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Comparative Study
. 2019 Jun 10;35(6):851-867.e7.
doi: 10.1016/j.ccell.2019.05.001.

Sequential Therapy with PARP and WEE1 Inhibitors Minimizes Toxicity while Maintaining Efficacy

Yong Fang  1 , Daniel J McGrail  2 , Chaoyang Sun  3 , Marilyne Labrie  4 , Xiaohua Chen  2 , Dong Zhang  4 , Zhenlin Ju  2 , Christopher P Vellano  2 , Yiling Lu  2 , Yongsheng Li  2 , Kang Jin Jeong  4 , Zhiyong Ding  2 , Jiyong Liang  2 , Steven W Wang  2 , Hui Dai  2 , Sanghoon Lee  2 , Nidhi Sahni  5 , Imelda Mercado-Uribe  6 , Tae-Beom Kim  7 , Ken Chen  7 , Shiaw-Yih Lin  2 , Guang Peng  8 , Shannon N Westin  9 , Jinsong Liu  6 , Mark J O'Connor  10 , Timothy A Yap  11 , Gordon B Mills  4
Affiliations
Comparative Study

Sequential Therapy with PARP and WEE1 Inhibitors Minimizes Toxicity while Maintaining Efficacy

Yong Fang et al. Cancer Cell. .

Abstract

We demonstrate that concurrent administration of poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors is effective in inhibiting tumor growth but poorly tolerated. Concurrent treatment with PARP and WEE1 inhibitors induces replication stress, DNA damage, and abrogates the G2 DNA damage checkpoint in both normal and malignant cells. Following cessation of monotherapy with PARP or WEE1 inhibitors, effects of these inhibitors persist suggesting that sequential administration of PARP and WEE1 inhibitors could maintain efficacy while ameliorating toxicity. Strikingly, while sequential administration mirrored concurrent therapy in cancer cells that have high basal replication stress, low basal replication stress in normal cells protected them from DNA damage and toxicity, thus improving tolerability while preserving efficacy in ovarian cancer xenograft and patient-derived xenograft models.

Keywords: PARP inhibitor; WEE1 inhibitor; replication stress; sequential therapy.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests: GBM consults with AstraZeneca, ImmunoMET, Ionis, Nuevolution, PDX bio, Signalchem, Symphogen, and Tarveda, has stock options with Catena Pharmaceuticals, ImmunoMet, SignalChem, Spindle Top Ventures and Tarveda, sponsored research from AstraZeneca, Immunomet, Pfizer, Nanostring, and Tesaro, travel support from Chrysallis Bio, and has licensed technology to Nanostring and Myriad Genetics. TAY consults with AstraZeneca, Pfizer, EMD Serono, Clovis, BMS, Ignyta, Roche, Janssen, Atrin, Aduro, and Almac, has sponsored research from AstraZeneca and Vertex and travel support from AstraZeneca, BMS, MSD, Vertex, GSK, EMD Serono and Tesaro. GP has sponsored research from Pfizer. SNW consults for AstraZeneca, Clovis Oncology, Medivation, Merck, Ovation, Pfizer, Roche/Genentech, Takeda, Tesaro and Vermillion. SNW has sponsored research from AstraZeneca, ArQule, Bayer, Clovis Oncology, Cotinga, Novartis, Roche/Genetech, and Tesaro. MO’C is an AstraZeneca full-time employee and shareholder.

Figures

Figure 1.
Figure 1.. PARPi and WEE1i alter protein expression.
(A) Cells were treated with olaparib or DMSO for 3 or 7 days, and protein lysates analyzed for 218 total and phosphoproteins by RPPA. Heatmap represents “rank ordered” changes induced by olaparib by summing median centered protein levels normalized to control. Proteins with consistent decreases are at top (green) and increases are at bottom (red) of y-axis of the heat map. (B) Cells were treated with DMSO or adavosertib and protein lysates analyzed for 305 total and phosphoproteins by RPPA. Heatmap representation is as in (A). (C) Representative cell lines were treated with talazoparib for 24 hr and analyzed for cell cycle progression. (D) OVCAR8 were treated with adavosertib for 24 hr and analyzed for cdc2Y15 and FOXM1 by western blot. Bands were quantified by ImageJ and normalized by ERK2 protein. Intensity values are relative to DMSO. (E) Cells were treated with indicated compounds for 24 hr and analyzed as in (D). See also Figures S1-S3.
Figure 2.
Figure 2.. PARPi and WEE1i alter cell cycle progression and death.
(A) OVCAR8 were treated with DMSO, PARPi, adavosertib, or concurrent treatment for 24 hr and subjected to pH3 and PI flow cytometric analysis. (B) Cells were treated with DMSO, talazoparib, adavosertib, or concurrent treatment for 24 hr, and western blotted with Cyclin B1 and pH3. Bands were quantified by ImageJ and normalized by ERK2. Intensity values are relative to DMSO. (C, D) OVCAR8 were exposed to DMSO, talazoparib (0.5 μM), adavosertib (0.5 μM), or concurrent treatment for 24 hr. Cells were stained for γH2AX, pH3 and DAPI (C), or cleaved-caspase3 (clv-caspase3), pH3 and DAPI (D). Scale bar, 25 μM. (E) Quantification of pH3+, γH2AX+/pH3+, or cleaved-caspase 3+/pH3+cells. For each condition, at least 300 cells were counted. Results are mean±SEM of three independent experiments. p value: Student’s t test. (F) Representative images of OVCAR8 exposed to DMSO, talazoparib (0.5 μM), adavosertib (0.5 μM), or combination for 0 or 12 hr with medium containing IncuCyte Caspase-3/7 reagent. Red arrowheads indicate mitotic cells. Scale bar, 50 μm. See also Figure S4 and Movie S1.
Figure 3.
Figure 3.. PARP and WEE1 inhibition is synergistic.
(A) Drug response curves for cells treated with talazoparib and adavosertib combinations for 7 days in 3D culture. Data are presented as relative PrestoBlue signal to control. Error bars represent SEM of three independent experiments. (B) CI values of ovarian cancer cell lines with selected mutations. Black indicates mutation; gray no mutation. Cell lines were divided into synergistic (CI<0.5) and non-synergistic (CI>0.5) groups. Presence or absence of mutation was compared by Student’s t test. *p<0.05. (C) 2×106 OVCAR8 were injected subcutaneously (s.c.) and grown for 2 weeks in nude mice. Mice were randomized with 6 in each group and treated as indicated. Average tumor volume±SEM (left) and mouse weight±SEM (right) are displayed. p value: one-way ANOVA. **p<0.01.
Figure 4.
Figure 4.. PARPi induced DNA damage and G2-M DNA damage checkpoint activation persists.
(A-B) OVCAR8 in 3D culture were treated with 30 nM or 60 nM talazoparib for indicated times (A) or treated for 7 days before drug wash out and re-culture without drugs for indicated time (B) before analysis by RPPA. Heat map is represented as in Figure 1A. (C) Protein changes ordered by talazoparib treatment (left column) from (A) and after talazoparib removal (right column) from (B). (D-E) Expression ratios (treated vs control) of Cyclin B1 (D) and CDK1 (E) at each concentration and time point of talazoparib treatment from RPPA data. “p” represents post-treatment. (F) OVCAR8 were treated with talazoparib, veliparib, or DMSO in 3D matrigel culture for 7 days, then washed out and re-cultured for 7 days, and western blotted with Cyclin B1 and γH2AX. (G) 2×106 OVCAR8 were injected s.c. and grown for 3 weeks in nude mice. Vehicle, talazoparib, olaparib, adavosertib were dosed daily to randomized cohorts for 7 days and then stopped. Tumor was obtained from 5-6 mice per group at indicated time points. (H) IHC with FFPE whole-tumor sections for Cyclin B1. Representative images are from indicated time point. (I) IHC protein levels from (H) are displayed as fold change for treatment groups compared to vehicle from each time point. (J) IHC performed from FFPE whole-tumor sections for γH2AX. Representative images are from indicated time points. (K) Protein levels from IHC from (J) are displayed as fold change for treatment groups compared to vehicle from each time point. All data are mean±SEM and Scale bar, 50 μm.
Figure 5.
Figure 5.. Sequential treatment with PARPi and WEE1i is effective in vitro.
(A) Cells were treated with 1-vehicle, 2-talazoparib (250 nM), 3-adavosertib (250 nM), 4-sequential and 5-concurrent treatment and subjected to pH3 and PI flow cytometric analysis. (B, C) Representative images (B) and quantification (C) of OVCAR8 treated as in (A) stained for γH2AX, pH3 and DAPI. Scale bar, 50 μm. For each condition, at least 300 cells were counted. Data are mean±SEM. (D) OVCAR8 were treated as in (A) and subjected to Comet analysis. DNA damage is quantified as percentage DNA in tails from 3 independent experiments. Scale bar, 10 μm. Data are mean±SD. (E, F) OVCAR8 (E) and U2OS (F) cells were treated as in (A) and subjected to DNA fiber analysis. Scale bar, 10 μm. Mean fork speed (kb min−1) is indicated. Data are mean±SD. (G) Cells were treated with indicated drugs for 14 days, except for sequential therapy that consisted of PARPi for 7 days followed by the other drug for 7 days. Representative photos of colonies stained with crystal violet. Data are mean±SEM. (H) Cells were pretreated with DMSO or 30 nM talazoparib for 7 days followed by adavosertib (upper), or pretreated with vehicle, 120 nM adavosertib or 240 nM adavosertib for 7 days followed by talazoparib (lower). Data are mean±SEM. Data across panels are Student’s t test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.: not significant. See also Figure S5.
Figure 6.
Figure 6.. Sequential treatment of PARPi and WEE1i is less toxic to normal cells in vitro.
(A) Representative photos of colony formation of MCF-10A and BM-MSC treated with different drugs as in Figure 5G stained with crystal violet (upper) or sulforhodamine B (lower). (B) MCF-10A and BM-MSC were treated as in Figure 5A and subjected to pH3 and PI flow cytometric analysis. (C, D) Representative images (C) and quantification (D) of MCF-10A cells treated as in Figure 5A and then stained for γH2AX, pH3 and DAPI. Scale bar, 50 μm. Data are mean±SEM. (E) MCF-10A were treated as in Figure 5A and subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. Scale bar, 10 μm. Data are mean±SD. (F, G) MCF-10A (F) and BM-MSC (G) were treated as in Figure 5A and subjected to DNA fiber analysis. Scale bar, 10 μm. Mean fork speed (kb min−1) is indicated. Data are mean±SD. (H) MCF-10A were treated with HU for 24 hr before incubation with EdU. Each dot represents a single cell, inset is %EdU+. (I) MCF-10A were treated as in Figure 5A with 50 μM HU for 24 hr and subjected to DNA fiber analysis. Mean fork speed (kb min−1) is indicated. Data are mean±SD. (J) BM-MSC were treated with HU for 24 hr before incubation with EdU. Each dot represents a single cell, inset is %EdU+. (K) BM-MSC were treated as in Figure 5A with 50 μM HU for 24 hr and subjected to DNA fiber analysis. Mean fork speed (kb min−1) is indicated. Data are mean±SD. (L) MCF-10A and MCF-10A with doxycycline inducible Cyclin E expression (MCF10A-iCCNE1) were incubated with or without doxycycline (Dox) for 24 hr and subjected to Western blot analysis. (M) MCF10A-iCCNE1 were incubated with or without Dox for 24 hr and analyzed by EdU flow cytometry. Each dot represents a single cell, inset is %EdU+. (N, O) MCF10A-iCCNE1 were incubated without Dox (N) or with Dox (O) for 24 hr and then treated as in Figure 5A, and subjected to DNA fiber analysis. Mean fork speed (kb min−1) is indicated. Data are mean±SD. Data across panels are Student’s ttest: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.: not significant. See also Figure S5.
Figure 7.
Figure 7.. Differential effects of sequential and concurrent PARPi and WEE1i treatment.
(A) Cells were treated with DMSO, talazoparib (250 nM), adavosertib (250 nM), sequentially or concurrently at indicated concentrations for 24 hr and western blotted. (B) MCF-10A were treated for 24 hr, drugs washed out, cells re-cultured and cell cycle assessed by flow cytometry. (C-E) MCF-10A were transfected with control or p53 siRNA for 48 hr. Cells were trypsinized and siRNA effect was assessed by western blot (C). Cell cycle was assessed by FITC BrdU as treated in Figure 6B (D), or for cell viability after 6 days (E). (F) OVCAR8 xenografts were treated as indicated. p values: one-way ANOVA. **p<0.01. (G) Analysis of talazoparib and olaparib combined (day 28) or sequential with WEE1i in OVCAR8 xenograft mice from day 42. Hemoglobin and red blood cells (RBC) from peripheral blood. Bone marrow cell count from bone marrow smear (Wright’s stain) per oil immersion field (100x). Student’s t test: n.s.: not significant, *p<0.05, **p<0.01. (H) Different drug-treated tumors were re-cultured in vitro for 5-7 days and then analyzed for sensitivity to PARP or WEE1 inhibition alone or in combination. Data across panels are mean±SEM.
Figure 8.
Figure 8.. Sequential administration of PARPi and WEE1i is effective and tolerable.
(A) Tumor volume and mouse body weight of PDX1 receiving indicated treatments (n=6 for each group). Group 4 and 7 were stopped on day 28 due to weight loss; group 3 and 6 were stopped on day 42 due to limited efficacy; group 2 was stopped on day 49 due to weight loss. Group 5 was continued throughout the study. Tumors from group 2, 3, 6, 7 were treated with group 5 sequential treatment strategy starting on day 70. (B) Tumor volume and mouse body weight of PDX2 receiving indicated treatments (n=5 for each group). Group 4 was stopped on day 21 for weight loss. (C) Tumor volume and mouse body weight of PDX3 receiving indicated treatments (n=5 for each group). On day 28, Group 1 was changed to intermittent dosing with adavosertib. Group 2 was changed to sequential treatment (5 days treatment and 2 days rest with each drug) on day 28. Group 3 was stopped due to weight loss on day 49. Because results from the two sequential regimens were similar, tumor volume data was combined and compared to adavosertib monotherapy. Differences in tumor volume data were determined by ANOVA at termination of study (p=0.041). Data across panels are mean±SEM. p values were determined by ANOVA. **p<0.01. See also Figure S6 and Tables S1-S5.
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