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. 2017 Feb 1;31(3):318-332.
doi: 10.1101/gad.290957.116. Epub 2017 Feb 27.

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells

Stephanie A Yazinski  1 , Valentine Comaills  1 , Rémi Buisson  1 , Marie-Michelle Genois  1 , Hai Dang Nguyen  1 , Chu Kwen Ho  1 , Tanya Todorova Kwan  1   2 , Robert Morris  1 , Sam Lauffer  1   3 , André Nussenzweig  4 , Sridhar Ramaswamy  1 , Cyril H Benes  1 , Daniel A Haber  1   2 , Shyamala Maheswaran  1 , Michael J Birrer  1   3 , Lee Zou  1   5
Affiliations

ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells

Stephanie A Yazinski et al. Genes Dev. .

Abstract

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.

Keywords: ATR; BRCA-deficient cancer; PARP inhibitor.

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Figures

Figure 1.
Figure 1.
ATRis have a unique ability to overcome the PARPi resistance of BRCA1-deficient cancer cells. (A) Schematic of derivation of PARPi-resistant cells from the parental BRCA1-deficient UWB1.289 ovarian cancer cell line. (B) Viability assay of UWB1 (parental), UWB + B1 (complemented with wild-type BRCA1), and derived PARPi-resistant cell lines after 6 d of increasing doses of PARPi (olaparib) treatment. n = 3 replicates. Error bars represent SD. (C) Gene set enrichment analysis of RNA sequencing data from UWB1, SYr12, and SYr13 cell lines. (D) Mini drug screen performed on UWB1 + B1, UWB1, and resistant lines (SYr12 and SYr13). Cells were treated with increasing doses of ATRi (VE-821, 0–0.625 µM), ATMi (KU55933, 0–0.625 µM), DNA-PKi (NU7441, 0–0.625 µM), Chk1i (MK-8776, 0–0.5 µM), or Wee1i (MK-1775, 0–0.25 µM) in the absence or presence of 2.0 µM PARPi (olaparib). Color-coding denotes the level of viability (green [100% viability] to red [0% cell viability]) relative to DMSO treatment. (E) IC50s of the indicated cell lines to PARPi (olaparib) were measured after 6 d of olaparib treatment in the presence of increasing concentrations of ATRi (VE-821). (F) The indicated cell lines were treated with ATRi (VE-821), PARPi (olaparib), or ATRi and PARPi for 7 d. Fractions of cells undergoing cell death (propidium iodide and/or annexin V-positive) were measured. n = 3 replicates. Error bars represent SD. (G) The indicated cell lines were treated with increasing doses of PARPi (0–25 µM) in combination with increasing doses of either ATRi (0–2.5 µM) or cisplatin (0–1.25 µM) in triplicate, and an overall Bliss score and P-value were calculated. Bliss score >0, synergistic; Bliss score = 0, additive; Bliss score <0, antagonistic.
Figure 2.
Figure 2.
ATRi broadly overcomes acquired and pre-existing PARPi resistance in multiple BRCA1-deficient cancer cell lines of distinct origins. (A) Colony formation assay of the indicated cell lines following 14 d of treatment with DMSO, 1 µM PARPi (olaparib), or PARPi and 0.313 µM ATRi (VE-821). Cells were stained with crystal violet. (B) Cell viability following 6 d of treatment across all cell lines using DMSO, PARPi (olaparib), ATRi (VE-821), or PARPi and ATRi. (C) Viability assay of mouse ovarian cancer cell lines ([T2] non-BRCA mutant; [BR5] BRCA1-deficient; [BR5-R1] BRCA1-deficient and PARPi-resistant) after 6 d of treatment with increasing doses of PARPi (olaparib) in the absence or presence of 1.0 µM ATRi (VE-821). n = 3 replicates. Error bars represent SD. (D) Viability assay of the BRCA1-deficient HCC1937 (de novo PARPi-resistant) and BRCA1-complemented HCC1937 + B1 cell lines after 6 d of treatment with increasing doses of PARPi (olaparib) and the indicated doses of ATRi (VE-821). n = 3 replicates. Error bars represent SD. (E) Colony formation assay of UWB1 + B1 and UWB1 cell lines after 45 d of treatment with ATMi, ATRi, PARPi, the ATMi + PARPi combination, or the ATRi + PARPi combination. Cells were stained with crystal violet.
Figure 3.
Figure 3.
The HR function of BRCA1, but not PALB2–BRCA2, is partially bypassed in BRCA1-deficient cancer cells. (A) Fractions of RAD51 focus-positive cells (more than five foci per cell) following 24 h of 10 µM PARPi (olaparib) treatment in the indicated cell lines. n = 3 replicates. Error bars represent SD. (B) The indicated cell lines were treated with siControl, siBRCA1, or siBRCA2 and irradiated with 10 Gy of IR, and fractions of RAD51 focus-positive cells were measured 4 h later. n = 3 replicates. Error bars represent SD. (C) The indicated cell lines were treated with siControl, siBRCA1, or siBRCA2 and irradiated with 10 Gy of IR, and fractions of RAD51 focus-positive cells were measured 4 h later. n = 3 replicates. Error bars represent SD. (D) The indicated cell lines were transfected with siControl, siPALB2, or siBRCA2 and treated with 10 µM PARPi (olaparib) for 24 h, and fractions of RAD51 focus-positive cells were determined. n = 3 replicates. Error bars represent SD. (E) The PARPi-resistant cell lines were transfected with siControl, siPALB2, or siBRCA2 and treated with increasing doses of PARPi (olaparib) for 6 d, and cell viability was measured. n = 3 replicates. Error bars represent SD.
Figure 4.
Figure 4.
ATR functions in HR downstream from BRCA1 and remains indispensable when BRCA1 is bypassed. (A) U2OS cells were treated with the indicated doses of ATMi (KU55933) or ATRi (VE821 or AZ20). The efficiency of HR was measured using the DR-GFP reporter 48 h after I-SceI transfection. n = 3 replicates. Error bars represent SD. (B) U2OS cells were treated with DMSO or ATRi and irradiated with 4 Gy of IR, and fractions of BRCA1 focus-positive cells were measured 2 h later. n = 3 replicates. Error bars represent SD. (C) Cells were treated with the indicated doses of ATRi (VE-821) and irradiated with 10 Gy of IR, and fractions of BRCA2 focus-positive cells (more than five foci per cell) were measured 4 h later. n = 3 replicates. Error bars represent SD. (D) U2OS cells were transfected with siControl, si53BP1, siBRCA1, or si53BP1 and siBRCA1. Transfected cells were treated with DMSO, 10 µM PARPi (olaparib), or PARPi and 0.3 µM ATRi (VE-821) for 24 h. Fractions of RAD51 focus-positive cells were determined. n = 3 replicates. Error bars represent SD. (*) P < 0.05; (**) P < 0.01. (E) U2OS cells or U2OS-derived 53BP1 knockout cells were transfected with siControl or siBRCA1 and treated with increasing doses of PARPi (olaparib) in the absence or presence of 62.6 nM ATRi (VE-821) for 6 d. Cell viability was measured. n = 3 replicates. Error bars represent SD.
Figure 5.
Figure 5.
ATRi blocks BRCA1-independent HR in PARPi-resistant cells by inhibiting PALB2–BRCA2 localization to DNA breaks. (A) The indicated cell lines were treated with ATRi (VE-821), PARPi (olaparib), or ATRi and PARPi for 24 h. Fractions of RAD51 focus-positive cells were measured. n = 4 replicates for SYr12; n = 3 replicates for other samples. Error bars represent SD. (B) The indicated cell lines were treated with DMSO or 10 µM ATRi (VE-821) and irradiated with ultraviolet (UV) laser, and fractions of cells showing BRCA2 staining in γH2AX stripes were measured 1 h later. n = 4 replicates for SYr12; n = 3 replicates for other samples. Error bars represent SD. (C) The indicated cell lines were treated with 1 µM camptothecin (CPT) for 2 h. The intensity of phosphorylated RPA32 (p-RPA) (S4/S8) was measured by immunofluorescence, with each cell plotted individually. n = 550 cells for each condition. (D) The indicated cell lines were treated with 1 µM CPT or CPT and 10 µM ATRi for 2 h. Levels of p-RPA (S4/S8) were analyzed by Western blot.
Figure 6.
Figure 6.
ATRi reactivates degradation of stalled forks in PARPi-resistant cells. (A) DNA fiber analysis of stalled replication forks. Newly synthesized DNA was sequentially labeled with 50 µM CldU for 30 min and 100 µM IdU for 30 min. Cells were subsequently treated with 4 mM HU for 5 h, and lengths of CIdU- and IdU-labeled DNA fibers were measured. The IdU/CldU ratio was binned in increments of 0.2 and fit to a Gausian curve using Prism software. At least n = 100 fibers were measured for each condition; experiments were completed in triplicate. (B) DNA fiber analysis of stalled forks as in A, with each fiber plotted individually. The indicated cell lines were untreated or treated with 4 mM HU, HU and 10 µM ATRi (VE-821), or HU, ATRi, and 50 µM mirin for 5 h. Bars represent the median IdU/CldU ratios. n = 150 fibers for each condition; experiments were completed in triplicate. Significance was determined by Mann-Whitney test. (****) P < 0.0001. (C) The indicated cell lines were treated with 4 mM HU in the absence or presence of 10 µM ATRi (VE-821) for 5 h. The resulting genomic DNA from 5 × 105 cells for each condition was subjected to pulsed-field gel electrophoresis (PFGE) to measure DNA fragmentation. (D) The indicated PARPi-resistant cell lines were treated with 4 mM HU for 5 h in the absence or presence of 10 µM ATRi (VE-821). The levels of chromatin-bound RAD51 and RPA32 were analyzed with KU70 as a loading control. (E) The indicated cell lines were pulsed with EdU for 30 min and then collected or treated with 4 mM HU in the absence or presence of 10 µM ATRi (VE-821) for 5 h. The association of RAD51 with nascent DNA was analyzed by iPOND (isolation of proteins on nascent DNA) with histone H4 as a loading control. (F) SYr12 cells were transfected with siControl, siXRCC2, or siXRCC3 and treated with 4 mM HU for 5 h in the absence or presence of 10 µM ATRi (VE-821). The levels of chromatin-bound RAD51 were analyzed with KU70 as a loading control.
Figure 7.
Figure 7.
ATRi reactivates fork degradation in tumor cells derived from PARPi-resistant BRCA-deficient patients. (A) DNA fiber analysis of tumor cells from a BRCA1-deficient PARPi-resistant ovarian cancer patient and a non-BRCA ovarian cancer patient, with each fiber plotted individually after no treatment, treatment with 4 mM HU, or treatment with HU and 10 µM ATRi (VE-821). Red bars represent the median IdU/CldU ratios. n = 125 for non-BRCA tumor cells; n = 215 for BRCA1-deficient PARPi-resistant tumor cells; experiments were performed in duplicate. Significance was determined by Mann-Whitney test. (**) P < 0.01. (B) DNA fiber analysis of PDX tumor cells derived from patients in A. Each fiber was plotted individually after no treatment, treatment with 4 mM HU, or treatment with HU and 10 µM ATRi (VE-821). Red bars represent the median IdU/CldU ratios. n = 185. Experiments were performed in duplicate. Significance was determined by Mann-Whitney test. (**) P < 0.01; (****) P < 0.0001. (C) DNA fiber analysis of circulating tumor cells (CTCs) from a BRCA2-deficient breast cancer patient and a non-BRCA breast cancer patient as in A, with each fiber plotted individually after no treatment, treatment with 4 mM HU, or treatment with HU and 10 µM ATRi (VE-821). Significance was determined by Mann-Whitney test. (****) P < 0.0001. (D) Model of how PARPi-resistant BRCA1-deficient cancer cells bypass BRCA1 in two sequential steps. In step 1, the HR function of BRCA1 is partially bypassed. This partial bypass of BRCA1 allows cancer cells to survive the lack of BRCA1 but is not sufficient to confer PARPi resistance (e.g., UWB1). In step 2, when cancer cells are under the selective pressure of PARPis, the HR function of BRCA1 is further bypassed in some cells (e.g., SY12). Furthermore, the function of BRCA1 in fork protection is commonly bypassed (e.g., SYr9, SYr12, SYr13, and SYr14). Our findings show that the bypasses of both BRCA1 functions in HR and fork protection contribute to PARPi resistance in cancer cells. This model explains why ATRi, which blocks both BRCA1-independent HR and fork protection, has a unique ability to overcome PARPi resistance.
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