Human prostate tumor cell lines (22RV1 and DU145), breast cancer (MCF7) and lung cancer (H460) were obtained from American Type Culture Collection (Manassas, VA). Triple negative breast carcinoma (SUM149) was obtained from Asterand Biosciences (Detroit, MI). Chordoma cells (Ch22) were generously supplied by The Chordoma Foundation (Durham, NC). DU145 TNFRSF10B (TRAIL Receptor 2) CRISPR knockout and corresponding wild type cell pools were obtained from Synthego (Menlo Park, CA). Removal of TRAIL R2 in DU145 TNFRSF10B −/− cells was validated by Synthego via genome sequencing against wild type cells and confirmed by flow cytometry. All cell lines were passaged for less than 6 months, free of Mycoplasma and cultured at 37 °C/5% CO₂. 22RV1 and H460 were maintained in RPMI, DU145 were maintained in EMEM, Ch22 were maintained in DMEM, MCF7 were maintained in DMEM supplemented with insulin (2.5 μg/mL), and SUM149 were maintained in Ham’s F12 supplemented with insulin (2.5 μg/mL) and hydrocortisone (1 μg/mL). All media were supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 0.5% gentamicin, nonessential amino acids (final concentrations: L-alanine (8.9 mg/L), L-asparagine (15 mg/L), L-aspartic acid (13.3 mg/L), L-glutamic acid (14.7 mg/L), glycine (7.5 mg/L), L-proline (11.5 mg/L), L-serine (10.5 mg/L)), and L-glutamine (final concentration 2 mM).

Blood samples were obtained from normal healthy donors on the NCI IRB approved NIH protocol 99-CC-0168. Research blood donors were provided written informed consent. Donors were non-pregnant adults of 18 years or older, who met healthy blood donor criteria and tested negative for transfusion-transmissible diseases. Blood was collected by standard phlebotomy and apheresis techniques in ACD-A (Anticoagulant Citrate Dextrose Solution Formula A), and the cells were mixed with the donor’s plasma. All samples were de-identified. PBMCs were isolated from this apheresis product within 24 h of donor sample collection using gradient centrifugation with Lymphocyte Separation Medium (Mediatech, Manassas, VA). Cells were washed with PBS (Mediatech) and adjusted to a concentration of 5 × 10⁷ cells/ml with Fetal Bovine Serum (Atlanta Biologicals, Atlanta, GA) containing 10% DMSO prior to freezing. Median cell yield of PBMCs prior to freezing was typically 1 × 10⁹ total cells with 90–95% viability as determined by trypan blue exclusion. Cells were cryopreserved using CoolCell LX (Corning, Corning, NY) freezing containers at a cooling rate of 1 degree Celsius per minute in a − 80 degree Celsius freezer for 24 h, then placed in Liquid Nitrogen (vapor phase) for long-term storage. Median cell yield after thawing one 5 ml vial (frozen at 5 × 10⁷ cells/ml) was typically 1.5–2 × 10^8, with 90–98% viability. NK effector cells were isolated from PBMCs using the Human NK Cell Isolation (negative selection) Kit 130–092-657 (Miltenyi Biotech, San Diego, CA), according to the manufacturer’s protocol. Median NK cell yield after isolation was typically 0.5-1 × 10⁷ with 94–98% viability. Purified NK cells were incubated overnight in RPMI-1640 medium (Mediatech, Manassas, VA) containing 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), glutamine, and antibiotics (Mediatech) prior to use. Fetal bovine serum was pretested for support of NK proliferation and function. Median NK cell yield after overnight resting was typically 5-8 × 10⁶ with 90–96% viability.

High-affinity NK (haNK) cells are an NK cell line, NK-92, which has been engineered to express IL-2 and the high-affinity valine (V) CD16 allele as previously described [5–7]. haNK cells were supplied by NantBioScience (Culver City, CA) through a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI) and cultured in X-Vivo-10 medium (Lonza, Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA) at a concentration of 5 × 10⁵ cells/ml.

Avelumab and matching IgG1 isotype control were obtained from EMD Serono (Rockland, MA) as part of a CRADA with the NCI as previously described [8, 9], and used at 2 μg/ml final concentration. Matching IgG1 cetuximab was obtained from Bristol Myers-Squibb (New York, NY), and was used at 1 μg/ml final concentration. Anti-human CD16-neutralizing mAb was obtained from Thermo Fisher Scientific (Waltham, MA). Anti-TRAIL-R antibody (KillerTRAIL) was obtained from Enzo Life Sciences (Farmingdale, NY). For Western blot, cell lysates were stained with antibodies for phospho-TBK1 (Cell Signaling, Danvers, MA #4947 s), TBK1 (Cell signaling #3504), IRF-3 (Cell Signaling #3013), phospho-IRF3 (Cell Signaling #5483 s), cGAS (Sigma-Aldrich, HPA031700), phospho-STING (Cell Signaling #85735) and vinculin (Sigma Aldrich, v9131). Rabbit polyclonal antibody targeting residues 324–340 of human STING was a kind gift from Glen N. Barber, Ph.D., University of Miami, Miami, FL. Secondary antibodies used were IRDye 800CW goat anti-mouse (LI-COR Biosciences, Lincoln, NE) and IRDye 680RD goat anti-rabbit (LI-COR Biosciences).

Olaparib was obtained from Selleck Biochemical (Houston, TX). For cytotoxic assays, adherent tumor cells in log-growth phase based on real-time cell analysis were exposed for 24 h to olaparib (20 μM). For flow cytometry, Western blots, and NanoString, cells at 50–80% confluency were exposed to olaparib (20 μM) while incubating at 37 °C/5% CO₂. For select experiments, NK cells were treated with 50 ng/mL IL-15/IL-15Rα superagonist N-803 (formerly known as ALT-803) obtained from Altor BioScience (Miramar, FL, now NantBioScience) as part of a CRADA with the NCI. After drug exposure as described above, cells were harvested, and viable cells were counted by trypan blue exclusion using a Cellometer Auto T4 automated cell counter (Nexcelom Bioscience, Lawrence, MA).

Prostate carcinoma cell lines (22RV1 or DU145) received 24 h pretreatment with 20 μM olaparib. NK cells were plated at a 5:1 NK to target cell ratio, with or without ADCC-mediating antibodies avelumab (anti-PD-L1) and cetuximab (anti-EGFR). Cell lysis was monitored for 36 h on an xCelligence RTCA impedance-based assay. DU145 TNFRSF10B −/− and wild type cell pools were treated, plated with NK, and monitored using the method described above. In select experiments, NK cells were incubated for 2 h with anti-CD16 antibody (Thermo Fisher Scientific) or Concanamycin A (Sigma-Aldrich, St. Louis, MO).

Tumor cells were cultured as described and treated in the presence or absence of olaparib (20 μM) for 60 h (to replicate the conditions in the assays, 24 h pretreatment and 36 h data collection period). Cells were then harvested and stained with the following antibodies: TRAIL-R2-APC (BD Biosciences, Franklin Lakes, NJ), PD-L1-PE (BD Biosciences), EGFR-PE (BD Biosciences). TRAIL R2 knockout in DU145 TNFRSF10B −/− versus wild type cell pools was confirmed using the same treatment method and stained with TRAIL-R2-PE (BD Biosciences).

NK cells were isolated from PBMCs as described and treated in the presence or absence of olaparib (20 μM) overnight in RPMI-1640 supplemented with 10% FBS. Cells were then stained with the following antibodies: CD16-PE (BD Biosciences), CD56-PE (BD Biosciences), DNAM1-PE (BD Biosciences), NKG2D-PE (BD Biosciences), NKp44-PE (BD Biosciences), NKp46-PE (BD Biosciences), Granzyme-PE (BD Biosciences), Perforin-PE (BD Biosciences).

Cell viability of both tumor and NK cells was examined using trypan blue staining prior to data acquisition on a FACSCalibur flow cytometer. Live cells were gated via forward and side scatter. Data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR). Isotype control staining was < 5% for all samples analyzed. In selected experiments, target cells (22RV1 and DU145) were cultured as described and treated for 24 h with 20 μM olaparib. The tumor cells were then co-cultured at a 1:1 ratio with either NK cells or haNK cells for 24 h prior to staining using the Image-iT LIVE Green Poly Caspases Detection Kit for microscopy (Thermo Fisher Scientific). Per the manufacturer’s protocol, cells were additionally stained for DAPI (Perkin Elmer, Waltham, MA) and CD56-APC (BD Biosciences), which allowed for the exclusion of human NK cells and haNK cells from analysis. Data were acquired on the Amnis Imagestream (EMD Millipore, Burlington, MA).

Cells were treated with 20 μM olaparib for 12 h at 37 °C/5% CO₂. The Apoptosis RT² Profiler PCR Array (Qiagen, Germantown, MD) was used to identify modulation of genes related to apoptosis according to the manufacturer’s instructions. The raw data tables for the apoptotic array are depicted in Additional file 1: Table S2.

NanoString data are available in the GEO database under accession number GSE121682. The nCounter® PanCancer Pathways Panel (NanoString Technologies, Seattle, WA) was performed according to the manufacturer’s instructions on 22RV1 and DU145 cells after 6 h, 12 h, and 18 h of treatment with 20 μM olaparib. Seven hundred seventy genes were surveyed as part of this multi-gene, RNA-based analysis and a 3-fold change cutoff was used to identify initial genes of interest. Curated genes were chosen based on both NanoString and apoptotic array data for inclusion in a STRING analysis of protein-protein interactions [10, 11].

Significant differences in the distribution of cell populations by flow cytometry analyses were examined using the Kolmogorov-Smirnov test and considered biologically significant if they differed by ≥20% relative to respective controls or if mean fluorescence intensity (MFI) underwent a 3-fold increase. Significant differences between two treatment groups were determined by a 2-tailed t test using GraphPad Prism 7.0 software. Differences were considered significant when the p value was < 0.05.

Olaparib is currently FDA approved only for ovarian and HER2-negative breast cancer in patients with germline BRCA mutations [12]. We focused on two prostate carcinoma cell lines, BRCA mutant 22RV1 and BRCA WT DU145; both were exposed to olaparib in vitro. Olaparib exposure significantly increased NK-mediated lysis as assessed by real-time cell analysis (RTCA) (Fig. 1a). NK-mediated killing of treated cells was increased by 1.3-fold compared to olaparib treatment alone in the BRCA mutant cell line 22RV1 after 12 h (p < 0.0001) and 1.6-fold difference persisted at 36 h (p = 0.0001) (Fig. 1b). Surprisingly, olaparib exposure also significantly increased NK-mediated lysis of the BRCA WT line DU145 (Fig. 1c). Lysis was increased by 3.4-fold (p = 0.0071) at 12 h and by 1.6-fold at 36 h (p = 0.0011) (Fig. 1d).Fig1Fig. 1

NK-mediated lysis of prostate carcinoma cells is increased following exposure to olaparib. a Real-time impedance-based cell analysis of BRCA mutant prostate carcinoma (22RV1) cells in the presence or absence of olaparib (ola) and NK. b Lysis of BRCA mutant cells treated with or without olaparib and NK compared to control at 12 h and 36 h. c Real-time impedance-based cell analysis of BRCA WT prostate carcinoma (DU145) cells in the presence or absence of olaparib and NK. d Lysis of BRCA WT cells treated with olaparib and NK compared to control at 12 h and 36 h. These experiments were performed with four different human NK donors with similar results. p < 0.01**, p < 0.001***, p < 0.0001****

We next examined the effect of olaparib exposure on NK-mediated ADCC. The EGFR+ line 22RV1 was treated with cetuximab, and the EGFR+ and PD-L1+ line DU145 was treated with either cetuximab or avelumab (Fig. 2a). In the presence of cetuximab, olaparib exposure significantly increased NK-mediated lysis of 22RV1 compared to olaparib only treated cells as early as 12 h (1.2-fold increase, p < 0.0001), reaching a 2.8-fold increase at 36 h (p < 0.0001) (Fig. 2b). NK-mediated lysis of cetuximab-treated BRCA WT DU145 cells was significantly increased after olaparib exposure (Fig. 3a) by 2.1-fold at 12 h (p = 0.0034) and by 1.7-fold at 36 h (p < 0.0001) (Fig. 3b). Olaparib- and avelumab-treated cells demonstrated a 1.5-fold increase in NK-mediated lysis at 12 h (p = 0.0010) and a 1.3-fold increase at 36 h (p < 0.0001) compared to cells treated with only avelumab (Fig. 3c).Fig2Fig. 2

NK-mediated lysis of BRCA mutant prostate carcinoma cells treated with olaparib is further increased by the addition of an ADCC-mediating antibody. a Real-time impedance-based cell analysis of BRCA mutant (mut) prostate carcinoma (22RV1) cells in the presence or absence of olaparib (ola) and human NK cells, treated with either cetuximab (cet) or isotype control. b 22RV1 cells lysed with or without olaparib pretreatment and NK cells, treated with either cetuximab or isotype control at 12 h and 36 h. This experiment was performed with four different human NK donors with similar results. p < 0.001***, p < 0.0001****

To confirm that the increased NK-cell lysis of tumor cells exposed to olaparib in the presence of avelumab or cetuximab is specifically mediated by ADCC, NK effectors were exposed to a CD16-neutralizing antibody prior to being used in an in vitro ADCC assay. Lysis of both olaparib-treated and untreated cells was significantly inhibited in the presence of CD16-neutralizing antibody, confirming that the augmented NK-cell lysis of tumor cells exposed to olaparib in the presence of mAbs is mediated by ADCC in both BRCA mutant (p < 0.0001) (Fig. 4a) and BRCA wildtype (p < 0.0001) cells (Fig. 4a and d). Functional assays in which NK cells were treated overnight with olaparib and washed before plating were also performed; there was no improved killing of tumor cells, confirming that the PARPi was affecting the phenotype of the target cells rather than the NK cells (Additional file 2: Table S1).Fig4Fig. 4

Olaparib treatment enhances ADCC using both cetuximab and avelumab without modulation of mAb targets EGFR and PD-L1. a Treatment with cetuximab (cet) significantly increased NK-induced lysis of olaparib (ola)-treated BRCA mutant prostate carcinoma (22RV1) cells at 12 h. The addition of anti-CD16 antibody neutralized this increase, confirming that the increased lysis is attributable to ADCC. b STING is not expressed in 22RV1 either before or after olaparib treatment. c Olaparib treatment did not result in significant modulation of EGFR expression on 22RV1 cells as measured by flow cytometry. d Treatment with cetuximab increased NK-induced lysis of olaparib-treated BRCA WT prostate carcinoma cells (DU145) cells. Role of anti-CD16 antibody on increased lysis attributable to ADCC. e The PD-L1+ cell line DU145 also underwent NK-induced ADCC in the presence of the anti-PD-L1 antibody avelumab (ave). Lysis of DU145 cells after 12 h in the presence or absence of olaparib and NK, treated with either avelumab or isotype control is shown. f STING was upregulated in DU145 following exposure to olaparib. g Olaparib treatment did not result in significant modulation of EGFR expression in DU145 cells as measured by flow cytometry. h Olaparib treatment did not result in significant modulation of PD-L1 expression in DU145 cells as measured by flow cytometry. These experiments were performed twice with similar results. p < 0.05*, p < 0.0001****

To establish a potential mechanism of increased NK-mediated target cell lysis following olaparib exposure, we performed a NanoString RNA analysis on tumor cells treated with olaparib for 6, 12, and 18 h (Fig. 5a). The initial NanoString screen, utilizing a three-fold cutoff at any timepoint, identified 102 genes of interest in DU145 (67 downregulated and 35 upregulated) and 89 genes of interest in 22RV1 (52 downregulated and 37 upregulated) (Fig. 5b). In addition, an apoptotic array identified genes related to cell growth and apoptosis; 9 select genes (4 downregulated and 5 upregulated) of interest were identified in DU145 and 11 (5 downregulated and 6 upregulated) were identified in 22RV1 (Fig. 5c, Additional file 1: Table S2). Modulation of the tumor necrosis factor superfamily (TNFSF) was observed in both cell lines. Only one downregulated gene was identified, IL-10. IL-10 has a distinct role upregulating human NK function [14]. The apoptotic gene array also identified changes in the expression levels of various members of TNFSF in both cell lines following treatment with olaparib. These data, taken together, indicated that the nexus of these affected genes is death receptor TNFRSF10B (TRAIL-R2), which was found by NanoString to be upregulated by 2-fold in 22RV1 and by 20% in DU145 (Fig. 5d).Fig5Fig. 5

Olaparib treatment induces increased expression of gene TNFRSF10B (TRAIL-R2) in both BRCA mutant and wildtype prostate carcinoma. a Heatmap demonstrating genes modulated by at least 3-fold after olaparib treatment as identified by the NanoString nCounter® PanCancer Pathways Panel. TNFRSF10B (TRAIL-R2) was upregulated in both cell lines (by 2-fold in 22RV1 and by 20% in DU145). b Summary of modulated genes identified in NanoString analysis. c Summary of modulated genes identified in Apoptosis RT² Profiler PCR Array with fold-change denoted in parentheses. d STRING network of protein-protein interactions of a curated set of modulated genes identified by both NanoString and apoptotic array. TNFRSF10B (TRAIL-R2; red box) identified as the nexus of both sets

Death receptors activate a caspase cascade that ultimately results in apoptosis [15]. Using imaging flow cytometry, we interrogated the activation of caspases-1, − 3, − 4, − 5, − 6, − 7, − 8 and − 9 in target cells following treatment with olaparib and exposure to human NK cells from healthy donors. Olaparib-treated BRCA mutant 22RV1 cells demonstrated a 1.7-fold increase in caspase activation compared to untreated cells after exposure to two different healthy NK donors. Olaparib treatment increased caspase activation 1.9-fold in BRCA WT DU145 after exposure to the first donor (Fig. 6a), and 1.3-fold after exposure to NK cells from the second donor as shown by fluorochrome inhibitor of caspases (FLICA) reagent positivity. Representative images demonstrate an NK cell lysing a target cell by initiating a caspase cascade (Fig. 6a).Fig6Fig. 6

TRAIL-R2 is functionally upregulated upon exposure to olaparib and leads to increased caspase cascade activation. a Representative image demonstrated NK cell lysing olaparib-treated BRCA WT target cell by activating caspase cascade and a representative graph demonstrating increased caspase activation as identified by a FLICA reagent following administration of olaparib in both BRCA WT and BRCA mutant cells. b Expression of TRAIL-R2 on 22RV1 was upregulated from 36% (MFI 104) to 65% (MFI 151) following a 48 h treatment with olaparib. c BRCA mutant prostate carcinoma (22RV1) cell lysis with or without olaparib (ola) and KillerTRAIL antibodies after 36 h. d Expression of TRAIL-R2 on DU145 was upregulated from 3% (MFI 28) to 96% (MFI 103). e BRCA WT prostate carcinoma (DU145) cell lysis with or without olaparib and KillerTRAIL antibodies after 36 h. f Expression of TRAIL-R2 on Wild-Type and TRAIL-R2 CRISPR knockout DU145 cells. g Functional confirmation of the involvement of TRAIL-R2. DU145 and TRAIL-R2 CRISPR knockout DU145 cell lysis with or without olaparib and NK cells after 36 h. Depicted is fold-change in percent lysis. These experiments were performed twice with similar results. p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001***

We broadened our studies to include two other immunomodulatory agents, haNK cells and N-803 (formerly known as ALT-803). N-803 is an IL-15/IL-15Rα superagonist known to increase the killing capacity of human NK cells. Lysis mediated by N-803-treated NK cells was increased by 1.7-fold at 12 h (p = 0.0001) and by pretreating target cells with olaparib; a significant difference persisted at 36 h (Fig. 7a). DU145 cells were also exposed to high-affinity NK cells (haNK) in the presence and absence of olaparib. After 36 h, olaparib-treated cells had 1.7-fold increased lysis (p = 0.001) (Fig. 7b). When cells were also exposed to avelumab, lysis was increased 1.4-fold after 36 h (p < 0.0001) (Fig. 7b).Fig7Fig. 7

Lysis of target cells by natural killer (NK) cells treated with N-803 or high-affinity NK cells (haNK) can be further increased with the addition of olaparib. a Lysis of BRCA wildtype prostate carcinoma (DU145) cells at 12 h and 36 h with or without pretreatment with olaparib (ola) and NK cells incubated in the presence or absence of an IL-15/IL-15Rα superagonist (N-803). b Lysis of DU145 at 36 h by haNK cells in the presence or absence of olaparib and avelumab (ave). p < 0.05*, p < 0.01**, p < 0.0001****

The above studies indicate that olaparib may have an immunomodulatory role in prostate cancer as well as its approved indications of ovarian and breast cancer. We further expanded our studies to include additional human cancer cell lines. These included MCF7 (an ER+ breast cancer) and H460 (a non-small cell lung cancer), both of which showed increased NK-mediated lysis of olaparib-treated cells and further increased lysis with the addition of the ADCC-mediating antibody cetuximab (Fig. 8a and b). H460 expresses PD-L1 in addition to EGFR and therefore showed increased ADCC with both avelumab and cetuximab (Fig. 8b). We also investigated two cell lines for which there is no effective standard of care: SUM149 (triple negative breast cancer) and Ch22 (chordoma, a rare sarcoma that originates from the remnants of the notochord). We observed increased NK-mediated lysis of these cells after olaparib treatment (Fig. 8c and d). The EGFR+ line SUM149 showed further increased lysis after the addition of cetuximab (Fig. 8c). Ch22 is both EGFR and PD-L1 positive and demonstrated an increase in lysis after the addition of both cetuximab and avelumab (Fig. 8d).Fig8Fig. 8

Olaparib increases NK-mediated target cell lysis and ADCC in a diverse set of tumors. A variety of tumor types demonstrated increased NK cell-mediated lysis as measured by real-time impedance. a Lysis of estrogen receptor positive (ER+) breast carcinoma (MCF7) in the presence or absence of olaparib and NK, treated with cetuximab or isotype control. b Lysis of non-small cell lung carcinoma (NSCLC) (H460) in the presence or absence of olaparib and NK, treated with cetuximab, avelumab (ave) or isotype control. c Lysis of triple negative breast carcinoma (SUM149) in the presence or absence of olaparib and NK, treated with cetuximab (cet) or isotype control. d Lysis of chordoma (Ch22) in the presence or absence of olaparib and NK, treated with cetuximab, avelumab or isotype control. p < 0.01**, p < 0.001***, p < 0.0001****