PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial–mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.

Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742–57. ©2017 AACR.

The proto-oncogene MYC is amplified in several different cancer types, including triple-negative breast cancer (TNBC), pancreas, ovarian, and prostate cancers (1). The variety of cellular processes that MYC regulates (e.g., metabolism, cell cycle, metastasis, and DNA repair) suggests a crucial role in the oncogenic transformation of MYC-driven cancers, resulting in an addiction to MYC expression (2). One of the mechanisms by which MYC mediates oncogenic potential is induction of replication stress, causing increased γH2AX expression and subsequent upregulation of homologous recombination (HR) via increased RAD51 activity (3). However, MYC's specific role in regulating DNA repair mechanisms as a function of response to therapy has often been overlooked. Treatment with PARP inhibitors (PARPi) is associated with a favorable response in tumors lacking BRCA1/2 function. Several studies have recently demonstrated that BRCA mutant (BRCAmut) cancer cells frequently retain an intrinsic RAD51-dependent HR repair mechanism that imparts de novo resistance to PARPis and platinum therapeutic agents (4–6). In addition, upregulation of the DNA repair pathway is often overlooked as a sign of decreased response to chemotherapy. Moreover, because RAD51 expression is involved in several non-DNA repair pathways (e.g., increased metastasis of TNBC; ref. 7), we hypothesized that MYC-positive tumors upregulate the HR DNA repair pathway causing resistance to DNA-damaging agents including PARPis. Therefore, using RAD51 as a marker of de novo resistance to PARPis we classified TNBC breast cancer cell lines as either PARPi-sensitive or -resistant independent of BRCA status. Furthermore, we showed that MYC directly regulates HR via several DNA repair proteins including RAD51, whereas inhibition (or downregulation) of MYC expression induces PARPi-sensitivity independent of BRCA status. These findings suggest that TNBC patients with high c-myc and RAD51 expression, which have poor prognoses and are unresponsive to neoadjuvant chemotherapy, are likely to be sensitive to agents that downregulate c-myc (e.g., dinaciclib) and PARPis independent of BRCA mutational status.

Cell lines and culture conditions

All parental cancer cell lines used in this study were purchased from the ATCC. The TNBC cell lines MDA-MB-231, MDA-MB-468, HCC1937, HCC1806, SUM149, SUM1315, MDA-MB-436, and MDA-MB-157 and human mammary epithelial cell lines MCF-10A were cultured as described previously (8, 9), The non–small cell lung cancer cell lines PC3, DU145, A549, Calu-1, H1299, and H1993 were cultured in RPMI medium in the presence of 10% FBS. The head and neck squamous cell carcinoma cell lines OVCAR3, 59M, FUOV1, BxPC3, PANC-1, HCT116, and SW620 were cultured in DMEM in the presence of 10% FBS and growth factors. All cells were free of mycoplasma contamination. Cell lines were identified and authenticated according to karyotype and using short tandem repeat analysis in the MD Anderson Characterized Cell Line Core facility every 6 months.

Acquired treatment resistance

Cells were cultured in normal growth media supplemented with the PARPi niraparib at increasing concentrations (MDA-MB-436, 0.1 nmol/L–2.0 μmol/L; HCC1806, 0.5–15.0 μmol/L) for 6 months. At the final concentrations, cells were maintained in media supplemented with niraparib. All experiments were conducted in the absence of niraparib-supplemented media unless otherwise noted.

siRNA

In vitro cell transfections were carried out in 6-well plates seeded (5 × 104) and then transfected with 5 μmol/L MYC siRNA(4609) (SMART pool; Dharmacon; 5′-ACGGAACUUGUGCGUAA-3′, 5′-GAACACACAACGUCUUGGA-3′, 5′-AACGUUAGCUUCACCAACA-3′, and 5′-CGAUGUUGUUUCUGUGGAA-3′), 5 μmol/L RAD51 (5888), 5 μmol/L RAD51 siRNA (SMART pool; 5′-UAUCAUCGCCCAUGCAUCA-3′, 5′-CUAAUCAGGUGGUAGCUCA-3′, 5′-GCAGUGAUGUCCUGGAUAA-3′, and 5′-CCAACGAUGUGAAGAAAAUU-3′), or a nontargeting pool 5 μmol/L siRNA. Cells were incubated at 36°C in 5% CO2 for 48 hours, and the media were removed. Briefly, in vitro siRNA transfections were performed using the jetPRIME transfection reagent (Polyplus), following the manufacturer's protocol.

Short hairpin and open reading frame constructs and viral infection

The pGIPZ-shRNA and MYC overexpression plasmids were purchased from Dharmacon and used to produce lentiviruses (shBRCA1 and sh53BP1) by transfecting 293T cells shRNA plasmids. TNBC cells were infected with viral particles in complete media in the presence of hexadimethrine bromide (Polybrene, 8 mg/mL; EMD Millipore) overnight. The next day, media containing the viruses were washed and replaced with fresh media. Puromycin (Invitrogen) selection for infected cells was performed for 7 days.

High-throughput survival assay

Cells were seeded at a density of 1,000 cells/well in a 96-well plate and treated with single drugs or drug combinations at the indicated concentrations for 72 hours. Cells were then released into a complete drug-free medium for 9 days, and the medium was changed every 48 hours. On the day of cell harvest, 100 μL/well 2.5 mg/mL MTT (Sigma) was added to the serum-free medium and incubated at 37°C for 3 to 4 hours. After incubation, the medium was removed, and 100 μL of a solubilization solution (0.04 mmol/L HCl and 1% SDS in isopropyl alcohol) was added to each well. Plates were lightly rocked at room temperature for 1 hour and read using a plate reader (Gen5 Epoch Microplate Spectrophotometer and software program; BioTek) at a wavelength of 590 nm. Combination indices were calculated using the CalcuSyn software program.

Quantitative RT-PCR

Total RNA was isolated from cell cultures using an RNeasy Kit with DNase treatment according to the manufacturer's protocol (Qiagen). Two micrograms of the RNA samples was reverse-transcribed using a cDNA Synthesis Kit (Applied Biosystems). RT-PCR was done with aliquots of cDNA samples mixed with SYBR Green Master Mix (Sigma). Reactions were carried out in triplicate. The fold difference in transcripts was calculated using the ΔΔCt method with GAPDH as a control. The following primers were used: MYC forward, 5′- GGCTCCTGGCAAAAGGTCA; MYC reverse, 5′-CTGCGTAGTTGTGCTGATGT-3′; RAD51 forward, 5′-CAACCCATTTCACGGTTAGAGC-3′; RAD51 reverse, 5′-TTCTTTGGCGATAGGCAACA-3′; BRCA2 forward, 5′-CACCCACCCTTAGTTCTACTGT-3′; BRCA2 reverse, 5′-CCAATGTGGTCTTTGCAGCTAT-3′; RAD54L forward, 5′-TTGAGTCAGCTAACCAATCAACC-3′; RAD54L reverse, 5′-GGAGGCTCATACAGAACCAAGG-3′; E2F1 forward, 5′-CATCCCAGGAGGTCACTTCTG-3′; E2F1 reverse, 5′-GACAACAGCGGTTCTTGCTC-3′; BRCA1 forward, 5′ ACCTTGGAACTGTGAGAACTCT-3′; BRCA1 reverse, 5′-TCTTGATCTCCCACACTGCAATA-3′; RAD21 forward, 5′-GGATAAGAAGCTAACCAAAGCCC-3′; and RAD21 reverse, 5′-CTCCCAGTAAGAGATGTCCTGAT-3′. FEN1 forward, 5′-ATGACATCAAGAGCTACTTTGGC-3′; and FEN1 reverse, 5′-GGCGAACAGCAATCAGGAACT-3′. APEX1 forward, 5′-CAATACTGGTCAGCTCCTTCG-3′; and APEX1 reverse, 5′-TGCCGTAAGAAACTTTGAGTGG-3′. The following conditions were used for quantitative RT-PCR: denaturation: 95°C for 10 minutes; 40 cycles: 95°C for 30 seconds, 58°C for 10 seconds, and 72°C for 30 seconds; extension: 72°C for 10 minutes as described previously to be optimum for data analysis of quantitative real-time PCR using GAPDH as an internal control (10, 11).

Western blot analysis

Western blot analyses were performed as described previously (12) with the following modifications. The cell pellet was lysed in RIPA buffer with a cocktail of protease/phosphatase inhibitors (250 μg/mL leupeptin, 250 μg/mL aprotinin, 100 μg/mL pepstatin, 1 mmol/L benzamidine, 100 μg/mL soybean trypsin inhibitor, 5 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L sodium fluoride, and 0.5 mmol/L sodium orthovanadate). The primary antibodies used were 53BP1 (ab21083; Abcam), Axl (#8661; Cell Signaling Technology), β-actin (MAB1501R; EMD Millipore), BRCA1 (07-434; EMD Millipore), c-myc (Y69, ab32072), E-cadherin (#3195; Cell Signaling Technology), E2F1 (KH-92, sc-251; Santa Cruz Biotechnology), γH2AX (05-36; EMD Millipore), N-cadherin (#4061; Cell Signaling Technology), phospho c-myc (Ser62, 13748; Cell Signaling Technology), phospho c-myc (Thr58) (ab28842; Abcam), phospho S6 (Ser235/6, #2211; Cell Signaling Technology), RAD21 (ab992; Abcam), RAD51 (H-92, sc-8349), vimentin (#3932; Cell Signaling Technology), Zeb1 (#3396; Cell Signaling Technology), PARP (polyclonal, #9542; Cell Signaling Technology), caspase-3 (polyclonal, #9662; Cell Signaling Technology), caspase-7 (polyclonal, #9492; Cell Signaling Technology), Bcl2 (M-0887; Dako), Mcl1 (polyclonal, sc-819; Santa Cruz Biotechnology), p53 (OP43; Oncogene), and phospho p53 (S15, #9284; Cell Signaling Technology).

Immunofluorescence

To measure DNA damage repair using γH2AX staining, cells were plated at 1 × 104 cells/well in an eight-well chamber slide. After 24 hours, cells were treated with dinaciclib and/or niraparib, incubated for 72 hours, and fixed in 4% paraformaldehyde for 20 minutes before staining. For quantification of RAD51 foci, cells were plated at 10,000 cells/well in an eight-well chamber slide. After 72 hours, cells were treated with DMSO, dinaciclib, or niraparib and incubated for 48 hours. For immunofluorescence of MYC siRNA, 1 × 104 cells/well were plated in an 8-well chamber and treated with doxorubicin as described above. Following treatment, cells were fixed in 4% formaldehyde for 20 minutes before staining. For immunofluorescence staining, fixed cells were rinsed in PBS and permeabilized for 5 minutes in 0.1% Triton X-100. The cells were then blocked in 1% BSA for 1 hour at room temperature. Primary antibodies prepared in 1% BSA were added to the cells according to the antibody manufacturer's instructions, followed by a 2-hour incubation at room temperature in a moist chamber. Primary antibodies used were those against γH2AX (mouse monoclonal; EMD Millipore) and RAD51 (rabbit polyclonal; a gift from Dr. Junjie Chen, MD Anderson Cancer Center, Houston, TX). A secondary antibody was added after the cells were washed thoroughly in PBS. Cells were incubated with secondary antibodies tagged with Alexa Fluor dyes (goat-anti-mouse-Alexa Fluor-488 and goat-anti-rabbit-Alexa Fluor-594; Invitrogen) for 1 hour at room temperature. After being rinsed and washed thoroughly with PBS, slides were mounted using VECTASHIELD mounting medium (Vector Laboratories) containing DAPI and sealed. Cells were visualized using a 1 × 81 DSU confocal microscope (Olympus), and images were analyzed using the SlideBook software program (Intelligent Imaging Innovations). At least 150 cells were counted in each experiment. Each experiment was performed in triplicate, and the average count was taken. Statistical analysis was performed using the Student t test (significance, P = 0.05).

Immunohistochemistry

Five-micron-thick sections deparaffinized in xylene and hydrated in a series of graded alcohol dilutions. Slides were boiled in a microwave at 650 V for 20 minutes in 10 mmol/L sodium citrate (pH 6.5) for antigen retrieval and subsequently cooled at room temperature for 30 minutes. Three percent of hydrogen peroxide used to block endogenous peroxidase activity. Sections were incubated overnight with primary antibodies against Ki67 (mouse monoclonal, clone MIB-1; DAKO, 1:100 dilution), Rad51 (rabbit polyclonal, clone H92; Santa Cruz Biotechnology, 1:500 dilution), c-myc (rabbit polyclonal, clone N-262; Santa Cruz Biotechnology, 1:500 dilution), and γH2AX (Ser139) (mouse monoclonal, clone 05-636; Millipore, 1:100 dilution). After the sections were rinsed, antibodies were detected with a secondary antibody from the Vectastain Elite ABC Kit (PK6101 and PK6102; Vector Laboratories) Color development was performed using 3,3-diaminobenzidine. Counterstaining was provided by staining with hematoxylin. All washing steps were performed in PBS alone and PBS with 0.1% Tween.

Homologous recombination (DR-GFP) assay

To measure HR efficiency in cells, a two-plasmid HR assay was used as described previously (8). Briefly, cells were plated at about 0.4 × 106 cells/well in a 6-well plate. After overnight incubation, the plasmids pSce-I and pDR-GFP were transfected into the cells using jetPRIME transfection reagent. Twenty-four hours after transfection, cells were treated with dinaciclib, 10058-F4, or MYC siRNA. Cells were harvested 72 hours later via trypsinization, resuspended in PBS, and analyzed using a Gallios flow cytometer. The percentage of GFP-positive cells was used as a measure of HR efficiency.

Statistical analyses

Statistical analyses were performed using the Prism software program (version 5.04). Tumor volume, FACS, and Annexin V data were analyzed using one-way ANOVA and Tukey multiple comparison test. Survival curves were analyzed using a log-rank (Mantel–Cox) test. P values below 0.05 were considered statistically significant.

PARPi sensitivity is independent of BRCA status

We used an in vitro high-throughput survival assay (HTSA) to examine the long-term (i.e., 12 days) response of PARPi (Supplementary Fig. S1A), in PARPi-sensitive BRCAmut MDA-MB-436 (MB436), and resistant BRCA wild-type (BRCAwt) MDA-MB-MB231 (MB231) cell lines. In this assay, we treated the cells with a PARPi for 24 hours with 11-day release (24-hour HTSA), 72 hours with a 9-day release (72-hour HTSA) or continuously until day 12 (continuous HTSA), we then assessed survival. For both cell lines the response to PARPi-based treatment differed markedly in the 24- and 72-hour HTSAs, but the response did not differ significantly in 72-hour and continuous HTSAs (Supplementary Fig. S1B–G). Since the response was enhanced in the 72-hour HTSA (as compared to the 24-hour HTSA), it suggests that the effect of PARPis is maintained during assay's recovery phase (Supplementary Fig. S2A–C). Hence, the therapeutic effects of PARPis are sustained following drug removal and at least 72 hours of treatment is required to sustain response.

Next, we examined a panel of breast cancer cell lines treated with two PARPis (niraparib and olaparib) and cisplatin via 72-hour HTSA (72 hours of treatment followed by 9 days of recovery) and clonogenic assay (Fig. 1A and B). With the exception of MB436 cells, BRCA status did not dictate sensitivity to PARPis (<1 μmol/L). All BRCAmut cell lines demonstrated increased sensitivity to cisplatin (Fig. 1A and B; Supplementary Fig. S2B), supporting divergence in the response of TNBC to PARPis versus platinum agents as a function of BRCAmut status. Poly(ADP-ribose) levels in sensitive and resistant cells did not differ significantly under different treatment conditions (Fig. 1c). Apoptotic analysis also demonstrated a difference in sensitivity and resistance irrespective of BRCA status in cells treated with PARPis for 72 hours with a 72-hour recovery (Fig. 1D). We next used RAD51 foci formation as an indicator of HR to interrogate whether HR defects are associated with BRCA1 mutation status. Our results revealed that the PARPi sensitive, MB436 and HCC1806, cells had impaired RAD51 foci, whereas PARPi de novo resistant SUM149 and MB231 cells exhibited increased RAD51 foci in response to PARPi exposure independent of BRCA1 mutational status (Fig. 1E and F; Supplementary Fig. S3A–F). Thus, PARPi sensitivity is independent of BRCA status, whereas PARPi-sensitive cells have impaired RAD51 foci formation. Collectively, these experiments validate the sustained effect of PARPis following drug removal (Supplementary Figs. S3G and S4A–H) and suggest that PARPi sensitive cells have impaired RAD51 foci formation. Consistently, BRCA1 knockdown revealed PARPi resistance to be an independent function of BRCA1 loss (Supplementary Fig. S5A–M).

Figure 1.

RAD51 predicts PARP inhibitor sensitivity in TNBC. A, HTSA IC50 values of breast cancer cell lines screened for niraparib, olaparib, cisplatin, and DMSO (ctrl). Error bars represent mean ± SD (n ≥ 3 independent experiments). B–F, Analysis of PARPi-sensitive (MB436, HCC1806) and -resistant (SUM149, MB231) TNBC cell lines. B, Clonogenic assay of cells treated with 0.5, 1, and 5 μmol/L niraparib, olaparib, cisplatin, and DMSO (ctrl) for 72 hours, followed by 9-day release. C, Immunoblot analysis of breast cancer cells treated 72 hours with 1 μmol/L PARPi (niraparib, olaparib) for PAR and PARP. Actin was used as a loading control. D, Average cell viability using a FACS-based assay with Annexin V and propidium iodide staining of cells treated with 10 μmol/L niraparib, olaparib, DMSO for 72 hours, followed by 72-hour release. Error bars represent mean ± SD (n ≥ 3 independent experiments); two-way ANOVA with Sidak posttest correcting for multiple comparisons. E and F, Immunofluorescence analysis of cells treated with 1 μmol/L niraparib, olaparib, cisplatin, and DMSO (ctrl) for 72 hours. E, Immunofluorescence images of γH2AX (red), RAD51 (green), and nuclear DAPI (blue) in breast cancer cells. Scale bar, 10 μm. Representative images from three independent experiments. F, Quantification of γH2AX, RAD51. Data are mean ± SD of biological replicates and analyzed by unpaired two-sided t tests. G, TCGA analysis of MYC mRNA expression of sporadic versus BRCA 1/2–mutant TNBC breast cancer patient cohort; statistical analysis unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

RAD51 predicts PARP inhibitor sensitivity in TNBC. A, HTSA IC50 values of breast cancer cell lines screened for niraparib, olaparib, cisplatin, and DMSO (ctrl). Error bars represent mean ± SD (n ≥ 3 independent experiments). B–F, Analysis of PARPi-sensitive (MB436, HCC1806) and -resistant (SUM149, MB231) TNBC cell lines. B, Clonogenic assay of cells treated with 0.5, 1, and 5 μmol/L niraparib, olaparib, cisplatin, and DMSO (ctrl) for 72 hours, followed by 9-day release. C, Immunoblot analysis of breast cancer cells treated 72 hours with 1 μmol/L PARPi (niraparib, olaparib) for PAR and PARP. Actin was used as a loading control. D, Average cell viability using a FACS-based assay with Annexin V and propidium iodide staining of cells treated with 10 μmol/L niraparib, olaparib, DMSO for 72 hours, followed by 72-hour release. Error bars represent mean ± SD (n ≥ 3 independent experiments); two-way ANOVA with Sidak posttest correcting for multiple comparisons. E and F, Immunofluorescence analysis of cells treated with 1 μmol/L niraparib, olaparib, cisplatin, and DMSO (ctrl) for 72 hours. E, Immunofluorescence images of γH2AX (red), RAD51 (green), and nuclear DAPI (blue) in breast cancer cells. Scale bar, 10 μm. Representative images from three independent experiments. F, Quantification of γH2AX, RAD51. Data are mean ± SD of biological replicates and analyzed by unpaired two-sided t tests. G, TCGA analysis of MYC mRNA expression of sporadic versus BRCA 1/2–mutant TNBC breast cancer patient cohort; statistical analysis unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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MYC regulates DNA repair in TNBC

The oncogene MYC transcriptionally regulates several DNA repair genes and correlates with decreased overall survival rates in several types of cancer, therefore we next sought to determine the utility of MYC as a mediator of DNA repair in PARPi-resistant models of TNBC (13). MYC amplification was higher in the BRCAmut patients than in sporadic TNBC (P = 0.0554; Fig. 1G).

The prevalence of BRCA mutational status has been estimated at approximately 10%–20% of patients who present with TNBC (14–16). Furthermore, patients with BRCA1 mutational status have a 60%–80% chance of having high-grade TNBC and/or a basal subtype (17–20). Similarly, The Cancer Genome Atlas (TCGA) patient cohort analysis also revealed that patients with BRCAmut disease are typically presented with TNBC and/or a basal molecular subtype, with high MYC amplification and gene expression (Fig. 2A–C; Supplementary Fig. S6A). Specifically, MYC was amplified/gained in >85% of TNBC patient samples, which correlated with (69%) of BRCA1/2 and PALB2 (directly interacts with RAD51 in HR; ref. 21) mutation carriers (Fig. 2A).

Figure 2.

MYC regulates DNA repair in TNBC. A, Oncoprint analysis of MYC copy number gain, BRCA 1/2, PALB2 mutations in TCGA, TNBC breast cancer cohort. B and C, MYC mRNA expression in breast cancer subtypes as determined by ER/PR/HER-2 expression via IHC (B), or PAM50 signature (C). MYC mRNA correlation with RAD51 (D) and EIF4E (E) mRNA expression in TCGA breast cancer. F, IHC of TNBC patient cohort stained for c-myc/RAD51 from either pre- or post-neoadjuvant chemotherapy treatment samples. Kaplan–Meier curve of c-myc+/RAD51+ versus c-myc/RAD51 of pre (G)- and post (H)-neoadjuvant chemotherapy–treated TNBC breast cancer patients. I and J, MB231 cells treated with siMYC, siNT (ctrl) for 48 hours. I, Immunoblot for c-myc, RAD51, actin (ctrl). J, RT-PCR analysis of c-myc, RAD51 (n = 3). K and L, HEK293T and MCF10A cells transfected with MYC ORF cDNA. K, Immunoblot analysis for c-myc, RAD51, actin (ctrl). I, RT-PCR analysis of c-myc, RAD51. M, Annexin V and propidium iodide staining of MB231 and SUM149 cells treated with nontargeting, MYC, and RAD51 siRNA 48 hours, followed by niraparib/DMSO treatment 72 hours, followed by 72-hour release. Error bars represent mean ± SD (n ≥ 3 independent experiments). N, Clonogenic assay. MB231 and SUM149 cells treated with siNT (ctrl), siMYC, and siRAD51 for 48 hours, followed by 48-hour treatment with DMSO (ctrl), niraparib (1, 5 μmol/L) treatment for 48 hours, followed by release for 6 days. O, Cell viability assay, MB231, and SUM149 cells transfected with siNT, siMYC for 48 hours, followed by 72-hours niraparib (2.5 μmol/L), then drug release for 7 days. Error bars represent mean ± SD (n ≥ 3 independent experiments). P and Q, Immunofluorescence of MB231 cells treated with siNT (ctrl), siMYC for 48 hours, followed by 72 hours niraparib (5 μmol/L), DMSO (ctrl) treatment. P, Immunofluorescence of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 μm. Representative images of three independent experiments. Q, Quantification of γH2AX, RAD51 staining. Data are mean ± SD of two biological replicates. R, DR-GFP homologous recombination repair assay. MB231 and HCC1937 were treated with siNT, siMYC 48 hours, followed by transfection of DR-GFP reporter assay, then analyzed by FACS for GFP+ cells. Values are normalized by control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). In B, two-tailed unpaired t test was performed. In, C, the multiple t tests were performed. n.s., nonsignificant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

MYC regulates DNA repair in TNBC. A, Oncoprint analysis of MYC copy number gain, BRCA 1/2, PALB2 mutations in TCGA, TNBC breast cancer cohort. B and C, MYC mRNA expression in breast cancer subtypes as determined by ER/PR/HER-2 expression via IHC (B), or PAM50 signature (C). MYC mRNA correlation with RAD51 (D) and EIF4E (E) mRNA expression in TCGA breast cancer. F, IHC of TNBC patient cohort stained for c-myc/RAD51 from either pre- or post-neoadjuvant chemotherapy treatment samples. Kaplan–Meier curve of c-myc+/RAD51+ versus c-myc/RAD51 of pre (G)- and post (H)-neoadjuvant chemotherapy–treated TNBC breast cancer patients. I and J, MB231 cells treated with siMYC, siNT (ctrl) for 48 hours. I, Immunoblot for c-myc, RAD51, actin (ctrl). J, RT-PCR analysis of c-myc, RAD51 (n = 3). K and L, HEK293T and MCF10A cells transfected with MYC ORF cDNA. K, Immunoblot analysis for c-myc, RAD51, actin (ctrl). I, RT-PCR analysis of c-myc, RAD51. M, Annexin V and propidium iodide staining of MB231 and SUM149 cells treated with nontargeting, MYC, and RAD51 siRNA 48 hours, followed by niraparib/DMSO treatment 72 hours, followed by 72-hour release. Error bars represent mean ± SD (n ≥ 3 independent experiments). N, Clonogenic assay. MB231 and SUM149 cells treated with siNT (ctrl), siMYC, and siRAD51 for 48 hours, followed by 48-hour treatment with DMSO (ctrl), niraparib (1, 5 μmol/L) treatment for 48 hours, followed by release for 6 days. O, Cell viability assay, MB231, and SUM149 cells transfected with siNT, siMYC for 48 hours, followed by 72-hours niraparib (2.5 μmol/L), then drug release for 7 days. Error bars represent mean ± SD (n ≥ 3 independent experiments). P and Q, Immunofluorescence of MB231 cells treated with siNT (ctrl), siMYC for 48 hours, followed by 72 hours niraparib (5 μmol/L), DMSO (ctrl) treatment. P, Immunofluorescence of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 μm. Representative images of three independent experiments. Q, Quantification of γH2AX, RAD51 staining. Data are mean ± SD of two biological replicates. R, DR-GFP homologous recombination repair assay. MB231 and HCC1937 were treated with siNT, siMYC 48 hours, followed by transfection of DR-GFP reporter assay, then analyzed by FACS for GFP+ cells. Values are normalized by control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). In B, two-tailed unpaired t test was performed. In, C, the multiple t tests were performed. n.s., nonsignificant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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MYC amplification also trended to an increased expression of several DNA repair genes, including RAD21, RAD54L, and RAD51, in both breast and ovarian cancers (Supplementary Fig. S6B). As an alternative objective to identify a secondary target in patients with BRCAmut breast cancer that can be used as a biomarker for PARPi response, we mined TCGA databases for BRCAmut breast (n = 27) and ovarian (n = 69) cancer patients, revealing that MYC was the most frequently amplified gene in these aggressive cancers (Supplementary Fig. S6C and 6D).

Pearson correlation analysis demonstrated that RAD51 was the third most significant DNA repair gene associated with MYC expression in TNBC breast tumor samples (Supplementary Fig. S6E, Pearson correlation = 1.022). Analysis of both TNBC and ovarian cancer tumors supported an upregulation of DNA repair genes including RAD21, RAD54L, and RAD51, in both breast and ovarian cancers (Supplementary Fig. S6F–I).Moreover, gene expression analysis of 817 human breast tumor samples demonstrated a significant correlation between MYC and RAD51 expression (R2 = 0.2294, P ≤ 0.001; Fig. 2D, R2 = 0.2294, P ≤ 0.001), more so than MYC target gene EIF4E (Fig. 2E, R2 = 0.124, P ≤ 0.011). Collectively, these analyses point to MYC as a mediator of DNA repair via HR in TNBC.

To directly test this hypothesis in patient samples, we interrogated whether the correlation between c-myc and RAD51 persists at the protein level in human TNBC samples obtained before and after neoadjuvant chemotherapy using IHC (Fig. 2F). Fifty-three TNBC patients with stage II and III TNBC enrolled in a prospective study at MD Anderson Cancer Center received neoadjuvant chemotherapy with anthracyclins, taxanes, or a combination. Pretreatment core biopsy samples from 40 patients and postchemotherapy surgical samples from 37 of those patients were available for immunohistochemical staining for c-myc and RAD51 (Supplementary Fig. S7A). We recorded their clinical and pathologic treatment responses then evaluated and compared the patient, tumor, and treatment characteristics in the two groups as a function of c-myc and RAD51 staining (Supplementary Table S1). Pre- and posttreatment tumors expressing both c-myc and RAD51 (Fig. 2F) exhibited poor treatment response whereas absence of both markers indicated favorable response (Fig. 2G and H, P = 0.0001 and 0.0002, respectively). Although, c-myc and RAD51 expression individually predicted poor overall response, dual expression predicted the strongest correlation in both the pre- and posttreatment cohorts (Supplementary Fig. S7B–D, P = 0.0001). Of the 53 patients, 24 had residual disease following chemotherapy allowing for comparison of RAD51 and c-myc expression in matched pre-/post-chemotherapy samples. Although c-myc expression was rarely altered in response to treatment (Supplementary Fig. S7e), RAD51 expression was altered (in both directions; Supplementary Fig. S7F). However, all patients with c-myc/RAD51 tumors retained the phenotype in response to chemotherapy, whereas only 35% of c-myc+/RAD51+ tumors reverted to a c-myc/RAD51 phenotype (Supplementary Fig. S7G). Furthermore, assessment of pathologic complete response (pCR) in pretreatment tumor samples supported previous findings that the c-myc/RAD51 phenotype predicted the highest chemotherapy response rate (71%), whereas the c-myc+/RAD51+ phenotype predicted a lower pCR response (Supplementary Fig. S7H). These observations suggested that low c-myc/RAD51 expression predicts response of TNBC patients to standard-of-care therapy, whereas high expression can be used to identify tumors likely responsive to c-myc–targeted therapy.

We next investigated whether MYC regulates RAD51 expression and the DNA repair pathway in breast cancer. MYC siRNA downregulated RAD51 mRNA and protein expression in MB231 cells (Fig. 2I and J) consistent with previous observations of transcriptional regulation of RAD51 via MYC (2). Consistently, MYC overexpression in normal breast cells (MCF10A) increased RAD51 protein and mRNA expression (Fig. 2K and I). To examine if downregulation of MYC alters the sensitivity of cells to PARPis, we treated BRCAwt MDA231, and BRCAmut SUM149 cells with siRNAs for MYC and RAD51 for 48 hours and then cultured them with the PARPi niraparib for 72 hours followed by 72 hours of recovery. MYC and RAD51 siRNA in combination with PARPi increased apoptosis (Fig. 2M), decreased clonogenicity (Fig. 2N), and decreased cell proliferation (Fig. 2O) of MB231 and SUM149 cells. In addition, treatment of TNBC BRCAmut (SUM149, HCC1937) and BRCAwt (MDA231) with MYC siRNA in combination with either platinum DNA damaging agent, cisplatin or a taxane, docetaxel, revealed only increased efficacy of cisplatin in combination with MYC siRNA, but not with docetaxel. Furthermore, the immortalized MCF-10A cells were resistant to the combination treatments with MYC siRNA as revealed in minimal increased cell death (Supplementary Fig. S8A). Cell-cycle changes as a result of MYC siRNA treatments had no effect on PARPi response (Supplementary Fig. S8B). However, MYC siRNA markedly impaired RAD51 foci formation in PARPi-treated cells as compared with control (Fig. 2P and Q; Supplementary Fig. S8C–D). Finally, MYC downregulation impaired HR activity in MB231 and HCC1937 cells as measured using a DR-GFP reporter assay (Fig. 2R). Collectively, these results suggested that MYC inhibition of HR is comparable with RAD51 loss in both MB231 and HCC1937 cells, providing further evidence of the HR-regulatory role of MYC in TNBCs.

Pharmacologic inhibition of MYC expression induces PARPi sensitivity

The downstream oncogenic function of MYC is primarily dependent on its heterodimerization with basic helix-loop-helix protein MAX, resulting in the activation of transcriptional targets (22). The small-molecule 10058-F4 inhibited MYC-MAX binding (23), which resulted in the downregulation of RAD51 protein (Fig. 3A) and RNA (Fig. 3B) expression in MB231 and SUM149 cells. Treatment with 10058-F4 also resulted in transcriptional downregulation of MYC in both MB231 and SUM149 cells (Fig. 3B). Treatment of these cells with combination of 10058-F4 and niraparib induced synergistic growth inhibition (Fig. 3C; Supplementary Fig. S9A and S9B), increased apoptosis (Fig. 3D), and decreased DR-GFP HR activity (Fig. 3E), resulting in a decreased RAD51-γH2AX ratio (Fig. 3F–H; Supplementary Fig. S9C). Collectively, these results suggest the following criteria for identifying agents that when combined with PARPi could lead to synergistic activity in TNBC cells: (i) downregulates MYC/RAD51 expression, (ii) induces synergistic growth inhibition in HTSAs, and (iii) increases apoptosis in combination with PARPi (Fig. 3I–M; Supplementary Fig. S10). We tested several compounds including JQ1, a BRD4 inhibitor, and known MYC expression inhibitor (24), dinaciclib, a pan cyclin-dependent kinase (CDK) 1,2,5,9 inhibitor with potent antiproliferative activity (25), THZ1, a CDK7 inhibitor that transcriptionally regulates MYC expression (26) and nitazoxanide, an antiparasitic agent that inhibits c-myc expression (27). Of all these agents, the only one that met all three criteria—reducing the expression of both MYC and RAD51 in both MB231 and SUM149 cells (Fig. 3I and J), exhibiting synergism with niraparib HTSAs (Fig. 3K), and significantly inducing apoptosis (Fig. 3L and M) in combination therapy was dinaciclib. Dinaciclib has an extensive clinical profile in several types of cancer including breast cancer (28).

Figure 3.

Inhibition of MYC in TNBC induces PARP inhibitor sensitivity. A, Immunoblot analysis for c-myc, RAD51, actin (ctrl) of MB231 and SUM149 cells treated with DMSO, MYC inhibitor 10058-F4 25–150μmol/L for 72 hours. B, RT-PCR analysis of c-myc, RAD51 in cells treated with 10058-F4, MB231 (100 μmol/L) SUM149 (50 μmol/L). Values are normalized to the levels of actin RNA expression in control (DMSO) cells (n = 3). Statistical analysis via unpaired two-sided t test. C, Clonogenic assay. MB231 cells treated 72 hours with DMSO (ctrl), 10058-F4 (25–150 μmol/L), and niraparib (1 and 5 mmol/L) with 9-day release. D, Annexin V and propidium iodide staining of cells treated for 72 hours with 10058-F4 (MB231, 100 μmol/L; SUM149, 50 μmol/L), niraparib 1 μmol/L, followed by drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). E, DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells treated with 10058-F4 for 72 hours, followed by transfection of DR-GFP and analyzed by FACS. Values are normalized with those from control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). F–H, Immunofluorescence analysis of MB231 cells treated 72 hours with DMSO (ctrl), 10058-F4 (100 μmol/L), and niraparib (5 mmol/L). F, Immunofluorescence images of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 μm. Representative images of three independent experiments. G and H, Quantification of γH2AX, RAD51 staining. Representative images of three different experiments. Data are mean ± SD of biological replicates. I and J, Immunoblot analysis for c-myc, RAD51, and actin of MB231 (I) and SUM149 cells treated for 72 hours with DMSO, JQ1 (1 μmol/L), dinaciclib (25 nmol/L), THZ1 (50 nmol/L), vorinostat (2.5 μmol/L), GSK343 (10 μmol/L), GSK772983 (1 μmol/L), AZD6244 (10 μmol/L), nitazoxanide (10 μmol/L; J). K, Synergistic analysis of MB231 and SUM149 cells treated with niraparib (0.1–20 μmol/L) in combination with the panel of drugs from I–J. Seventy-two hour HTSA was performed and synergism was determined using CalcuSyn (<0.9 = synergism; 0.9–1.1 = additive; >1.1 + antagonistic). L and M, Annexin V and propidium iodide staining of cells treated for 72 hours with niraparib 1 μmol/L, in combination with panel of drugs, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). *, P < 0.05; **, P < 0.01 two-way ANOVA with Sidak posttest correcting for multiple comparisons.

Figure 3.

Inhibition of MYC in TNBC induces PARP inhibitor sensitivity. A, Immunoblot analysis for c-myc, RAD51, actin (ctrl) of MB231 and SUM149 cells treated with DMSO, MYC inhibitor 10058-F4 25–150μmol/L for 72 hours. B, RT-PCR analysis of c-myc, RAD51 in cells treated with 10058-F4, MB231 (100 μmol/L) SUM149 (50 μmol/L). Values are normalized to the levels of actin RNA expression in control (DMSO) cells (n = 3). Statistical analysis via unpaired two-sided t test. C, Clonogenic assay. MB231 cells treated 72 hours with DMSO (ctrl), 10058-F4 (25–150 μmol/L), and niraparib (1 and 5 mmol/L) with 9-day release. D, Annexin V and propidium iodide staining of cells treated for 72 hours with 10058-F4 (MB231, 100 μmol/L; SUM149, 50 μmol/L), niraparib 1 μmol/L, followed by drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). E, DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells treated with 10058-F4 for 72 hours, followed by transfection of DR-GFP and analyzed by FACS. Values are normalized with those from control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). F–H, Immunofluorescence analysis of MB231 cells treated 72 hours with DMSO (ctrl), 10058-F4 (100 μmol/L), and niraparib (5 mmol/L). F, Immunofluorescence images of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 μm. Representative images of three independent experiments. G and H, Quantification of γH2AX, RAD51 staining. Representative images of three different experiments. Data are mean ± SD of biological replicates. I and J, Immunoblot analysis for c-myc, RAD51, and actin of MB231 (I) and SUM149 cells treated for 72 hours with DMSO, JQ1 (1 μmol/L), dinaciclib (25 nmol/L), THZ1 (50 nmol/L), vorinostat (2.5 μmol/L), GSK343 (10 μmol/L), GSK772983 (1 μmol/L), AZD6244 (10 μmol/L), nitazoxanide (10 μmol/L; J). K, Synergistic analysis of MB231 and SUM149 cells treated with niraparib (0.1–20 μmol/L) in combination with the panel of drugs from I–J. Seventy-two hour HTSA was performed and synergism was determined using CalcuSyn (<0.9 = synergism; 0.9–1.1 = additive; >1.1 + antagonistic). L and M, Annexin V and propidium iodide staining of cells treated for 72 hours with niraparib 1 μmol/L, in combination with panel of drugs, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). *, P < 0.05; **, P < 0.01 two-way ANOVA with Sidak posttest correcting for multiple comparisons.

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Dinaciclib induces TNBC sensitivity to PARPis

Treatment of a panel of TNBC BRCAmut and BRCAwt TNBC cell lines with dinaciclib and niraparib revealed that the synergism of this combination therapy (assessed via HTSA) increased proportionally with the dinaciclib concentration (Fig. 4A and B) that resulted in c-myc downregulation (Fig. 4C), increased apoptosis (Fig. 4D), a decrease in cell viability (Fig. 4E), and an increase sub-G1 cell-cycle population (Fig. 4F). Other PARPis such as olaparib and velaparib exhibited similar synergism in TNBC cell lines when combined with dinaciclib (Supplementary Fig. S11A–C). The combination therapy was not synthetically lethal in the immortalized mammary epithelial cell line MCF10A, suggesting that induction of apoptosis by this combination strategy is tumor specific and dependent upon MYC expression (Fig. 4D). Dinaciclib + niraparib combination therapy consistently inhibited growth in both BRCA wild-type (MB231, MB157) and BRCA mutant (SUM149, HCC1937; Supplementary Fig. S11D). Induced MYC expression in MB231 cells partially reversed the effect of dinaciclib and niraparib combination therapy (Supplementary Fig. S11E and S11F). Protein expression for several proapoptotic and anti-apoptotic markers including cleaved PARP, caspase-3, caspase-7, Mcl-1, and Bcl-2 did not delineate activation of a conventional apoptotic pathway in the cells treated with monotherapy versus combination therapy (Supplementary Fig. S11G). However, the DR-GFP assay demonstrated impaired HR activity in the dinaciclib-treated cells (Fig. 4G). Dinaciclib inhibited RAD51 foci formation in MB231, HCC1937, and SUM149 cells (Fig. 4G–J; Supplementary Fig. S12A–S12D). Combination therapy with dinaciclib and niraparib further increased the number of γH2AX-positive cells (Fig. 4H and I; Supplementary Fig. S12A–S12G). Therefore, the mechanism of synthetic lethality of dinaciclib and niraparib is manifested through increased γH2AX DNA damage concomitant with downregulation of RAD51 foci formation. BRCA1 foci formation was also impaired by dinaciclib and niraparib in MB231 cells combination (Supplementary Fig. S12H). Therefore, dinaciclib can impair the HR pathway by inhibiting BRCA1 and RAD51 activity via c-myc in BRCAwt cells. However, in cells with retained RAD51 functionality in the absence of BRCA1, dinaciclib and niraparib also induce synthetic lethality via RAD51 inhibition. Downregulation of MYC target gene expression in response to treatment with dinaciclib alone or combined with niraparib supports the effect of dinaciclib on c-myc and transcriptional regulation of the DNA repair pathway (Fig. 4K).

Figure 4.

CDK inhibitor dinaciclib induces PARP inhibitor sensitivity in TNBC. A and B, For seventy-two hours HTSA was performed on a panel of TNBC cell lines that was treated with dinaciclib in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 = synergism; 0.9–1.1 = additive; >1.1 + antagonistic). A, Synergistic analysis of TNBC cells treated with niraparib (0.1–20 μmol/L) in combination with a panel of drugs. B, CalcuSyn combination index averages of dinaciclib (5, 10, and 25 nmol/L) treatment with niraparib (0.1–20 μmol/L). C, Immunoblot analysis for E2F1, c-myc, p-c-myc (S62), p-c-myc (T58), RAD51, and actin of TNBC cells treated for 72 hours with DMSO, dinaciclib (5, 10, and 25 nmol/L). D, Annexin V and propidium iodide staining of MCF10A, MB231, and SUM149 cells treated for 72 hours with dinaciclib (10 nmol/L) and niraparib (1 μmol/L). Error bars represent mean ± SD (n ≥ 3 independent experiments). E, Clonogenic assay. TNBC cells treated for 72 hours with DMSO (ctrl), niraparib (1 μmol/L), dinaciclib (10 nmol/L), or combo, followed by with 9-day release. F, FACS cell-cycle analysis of TNBC cells treated for 72 hours with dinaciclib 10 nmol/L, niraparib 1 μmol/L, followed by 72-hour drug release. G, DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells were transfected 24 hours with DR-GFP reporter assay, then treated 72 hours with dinaciclib (10 and 25 nmol/L), followed by FACS analysis for GFP+ cells. Values are normalized by control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). H–J, Immunofluorescence analysis of MB231 cells treated for 72 hours with DMSO (ctrl), niraparib (5 μmol/L), dinaciclib (10 nmol/L), or combo. H, Immunofluorescence images of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 2.5 μm. Representative images of three independent experiments. I, Quantification of γH2AX, RAD51 staining. Representative images of three different experiments. Data are mean ± SD of biological replicates. unpaired two-sided t tests. J, Quantification of average γH2AX foci per cell. A minimum of 250 cells were counted. K, RT-PCR analysis of DNA repair genes in MB231 cells treated with DMSO, niraparib (1 μmol/L), dinaciclib (10 nmol/L), combo. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparisons.

Figure 4.

CDK inhibitor dinaciclib induces PARP inhibitor sensitivity in TNBC. A and B, For seventy-two hours HTSA was performed on a panel of TNBC cell lines that was treated with dinaciclib in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 = synergism; 0.9–1.1 = additive; >1.1 + antagonistic). A, Synergistic analysis of TNBC cells treated with niraparib (0.1–20 μmol/L) in combination with a panel of drugs. B, CalcuSyn combination index averages of dinaciclib (5, 10, and 25 nmol/L) treatment with niraparib (0.1–20 μmol/L). C, Immunoblot analysis for E2F1, c-myc, p-c-myc (S62), p-c-myc (T58), RAD51, and actin of TNBC cells treated for 72 hours with DMSO, dinaciclib (5, 10, and 25 nmol/L). D, Annexin V and propidium iodide staining of MCF10A, MB231, and SUM149 cells treated for 72 hours with dinaciclib (10 nmol/L) and niraparib (1 μmol/L). Error bars represent mean ± SD (n ≥ 3 independent experiments). E, Clonogenic assay. TNBC cells treated for 72 hours with DMSO (ctrl), niraparib (1 μmol/L), dinaciclib (10 nmol/L), or combo, followed by with 9-day release. F, FACS cell-cycle analysis of TNBC cells treated for 72 hours with dinaciclib 10 nmol/L, niraparib 1 μmol/L, followed by 72-hour drug release. G, DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells were transfected 24 hours with DR-GFP reporter assay, then treated 72 hours with dinaciclib (10 and 25 nmol/L), followed by FACS analysis for GFP+ cells. Values are normalized by control group. Error bars represent mean ± SD (n ≥ 3 independent experiments). H–J, Immunofluorescence analysis of MB231 cells treated for 72 hours with DMSO (ctrl), niraparib (5 μmol/L), dinaciclib (10 nmol/L), or combo. H, Immunofluorescence images of γH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 2.5 μm. Representative images of three independent experiments. I, Quantification of γH2AX, RAD51 staining. Representative images of three different experiments. Data are mean ± SD of biological replicates. unpaired two-sided t tests. J, Quantification of average γH2AX foci per cell. A minimum of 250 cells were counted. K, RT-PCR analysis of DNA repair genes in MB231 cells treated with DMSO, niraparib (1 μmol/L), dinaciclib (10 nmol/L), combo. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparisons.

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We next examined the in vivo efficacy of dinaciclib and niraparib against de novo treatment-resistant BRCAmut SUM149 (Fig. 5A–C; Supplementary Fig. S13A–S13C), and BRCAwt MB231T cells (Fig. 5D–F; Supplementary Fig. S13D and S13E). In both cell types, combination therapy produced superior growth inhibition versus either agent alone (Fig. 5A–F). We also determined whether growth inhibition during this combination therapy (four cycles) was sustained in BRCAmut SUM149 xenografts after treatment (Fig. 5B; Supplementary Fig. S13A). Only the dinaciclib + niraparib combination therapy arm showed significant increases in median survival duration of 43 days as compared with vehicle, niraparib, and dinaciclib arms with 15-, 26-, and 23-day median survival times, respectively (P = 0.0115; Fig. 5B).

Figure 5.

Dinaciclib + niraparib treatment in vivo. A–C, SUM149 cells were injected into immunocompromised mice and allowed to grow to approximately 200 mm3; xenografts were treated with either vehicle, dinaciclib 25 mg/kg three times weekly, niraparib 5 mg/kg five times weekly, or dinaciclib + niraparib (combo) therapy for 4 weeks. Tumor volume measurements (A) and Kaplan–Meier survival analysis (B) of mice treated with indicated drug treatment arms for 4 weeks (28 days), followed by release without treatment until mice reached maximum allowed tumor size. C, Percent change in tumor volume normalized by day 1 of treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparison. D–J, MB231T cells were injected into nude mice and cells were allowed to grow to >500 mm3 before treatment was initiated. Mice were treated with vehicle, dinaciclib 25 mg/kg three times weekly, niraparib 5 mg/kg five times weekly, or dinaciclib + niraparib (combo) therapy for 3 weeks. Upon completion, tumors were extracted and analyzed by IHC and RPPA. Tumor volume measurements (D) and Kaplan–Meier survival analysis (E) of mice treated with indicated drug arms for 3 weeks. F, Percent change in tumor volume normalized by day 1 of treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparison. G–K Tumors from mice treated with each of the treatment arms were extracted and weighed (G and H) and subjected to IHC analysis of treated tumors for hematoxylin and eosin (H&E), Ki67, c-myc, RAD51, and γH2AX (I), resulting in a comparison of the H-score of c-myc with RAD51 expression in MB231T–treated xenografts (J), and immunoblot analysis for c-myc, RAD51, γH2AX, Zeb1, Axl, E-cadherin, N-cadherin, and actin (K).

Figure 5.

Dinaciclib + niraparib treatment in vivo. A–C, SUM149 cells were injected into immunocompromised mice and allowed to grow to approximately 200 mm3; xenografts were treated with either vehicle, dinaciclib 25 mg/kg three times weekly, niraparib 5 mg/kg five times weekly, or dinaciclib + niraparib (combo) therapy for 4 weeks. Tumor volume measurements (A) and Kaplan–Meier survival analysis (B) of mice treated with indicated drug treatment arms for 4 weeks (28 days), followed by release without treatment until mice reached maximum allowed tumor size. C, Percent change in tumor volume normalized by day 1 of treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparison. D–J, MB231T cells were injected into nude mice and cells were allowed to grow to >500 mm3 before treatment was initiated. Mice were treated with vehicle, dinaciclib 25 mg/kg three times weekly, niraparib 5 mg/kg five times weekly, or dinaciclib + niraparib (combo) therapy for 3 weeks. Upon completion, tumors were extracted and analyzed by IHC and RPPA. Tumor volume measurements (D) and Kaplan–Meier survival analysis (E) of mice treated with indicated drug arms for 3 weeks. F, Percent change in tumor volume normalized by day 1 of treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparison. G–K Tumors from mice treated with each of the treatment arms were extracted and weighed (G and H) and subjected to IHC analysis of treated tumors for hematoxylin and eosin (H&E), Ki67, c-myc, RAD51, and γH2AX (I), resulting in a comparison of the H-score of c-myc with RAD51 expression in MB231T–treated xenografts (J), and immunoblot analysis for c-myc, RAD51, γH2AX, Zeb1, Axl, E-cadherin, N-cadherin, and actin (K).

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We injected BRCAwt MB231T cells into nude mice and allowed the resulting tumors to grow >500 mm3 before initiating treatment with dinaciclib and niraparib to determine whether the initial tumor size had any bearing on therapeutic efficacy (Fig. 5D). Treatment with dinaciclib and niraparib resulted in significant tumor growth inhibition (Fig. 5D; Supplementary Fig. S13D), increased overall survival (Fig. 5E), decreased percent change in tumor volume (Fig. 5F; Supplementary Fig. S13F), and a reduced tumor weight (Fig. 5G and H). IHC analysis demonstrated lower c-myc and RAD51 in dinaciclib- and combination-treated tumors than in the niraparib and vehicle treatment arms (Fig. 5I; Supplementary S13G–S13I). Tumors from the combination treatment arm also demonstrated the greatest upregulation of γH2AX expression (Fig. 5I; Supplementary Fig. S13J). Downregulation of c-myc expression correlated with RAD51 and Ki67 downregulation in treated xenografts (Fig. 5J; Supplementary Fig. S13G and S13K). Western blot analysis revealed downregulation of c-myc and RAD51 in both dinaciclib and combination treatment arms (Fig. 5K). In addition, we observed loss of programmed cell death ligand 1 (PD-L1) expression in treated tumors (Supplementary Fig. S13L). Consistent with downregulation of EMT markers, dinaciclib given either alone or in combination therapy inhibited the CD44hi/CD24lo cancer stem cell (CSC) population in vitro, whereas niraparib alone had no effect on it (Supplementary Fig. S13M). In addition, we observed a reduction of mammosphere formation of MB231 cells treated with siMYC and dinaciclib compared with control, supporting a role of MYC on the CSC phenotype in TNBC (Supplementary Fig. S13N). Although MYC itself is a marker of the CSC phenotype, several others (e.g., Vimentin, ALDH) were downregulated at the protein level. Reverse phase protein array (RPPA) analysis demonstrated upregulation of the proapoptotic pathway but downregulation of the DNA repair, antiapoptotic, and epithelial–mesenchymal transition proteins (Supplementary Fig. S13O–S13Q). Downregulation of the EMT pathway was confirmed by protein downregulation of Axl, Vimentin, Zeb1, and N-cadherin protein expression and upregulation of E-cadherin expression (Fig. 5K). These alterations correlated with downregulation of c-myc and RAD51 expression (Fig. 5J and I). Collectively, our in vivo analysis suggested that c-myc is a useful predictive biomarker for treatment with the combination of dinaciclib and niraparib having synthetic lethality via HR repair and dinaciclib inhibits the EMT and CSC pathways in TNBC xenografts.

Dinaciclib resensitizes PARPi-resistant cells to PARP inhibition

Next, we sought to determine whether therapy with dinaciclib and niraparib is also effective against TNBC cells with acquired resistance to PARP inhibition. We generated PARPi-sensitive BRCAmut, MB436 and BRCAwt, HCC1806 TNBC cell lines (Fig. 1A), resistant to increasing concentrations of the PARPi niraparib in a step-wise fashion, resulting in 5- to 18-fold increased resistance according to IC50 values (Fig. 6A). Dinaciclib alone reversed the resistance of these cells to PARP inhibition, while inducing a synthetically lethal increase in apoptosis incidence (Fig. 6B). Also, the combination therapy induced synergistic growth inhibition (Fig. 6C and D). To further analyze the efficacy of combination therapy of dinaciclib and niraparib, we induced PARPi resistance via 53BP1 loss. Knockdown of 53BP1 expression in parental HCC1806 cells induced PARPi resistance by two-fold (Supplementary Fig. S14A and S14b). The combination treatment also induced synergistic growth inhibition response in 53BP1 knockdown cells (Supplementary Fig. S14C and S14D). Conversely, 53BP1 knockdown by short hairpin RNA did not induce PARP inhibitor resistance in MB436 cells (data not shown). Thus, treatment with dinaciclib and niraparib can overcome both acquired and induced resistance to PARPi.

Figure 6.

Dinaciclib induces synthetic lethality in niraparib acquired resistant and high MYC cancers. A, Seventy-two hour HTSA for niraparib was performed on acquired resistance and parental MB436 and HCC8106 cells. B, Annexin V and propidium iodide staining of MB436 (parental), MB436 (resistant), HCC1806, and HCC1806 (resistant) cells treated 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). C, Seventy-two hour HTSA was performed on MB436, MB436 (R), HCC1806, and HCC1806 (R) treated with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 = synergism; 0.9–1.1 = additive; >1.1 antagonistic). D, Clonogenic assay of MB436, MB436 (R), HCC1806, and HCC1806 (R) cells treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 72-hour drug release. E, TCGA analysis of MYC amplification across several cancer types. F and G, Seventy-two hour HTSA was performed on c-myc–high cancers with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSyn software. H, Clonogenic assay of MYC-high cancer cell lines treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 9-hour drug release. I, Annexin V and propidium iodide staining of MYC-high cancer cell lines treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 mmol/L, and combo, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparisons.

Figure 6.

Dinaciclib induces synthetic lethality in niraparib acquired resistant and high MYC cancers. A, Seventy-two hour HTSA for niraparib was performed on acquired resistance and parental MB436 and HCC8106 cells. B, Annexin V and propidium iodide staining of MB436 (parental), MB436 (resistant), HCC1806, and HCC1806 (resistant) cells treated 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). C, Seventy-two hour HTSA was performed on MB436, MB436 (R), HCC1806, and HCC1806 (R) treated with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 = synergism; 0.9–1.1 = additive; >1.1 antagonistic). D, Clonogenic assay of MB436, MB436 (R), HCC1806, and HCC1806 (R) cells treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 72-hour drug release. E, TCGA analysis of MYC amplification across several cancer types. F and G, Seventy-two hour HTSA was performed on c-myc–high cancers with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSyn software. H, Clonogenic assay of MYC-high cancer cell lines treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 μmol/L, and combo, followed by 9-hour drug release. I, Annexin V and propidium iodide staining of MYC-high cancer cell lines treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 mmol/L, and combo, followed by 72-hour drug release. Error bars represent mean ± SD (n ≥ 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA with Sidak posttest correcting for multiple comparisons.

Close modal

Dinaciclib and PARP inhibition are effective against MYC-driven cancers

MYC is a potent oncogene that is amplified in several cancer types. Analysis of several TCGA patient cohorts revealed high MYC gene expression in uveal, colorectal, head and neck, lung, and ovarian tumors (Fig. 6E; Supplementary Fig. S15). Often, patients with these high MYC-expressing cancers have very poor response rates to standard of care therapies, indicating a need for novel therapeutic strategies (13). Therefore, we sought to determine the applicability of c-myc and PARP inhibition for other aggressive cancer types besides TNBC. We selected a panel of high MYC-expressing ovarian, prostate, pancreatic, lung, and colon cancer cell lines (Fig. 6F). In vitro analysis of dinaciclib and niraparib revealed synergistic growth inhibition (i.e., combination index < 1) in all cell lines examined (Fig. 6F–H). In addition, the combination therapy significantly increased apoptosis incidence, as compared with either single agent, in several cell lines tested irrespective of cancer type (Fig. 6I). These results suggested that the combination of dinaciclib and niraparib is an effective treatment strategy extending to many tumors with high MYC expression.

Clinical PARPi use is steadily advancing with three drugs receiving FDA approval thus far and a high probability of more approvals to come. As the first therapeutics approved for BRCAmut tumors, their safe clinical profiles render them a paradigm-changing modality versus conventional chemotherapy. However, most cancer patients do not benefit from PARPi use, as the BRCAmut population represents only a small percentage of cancer patients overall. De novo and acquired resistance mechanisms further limit PARPi efficacy (29), demonstrating the need for increased clinical PARPi use in combination therapy. We developed and tested a promising treatment strategy based on MYC oncogene addiction and DNA repair. Oncogene MYC induction induces replication stress, causing double-strand DNA breaks, which require the activation of HR pathway to repair lesions (3). Our study demonstrates that MYC expression directly invokes HR activation in response to DNA damage and can potentially be used as a predictor of response of TNBC to therapy with PARPi combination.

The connection between MYC and RAD51 (as an indicator of HR activity) is based upon (i) correlation between TNBC and ovarian cancer tumor tissue gene expression of MYC and several DNA repair genes including RAD51 in the TCGA cohort (Supplementray Fig. 6E–I); (ii) IHC analysis of MYC and RAD51 in TNBC patient cohorts accrued at MD Anderson Cancer Center (Fig. 2G and H; Supplementary Fig. S7A–S7D); (iii) downregulation of RAD51 in response to MYC knockdown in MDA231 cells (Fig. 2I and J); (iv) MYC regulation of RAD51 in knock-in experiments (Fig. 2K and L). Collectively these results, which are consistent with published reports showing MYC is a transcriptional regulator of RAD51 (30–33), provide experimental evidence for the functional relationship between MYC and RAD51.

Although, oncogenic MYC transformation embodies several aspects of the “Hallmarks of Cancer,” it is designated as an undruggable target (1). Therefore, targeting of MYC-driven cancers through a synthetically lethal approach has therapeutic potential. MYC expression has long been associated with BRCA mutation frequency in breast cancer cases (34, 35). Studies of transgenic mouse models have demonstrated that MYC facilitates BRCAmut progression of ovarian cancer (36). The frequency of MYC upregulation in BRCAmut tumors and correlation with HR repair genes suggests that MYC compensates for BRCA loss via upregulation of a compensatory HR pathway, via increased RAD51 expression (37). The correlation of MYC and RAD51 gene expression in sporadic TNBCs (Fig. 2D) supports MYC dependence on HR repair as a function of MYC-driven tumors. RAD51 also regulates TNBC resistance to PARPis via the CSC resistance phenotype in TNBC (38). Inhibition of RAD51 expression in combination with PARPi-based treatment abrogates this CSC resistance phenotype, supporting our use of RAD51 as a marker of response to PARPis. Although RAD51 is classified as a tumor suppressor, high RAD51 expression compensates for BRCA loss in tumors, causing decreased sensitivity to irradiation and chemotherapy (37). Independent of MYC, increased RAD51 expression promotes TNBC metastasis, supporting high MYC, and RAD51 individual expression as negative predictors of poor outcome of TNBC (7).

The clinical use of CDK inhibitors to treat cancer has yielded minimal success until recently with the approval of the CDK4/6 inhibitors palbociclib and riibociclib (39). Dinaciclib, a pan CDK 1/2/5/9 inhibitor, has undergone several rounds of clinical investigation for several types of cancer including breast cancer (28, 40). However, the lack of a suitable biomarker for patient selection has plagued the advancement of dinaciclib beyond phase II clinical trials. In addition, dinaciclib combined with conventional chemotherapy has compounded patient toxicity, leading to treatment discontinuation, which further supports the use of dinaciclib in combination with targeted therapy to minimize patient toxicity profiles (28). Although dinaciclib was not developed as an agent targeted against MYC, several studies have corroborated our observation of dinaciclib's regulation of MYC expression (41, 42). Ideally, MYC downregulation would induce synthetic lethality in MYC-driven tumors, however studies have demonstrated that MYC loss alone is insufficiently lethal (31–33) further strengthening the need for combination therapy.

The precise mechanism of dinaciclib-induced MYC downregulation remains unclear. CDK1 and CDK9 inhibition are both synthetically lethal in high MYC-expressing cancer cells (43). Several CDKs (CDK2, CDK1, CDK5, CDK12) have been linked to DNA repair regulation, with many CDKs phosphorylating DNA repair genes directly (BRCA1, BRCA2; refs. 44–48). CDK5 has been directly linked to regulation of DNA repair in cancer (49). Transcriptional downregulation of MYC in response to dinaciclib exposure points to a transcriptional CDK-regulating MYC expression. Recently, Johnson and colleagues have suggested that CDK12 mediates dinaciclib-induced sensitivity to PARP inhibition in TNBC (50). In support of these findings, loss of CDK12 expression is synthetic lethal in c-myc–overexpressing cells, thus characterizing CDK12 as an upstream effector of MYC expression via RNA polymerase phosphorylation (50). Loss of CDK12 expression also predicts sensitivity of ovarian cancer to PARPis (51). Traditionally, the degree of PARP trapping by PARPis plays a significant role in the efficacy of PARPis against HR-deficient cells (52). Our study demonstrates a synergistic growth inhibitory effect of dinaciclib on TNBC cells when given with several different PARPis, suggesting that PARP trapping does not dictate sensitivity to this combination therapy.

We have also presented a novel mechanism of stratifying TNBC patients for therapy with PARPis based upon MYC expression (Supplementary Fig. S16). This model corrects a number of deficiencies in treatment options for TNBC patients by making MYC druggable, enhancing the therapeutic effect of PARPis in non-BRCAmut TNBC patients while targeting several cancer hallmarks including DNA repair, EMT, and the CSC phenotype, which lie at the crux of conventional chemotherapeutic efficacy (Fig. 7).

Figure 7.

A graphical representation of targeting both MYC and PARP in MYC-high TNBC could elicit antitumor effects.

Figure 7.

A graphical representation of targeting both MYC and PARP in MYC-high TNBC could elicit antitumor effects.

Close modal

Predictive IHC analysis of c-myc/RAD51 expression points to a less favorable response to chemotherapy of tumors that express both c-myc and RAD51 (Fig. 2f–h). By harnessing this knowledge we seek to improve patient outcomes in both c-myc-/RAD51- (via chemotherapy) and c-myc+/RAD51+ (via treatment with dinaciclib and niraparib) patient cohorts. In addition, implementation of dinaciclib and niraparib as a neoadjuvant therapeutic treatment that downregulates both c-myc and RAD51 expression infers subsequent response to chemotherapy and/or radiation therapy, if necessary. As a proto-oncogene, upregulation of MYC can drive therapeutic resistance to other targeted therapeutic agents (53, 54). Therefore, the combination strategy with dinaciclib and niraparib may be a secondary treatment option for patients who have developed MYC-dependent resistance to therapy.

J.K. Litton reports receiving a commercial research grant from Pfizer and Astra-Zeneca and also is a consultant/advisory board member for Pfizer and Astra-Zeneca. K.K. Hunt reports receiving a commercial research grant from Endomagnetics and Lumicell and is also a consultant/advisory board member for Lumicell and Armada Health. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.P.W. Carey, K. Mikule, K.K. Hunt, K. Keyomarsi

Development of methodology: J.P.W. Carey, Y. Zhao, K. Keyomarsi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.P.W. Carey, C. Karakas, T. Bui, X. Chen, S. Vijayaraghavan, K.K. Hunt, K. Keyomarsi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.P.W. Carey, C. Karakas, S. Vijayaraghavan, Y. Zhao, J. Wang, K.K. Hunt, K. Keyomarsi

Writing, review, and/or revision of the manuscript: J.P.W. Carey, Y. Zhao, K. Mikule, J.K. Litton, K.K. Hunt, K. Keyomarsi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.P.W. Carey,

Study supervision: J.P.W. Carey, K.K. Hunt, K. Keyomarsi

We thank Dr. Debu Tripathy, Dr. Banu Arun, and Dr. Gabriel Hortobagyi for their insightful comments during the course of this study. Research reported in this publication was supported by the National Cancer Institute of the NIH under award (P30CA016672) to The University of Texas MD Anderson Cancer Center, R01 grants CA87548 and CA1522218 and Cancer Prevention and Research Institute of Texas (CPRIT) RP170079 grants (to K. Keyomarsi), the Susan G. Komen for the Cure grant KG100521 (to K.K. Hunt), the Susan G. Komen postdoctoral fellowship grant PDF14302675 (to J.P.W. Carey), and a CPRIT training award RP170067 (to S. Vijayaraghavan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data