co-cultures. C. Bar graph illustrating the increase in secondary mutant clone frequency following 14 days of drug exposure. D. Graphs showing the frequency of CAPAN1B2*S cells in CAPAN1/CAPAN1B2* co-cultures exposed to 500 nM olaparib. Clone frequency was estimated by ddPCR at the time points shown. Error bars represent SEM from three independent measurements.
Figure 3
Figure 3. PARPi-induced selectivity operates in BRCA2 isogenic DLD1 tumour cells.
A. Dose-response curves illustrating 6-well clonogenic survival data for BRCA2 isogenic cells, DLD1.BRCA2WT/WT and DLD1.BRCA2–/–, exposed to olaparib over 14 days. Error bars represent SEM from triplicate experiments. B. Bar graph illustrating the increase in DLD1.BRCA2WT/WT frequency in a 1:100 starting DLD1.BRCA2WT/WT:DLD1.BRCA2–/– ratio co-culture following 13 days of drug exposure. C. Bar graph showing starting ratio of DLD1.BRCA2–/– to DLD1.BRCA2WT/WT influences time (days) for DLD1.BRCA2WT/WT cells to reach clonal dominance (75% of cell population). D-E. Graphs showing time required for DLD1.BRCA2WT/WT-GFP to reach clonal dominance (75%, dotted line) when exposed to either 25 nM or 100 nM of olaparib (D) or 10 nM of talazoparib (E) in DLD1.BRCA2WT/WT :DLD1.BRCA2–/– co-cultures.
Figure 4
Figure 4. PARPi-induced Darwinian selection of BRCA proficient tumour cells in vivo.
A. Experimental schematic of mixed CAPAN1:CAPAN1.B2.S* xenografts treated with olaparib. B. Bar chart illustrating CAPAN-1 (white) and CAPAN1.B2.S* (red) cell frequency in tumour xenografts prior to drug treatment. Values shown for each animal were derived from three tumour sections with mean ± SEM shown. C. Bar graph illustrating the increase in secondary mutant clone frequency in tumours following 28-day olaparib treatment (n=6, mean ± SEM shown). p values were calculated by Student’s t test. D. Correlation between fold increase in the frequency of the secondary mutant clone and the periodicity of olaparib administration.
Figure 5
Figure 5. PARPi-resistant, secondary mutant clones and parental tumour cells are sensitive to AZD-1775 in vitro and in vivo.
A. Waterfall plot comparing AUC values collated from a five day exposure to AZD-1775 from 146 cancer cell lines. B. Dose-response survival curves illustrating 6-well clonogenic survival data in CAPAN1, CAPAN1.B2.S*, SUM149, SUM149.B1.S*, MCF-10A and MCF-12A cells exposed to AZD-1775. C. Bar graph illustrating the increase in secondary mutant clone frequency following 14 days of drug exposure. D. Graph showing the frequency of CAPAN1B2*S cells in CAPAN1/CAPAN1B2* co-cultures exposed to AZD-1775. Clone frequency was estimated by ddPCR and the time points shown. Error bars represent SEM from three independent measurements. This experiment was conducted alongside the experiment described in Figure 2D; to allow comparison, the response to olaparib and DMSO exposure from Figure 2D is re-plotted here. E. Western blot for CAPAN1 and CAPAN1.B2.S* cells lysates probed for pCDC2(Y15), γ-H2AX (a DNA damage marker), and cleaved PARP1 (a marker of apoptosis). Tubulin was used as a loading control. F. Experimental schematic of mixed CAPAN1:CAPAN1.B2.S* xenografts treated with olaparib or AZD-1775. G. Bar chart illustrating CAPAN-1 (white) to CAPAN-1.B2.S* (red) clone ratio in in tumour xenografts prior to drug treatment. Values shown from six sentinel animals with mean ± SEM shown. H. Tumour volume plotted against length of treatment for individual xenografts comprised of CAPAN1:CAPAN1.B2.S* mixed tumour cells over 150 days (n=18 total, n=6 in each cohort). I. Survival curves using maximum tumour size (1500 mm3) as a surrogate for survival from the experiment shown in E. J. Bar chart showing proportion of CAPAN1.B2.S* tumour cells following treatment from the experiment shown in E (n=6, mean ± SEM). P values were calculated by Student’s t test.
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