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Synthetic Lethality of PARP Inhibitors inCombination with MYC Blockade Is Independentof BRCA Status in Triple-Negative Breast CancerJason. P.W. Carey1, Cansu Karakas1, Tuyen Bui1, Xian Chen1, Smruthi Vijayaraghavan1,Yang Zhao2, Jing Wang2, Keith Mikule3, Jennifer K. Litton4, Kelly K. Hunt5, andKhandan Keyomarsi1

Abstract

PARP inhibitors (PARPi) benefit only a fraction of breast cancerpatients. Several of those patients exhibit intrinsic/acquired resis-tance mechanisms that limit efficacy of PARPi monotherapy. Herewe show how the efficacy of PARPi in triple-negative breast cancers(TNBC) can be expanded by targeting MYC-induced oncogenicaddiction. In BRCA-mutant/sporadic TNBC patients, amplificationof the MYC gene is correlated with increased expression of thehomologous DNA recombination enzyme RAD51 and tumorsoverexpressing both genes are associated with worse overall sur-vival. Combining MYC blockade with PARPi yielded syntheticlethality in MYC-driven TNBC cells. Using the cyclin-dependentkinase inhibitor dinaciclib, which downregulates MYC expression,we found that combination with the PARPi niraparib increasedDNA damage and downregulated homologous recombination,

leading to subsequent downregulation of the epithelial–mesen-chymal transition and cancer stem-like cell phenotypes. Notably,dinaciclib resensitized TBNC cells, which had acquired resistanceto niraparib. We found that the synthetic lethal strategy employingdinaciclib 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 canleverage cancer cell sensitivity to PARPi, facilitating the clinicaluse of c-myc as a predictive biomarker for this treatment.

Significance: Dual targeting of MYC-regulated homologousrecombination and PARP-mediated DNA repair yields potentsynthetic lethality in triple-negative breast tumors and otheraggressive tumors characterized by MYC overexpression. CancerRes; 78(3); 742–57. �2017 AACR.

IntroductionThe 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 cellularprocesses that MYC regulates (e.g., metabolism, cell cycle, metas-tasis, and DNA repair) suggests a crucial role in the oncogenictransformation of MYC-driven cancers, resulting in an addictionto MYC expression (2). One of the mechanisms by which MYCmediates oncogenic potential is induction of replication stress,causing increased gH2AX expression and subsequent upregula-tion of homologous recombination (HR) via increased RAD51activity (3).However,MYC's specific role in regulatingDNA repairmechanisms as a function of response to therapy has often been

overlooked. Treatment with PARP inhibitors (PARPi) is associat-edwith 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 resistanceto PARPis and platinum therapeutic agents (4–6). In addition,upregulation of the DNA repair pathway is often overlooked as asign of decreased response to chemotherapy. Moreover, becauseRAD51 expression is involved in several non-DNA repair path-ways (e.g., increasedmetastasis of TNBC; ref. 7), we hypothesizedthatMYC-positive tumors upregulate theHRDNA repair pathwaycausing resistance to DNA-damaging agents including PARPis.Therefore, using RAD51as amarker of de novo resistance to PARPiswe classified TNBC breast cancer cell lines as either PARPi-sensi-tive or -resistant independent of BRCA status. Furthermore, weshowed that MYC directly regulates HR via several DNA repairproteins including RAD51, whereas inhibition (or downregula-tion) of MYC expression induces PARPi-sensitivity independentof BRCA status. These findings suggest that TNBC patients withhigh c-myc and RAD51 expression, which have poor prognosesand are unresponsive to neoadjuvant chemotherapy, are likely tobe sensitive to agents that downregulate c-myc (e.g., dinaciclib)and PARPis independent of BRCA mutational status.

Materials and MethodsCell lines and culture conditions

All parental cancer cell lines used in this study were purchasedfrom the ATCC. The TNBC cell lines MDA-MB-231, MDA-MB-468, HCC1937, HCC1806, SUM149, SUM1315, MDA-MB-436,

1Department of Experimental Radiation Oncology, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Department of Bioinformatics andComputational Biology, The University of Texas MD Anderson Cancer Center,Houston, Texas. 3Tesaro Biopharmaceuticals, Waltham, Massacheusetts.4Department of Breast Medical Oncology, The University of Texas MDAndersonCancer Center, Houston, Texas. 5Department of Breast Surgical Oncology, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Khandan Keyomarsi, The University of Texas MDAnderson Cancer Center, 6565 MD Anderson Blvd, Unit 1052, Houston, TX77030. Phone: 713-792-4845; Fax: 713-794-5369; E-mail:[email protected]; and Jason P.W. Carey, [email protected]

doi: 10.1158/0008-5472.CAN-17-1494

�2017 American Association for Cancer Research.

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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) TNBCcell lines. B, Clonogenic assay of cells treated with 0.5, 1, and 5 mmol/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 mmol/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 mmol/L niraparib, olaparib, DMSO for72 hours, followedby 72-hour release. Error bars representmean�SD (n� 3 independent experiments); two-wayANOVAwith Sidak posttest correcting formultiplecomparisons. E and F, Immunofluorescence analysis of cells treated with 1 mmol/L niraparib, olaparib, cisplatin, and DMSO (ctrl) for 72 hours. E, Immunofluorescenceimages of gH2AX (red), RAD51 (green), and nuclear DAPI (blue) in breast cancer cells. Scale bar, 10 mm. Representative images from three independentexperiments. F, Quantification of gH2AX, RAD51. Data are mean � SD of biological replicates and analyzed by unpaired two-sided t tests. G, TCGA analysisof 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.

Dual MYC and PARP Inhibition Induces Synthetic Lethality

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Carey et al.

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and MDA-MB-157 and human mammary epithelial cell linesMCF-10A were cultured as described previously (8, 9), Thenon–small cell lung cancer cell lines PC3, DU145, A549, Calu-1,H1299, and H1993 were cultured in RPMI medium in thepresence of 10%FBS. Theheadandneck squamous cell carcinomacell lines OVCAR3, 59M, FUOV1, BxPC3, PANC-1, HCT116, andSW620 were cultured in DMEM in the presence of 10% FBS andgrowth factors. All cells were free of mycoplasma contamination.Cell lines were identified and authenticated according to karyo-type and using short tandem repeat analysis in the MD AndersonCharacterized Cell Line Core facility every 6 months.

Acquired treatment resistanceCells were cultured in normal growth media supplemented

with the PARPi niraparib at increasing concentrations (MDA-MB-436, 0.1 nmol/L–2.0 mmol/L; HCC1806, 0.5–15.0 mmol/L) for6 months. At the final concentrations, cells were maintained inmedia supplemented with niraparib. All experiments were con-ducted in the absence of niraparib-supplemented media unlessotherwise noted.

siRNAIn vitro cell transfections were carried out in 6-well plates

seeded (5 � 104) and then transfected with 5 mmol/L MYCsiRNA(4609) (SMART pool; Dharmacon; 50-ACGGAACUU-GUGCGUAA-30, 50-GAACACACAACGUCUUGGA-30, 50-AAC-GUUAGCUUCACCAACA-30, and 50-CGAUGUUGUUUCU-GUGGAA-30), 5 mmol/L RAD51 (5888), 5 mmol/L RAD51siRNA (SMART pool; 50-UAUCAUCGCCCAUGCAUCA-30, 50-CUAAUCAGGUGGUAGCUCA-30, 50-GCAGUGAUGUCCUG-GAUAA-30, and 50-CCAACGAUGUGAAGAAAAUU-30), or anontargeting pool 5 mmol/L siRNA. Cells were incubated at36�C in 5% CO2 for 48 hours, and the media were removed.Briefly, in vitro siRNA transfections were performed using thejetPRIME transfection reagent (Polyplus), following the man-ufacturer's protocol.

Short hairpin and open reading frame constructs and viralinfection

The pGIPZ-shRNA and MYC overexpression plasmids werepurchased from Dharmacon and used to produce lentiviruses(shBRCA1 and sh53BP1) by transfecting 293T cells shRNA

plasmids. TNBCcellswere infectedwith viral particles in completemedia in the presence of hexadimethrine bromide (Polybrene,8 mg/mL; EMD Millipore) overnight. The next day, media con-taining the viruses were washed and replaced with fresh media.Puromycin (Invitrogen) selection for infected cells was performedfor 7 days.

High-throughput survival assayCells were seeded at a density of 1,000 cells/well in a 96-well

plate and treated with single drugs or drug combinations at theindicated concentrations for 72 hours. Cells were then releasedinto a complete drug-free medium for 9 days, and the mediumwas changed every 48 hours. On the day of cell harvest, 100 mL/well 2.5 mg/mL MTT (Sigma) was added to the serum-freemedium and incubated at 37�C for 3 to 4 hours. After incubation,themediumwas removed, and100mLof a solubilization solution(0.04 mmol/L HCl and 1% SDS in isopropyl alcohol) was addedto each well. Plates were lightly rocked at room temperature for 1hour and read using a plate reader (Gen5 Epoch MicroplateSpectrophotometer and software program; BioTek) at a wave-length of 590 nm. Combination indices were calculated using theCalcuSyn software program.

Quantitative RT-PCRTotal 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 withSYBR Green Master Mix (Sigma). Reactions were carried out intriplicate. The fold difference in transcripts was calculated usingtheDDCtmethodwithGAPDHas a control. The following primerswere used: MYC forward, 50- GGCTCCTGGCAAAAGGTCA; MYCreverse, 50-CTGCGTAGTTGTGCTGATGT-30; RAD51 forward, 50-CAACCCATTTCACGGTTAGAGC-30; RAD51 reverse, 50-TTCTTT-GGCGATAGGCAACA-30; BRCA2 forward, 50-CACCCACCCT-TAGTTCTACTGT-30; BRCA2 reverse, 50-CCAATGTGGTCTTTG-CAGCTAT-30; RAD54L forward, 50-TTGAGTCAGCTAACCAAT-CAACC-30; RAD54L reverse, 50-GGAGGCTCATACAGAAC-CAAGG-30; E2F1 forward, 50-CATCCCAGGAGGTCACTTCTG-30;E2F1 reverse, 50-GACAACAGCGGTTCTTGCTC-30; BRCA1 for-ward, 50 ACCTTGGAACTGTGAGAACTCT-30; BRCA1 reverse,

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 mRNAcorrelation 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- orpost-neoadjuvant chemotherapy treatment samples. Kaplan–Meier curve of c-mycþ/RAD51þ versus c-myc�/RAD51� of pre (G)- and post (H)-neoadjuvantchemotherapy–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 ¼ 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 independentexperiments). 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 mmol/L) treatment for 48 hours, followed by release for 6 days.O, Cell viability assay, MB231, and SUM149 cells transfected with siNT, siMYC for48 hours, followed by 72-hours niraparib (2.5 mmol/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 mmol/L), DMSO (ctrl) treatment. P,Immunofluorescence of gH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 mm. Representative images of three independent experiments. Q,Quantification of gH2AX, RAD51 staining. Data are mean� SD of two biological replicates. R, DR-GFP homologous recombination repair assay. MB231 and HCC1937were treated with siNT, siMYC 48 hours, followed by transfection of DR-GFP reporter assay, then analyzed by FACS for GFPþ cells. Values are normalized bycontrol group. Error bars represent mean � SD (n � 3 independent experiments). In B, two-tailed unpaired t test was performed. In, C, the multiple t testswere performed. n.s., nonsignificant. �, P < 0.05; �� , P < 0.01; �� , P < 0.001.






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 treatedwith DMSO, MYC inhibitor 10058-F4 25–150mmol/L for 72 hours. (Continued on the following page.)

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50-TCTTGATCTCCCACACTGCAATA-30; RAD21 forward, 50-GGATAAGAAGCTAACCAAAGCCC-30; and RAD21 reverse, 50-CTCCCAGTAAGAGATGTCCTGAT-30. FEN1 forward, 50-ATGA-CATCAAGAGCTACTTTGGC-30; and FEN1 reverse, 50-GGCGAA-CAGCAATCAGGAACT-30. APEX1 forward, 50-CAATACTGGT-CAGCTCCTTCG-30; and APEX1 reverse, 50-TGCCGTAA-GAAACTTTGAGTGG-30. The following conditions were used forquantitative RT-PCR: denaturation: 95�C for 10 minutes; 40cycles: 95�C for 30 seconds, 58�C for 10 seconds, and 72�C for30 seconds; extension: 72�C for 10 minutes as described previ-ously to be optimum for data analysis of quantitative real-timePCR using GAPDH as an internal control (10, 11).

Western blot analysisWestern blot analyses were performed as described previously

(12) with the followingmodifications. The cell pellet was lysed inRIPA buffer with a cocktail of protease/phosphatase inhibitors(250 mg/mL leupeptin, 250 mg/mL aprotinin, 100 mg/mLpepstatin, 1 mmol/L benzamidine, 100 mg/mL soybean trypsininhibitor, 5 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/Lsodium fluoride, and 0.5 mmol/L sodium orthovanadate). Theprimary antibodies used were 53BP1 (ab21083; Abcam), Axl(#8661; Cell Signaling Technology), b-actin (MAB1501R; EMDMillipore), BRCA1 (07-434; EMD Millipore), c-myc (Y69,ab32072), E-cadherin (#3195; Cell Signaling Technology),E2F1 (KH-92, sc-251; Santa Cruz Biotechnology), gH2AX(05-36; EMDMillipore), N-cadherin (#4061; Cell Signaling Tech-nology), phospho c-myc (Ser62, 13748; Cell Signaling Technol-ogy), 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 Signal-ing Technology), Zeb1 (#3396; Cell Signaling Technology), PARP(polyclonal, #9542; Cell Signaling Technology), caspase-3 (poly-clonal, #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 SignalingTechnology).

ImmunofluorescenceTo measure DNA damage repair using gH2AX staining, cells

were plated at 1 � 104 cells/well in an eight-well chamber slide.After 24 hours, cells were treatedwith dinaciclib and/or niraparib,incubated for 72 hours, and fixed in 4% paraformaldehyde for 20minutes before staining. For quantification of RAD51 foci, cellswere plated at 10,000 cells/well in an eight-well chamber slide.

After 72 hours, cells were treated with DMSO, dinaciclib, orniraparib and incubated for 48 hours. For immunofluorescenceof MYC siRNA, 1 � 104 cells/well were plated in an 8-wellchamber and treated with doxorubicin as described above. Fol-lowing treatment, cells were fixed in 4% formaldehyde for 20minutes before staining. For immunofluorescence staining, fixedcells 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 atroom temperature. Primary antibodies prepared in 1% BSA wereadded to the cells according to the antibody manufacturer'sinstructions, followed by a 2-hour incubation at room temper-ature in a moist chamber. Primary antibodies used were thoseagainst gH2AX (mousemonoclonal; EMDMillipore) and RAD51(rabbit polyclonal; a gift from Dr. Junjie Chen, MD AndersonCancer Center, Houston, TX). A secondary antibody was addedafter the cells were washed thoroughly in PBS. Cells were incu-bated with secondary antibodies tagged with Alexa Fluor dyes(goat-anti-mouse-Alexa Fluor-488 and goat-anti-rabbit-AlexaFluor-594; Invitrogen) for 1 hour at room temperature. Afterbeing rinsed and washed thoroughly with PBS, slides weremounted using VECTASHIELD mounting medium (Vector Lab-oratories) containingDAPI and sealed. Cellswere visualized usinga 1 � 81 DSU confocal microscope (Olympus), and images wereanalyzed using the SlideBook software program (Intelligent Imag-ing Innovations). At least 150 cells were counted in each exper-iment. Each experiment was performed in triplicate, and theaverage count was taken. Statistical analysis was performed usingthe Student t test (significance, P ¼ 0.05).

ImmunohistochemistryFive-micron-thick sections deparaffinized in xylene andhydrat-

ed in a series of graded alcohol dilutions. Slides were boiled in amicrowave at 650 V for 20 minutes in 10mmol/L sodium citrate(pH 6.5) for antigen retrieval and subsequently cooled at roomtemperature for 30 minutes. Three percent of hydrogen peroxideused to block endogenous peroxidase activity. Sections wereincubated overnight with primary antibodies against Ki67(mousemonoclonal, cloneMIB-1;DAKO,1:100dilution), Rad51(rabbit polyclonal, clone H92; Santa Cruz Biotechnology, 1:500dilution), c-myc (rabbit polyclonal, clone N-262; Santa CruzBiotechnology, 1:500 dilution), and gH2AX (Ser139) (mousemonoclonal, clone 05-636; Millipore, 1:100 dilution). After thesections were rinsed, antibodies were detected with a secondaryantibody from the Vectastain Elite ABC Kit (PK6101 and PK6102;Vector Laboratories) Color development was performed using3,3-diaminobenzidine. Counterstainingwas provided by staining

(Continued.) B, RT-PCR analysis of c-myc, RAD51 in cells treated with 10058-F4, MB231 (100 mmol/L) SUM149 (50 mmol/L). Values are normalized to thelevels of actin RNA expression in control (DMSO) cells (n ¼ 3). Statistical analysis via unpaired two-sided t test. C, Clonogenic assay. MB231 cells treated 72 hourswith DMSO (ctrl), 10058-F4 (25–150 mmol/L), and niraparib (1 and 5 mmol/L) with 9-day release. D, Annexin V and propidium iodide staining of cells treated for72 hours with 10058-F4 (MB231, 100 mmol/L; SUM149, 50 mmol/L), niraparib 1 mmol/L, followed by drug release. Error bars represent mean� SD (n� 3 independentexperiments). E,DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells treatedwith 10058-F4 for 72 hours, followed by transfection of DR-GFPand 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 mmol/L), and niraparib (5 mmol/L). F, Immunofluorescenceimages of gH2AX (red), RAD51 (green), nuclear DAPI (blue). Scale bar, 10 mm. Representative images of three independent experiments. G and H, Quantificationof gH2AX, RAD51 staining. Representative images of three different experiments. Data aremean� 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 mmol/L), dinaciclib (25 nmol/L), THZ1 (50 nmol/L), vorinostat (2.5 mmol/L),GSK343 (10 mmol/L), GSK772983 (1 mmol/L), AZD6244 (10 mmol/L), nitazoxanide (10 mmol/L; J). K, Synergistic analysis of MB231 and SUM149 cells treatedwith niraparib (0.1–20 mmol/L) in combination with the panel of drugs from I–J. Seventy-two hour HTSA was performed and synergism was determinedusing CalcuSyn (<0.9 ¼ synergism; 0.9–1.1 ¼ additive; >1.1 þ antagonistic). L and M, Annexin V and propidium iodide staining of cells treated for 72 hours withniraparib 1 mmol/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 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 wastreated with dinaciclib in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 ¼ synergism; 0.9–1.1 ¼ additive; >1.1 þantagonistic). A, Synergistic analysis of TNBC cells treated with niraparib (0.1–20 mmol/L) in combination with a panel of drugs. B, CalcuSyn combinationindex averages of dinaciclib (5, 10, and 25 nmol/L) treatment with niraparib (0.1–20 mmol/L). (Continued on the following page.)

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with hematoxylin. All washing steps were performed in PBS aloneand PBS with 0.1% Tween.

Homologous recombination (DR-GFP) assayTo measure HR efficiency in cells, a two-plasmid HR assay was

used as described previously (8). Briefly, cells were plated at about0.4 � 106 cells/well in a 6-well plate. After overnight incubation,the plasmids pSce-I and pDR-GFP were transfected into the cellsusing jetPRIME transfection reagent. Twenty-four hours aftertransfection, cells were treated with dinaciclib, 10058-F4, or MYCsiRNA. 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 HRefficiency.

Statistical analysesStatistical analyses were performed using the Prism software

program (version 5.04). Tumor volume, FACS, and Annexin Vdata were analyzed using one-way ANOVA and Tukey multiplecomparison test. Survival curves were analyzed using a log-rank(Mantel–Cox) test. P values below 0.05 were considered statisti-cally significant.

ResultsPARPi sensitivity is independent of BRCA status

We used an in vitro high-throughput survival assay (HTSA) toexamine the long-term (i.e., 12 days) response of PARPi (Sup-plementary 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 witha PARPi for 24 hours with 11-day release (24-hour HTSA), 72hours with a 9-day release (72-hour HTSA) or continuously untilday 12 (continuous HTSA), we then assessed survival. For bothcell lines the response to PARPi-based treatment differedmarked-ly in the 24- and 72-hour HTSAs, but the response did not differsignificantly in 72-hour and continuous HTSAs (SupplementaryFig. S1B–G). Since the response was enhanced in the 72-hourHTSA (as compared to the 24-hour HTSA), it suggests that theeffect of PARPis is maintained during assay's recovery phase(Supplementary Fig. S2A–C). Hence, the therapeutic effects ofPARPis are sustained following drug removal and at least 72hoursof treatment is required to sustain response.

Next, we examined a panel of breast cancer cell lines treatedwith two PARPis (niraparib and olaparib) and cisplatin via72-hour HTSA (72 hours of treatment followed by 9 days ofrecovery) and clonogenic assay (Fig. 1A and B). With the excep-tion of MB436 cells, BRCA status did not dictate sensitivity to

PARPis (<1 mmol/L). All BRCAmut cell lines demonstratedincreased sensitivity to cisplatin (Fig. 1A and B; SupplementaryFig. S2B), supporting divergence in the response of TNBC toPARPis versus platinum agents as a function of BRCAmut status.Poly(ADP-ribose) levels in sensitive and resistant cells did notdiffer significantly under different treatment conditions (Fig. 1c).Apoptotic analysis also demonstrated a difference in sensitivityand resistance irrespective of BRCA status in cells treated withPARPis for 72 hours with a 72-hour recovery (Fig. 1D). We nextused RAD51 foci formation as an indicator of HR to interrogatewhether HR defects are associated with BRCA1 mutation status.Our results revealed that the PARPi sensitive, MB436 andHCC1806, cells had impaired RAD51 foci, whereas PARPide novo resistant SUM149 and MB231 cells exhibited increasedRAD51 foci in response to PARPi exposure independent of BRCA1mutational status (Fig. 1E and F; Supplementary Fig. S3A–F).Thus, PARPi sensitivity is independent of BRCA status, whereasPARPi-sensitive cells have impaired RAD51 foci formation.Collectively, these experiments validate the sustained effectof PARPis following drug removal (Supplementary Figs. S3Gand S4A–H) and suggest that PARPi sensitive cells haveimpaired RAD51 foci formation. Consistently, BRCA1 knock-down revealed PARPi resistance to be an independent functionof BRCA1 loss (Supplementary Fig. S5A–M).

MYC regulates DNA repair in TNBCThe oncogene MYC transcriptionally regulates several DNA

repair genes and correlates with decreased overall survival ratesin several types of cancer, therefore we next sought to determinethe utility of MYC as a mediator of DNA repair in PARPi-resistant models of TNBC (13). MYC amplification was higherin the BRCAmut patients than in sporadic TNBC (P ¼ 0.0554;Fig. 1G).

The prevalence of BRCA mutational status has been estimatedat approximately 10%–20% of patients who present with TNBC(14–16). Furthermore, patients with BRCA1 mutational statushave a 60%–80% chance of having high-grade TNBC and/or abasal subtype (17–20). Similarly, The Cancer Genome Atlas(TCGA) patient cohort analysis also revealed that patients withBRCAmut disease are typically presentedwith TNBC and/or a basalmolecular subtype,with highMYCamplification andgene expres-sion (Fig. 2A–C; Supplementary Fig. S6A). Specifically, MYC wasamplified/gained in >85% of TNBC patient samples, whichcorrelated with (69%) of BRCA1/2 and PALB2 (directly interactswith RAD51 in HR; ref. 21) mutation carriers (Fig. 2A).

MYC amplification also trended to an increased expression ofseveral DNA repair genes, including RAD21, RAD54L, andRAD51, in both breast and ovarian cancers (Supplementary

(Continued.) 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 mmol/L). Error bars represent mean� SD (n� 3 independent experiments). E, Clonogenic assay. TNBC cells treated for 72 hours with DMSO (ctrl),niraparib (1 mmol/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 withdinaciclib 10 nmol/L, niraparib 1 mmol/L, followed by 72-hour drug release. G, DR-GFP homologous recombination repair assay. MB231 and HCC1937 cells weretransfected 24 hours with DR-GFP reporter assay, then treated 72 hours with dinaciclib (10 and 25 nmol/L), followed by FACS analysis for GFPþ cells. Values arenormalized by control group. Error bars representmean�SD (n�3 independent experiments).H–J, Immunofluorescence analysis ofMB231 cells treated for 72hourswith DMSO (ctrl), niraparib (5 mmol/L), dinaciclib (10 nmol/L), or combo. H, Immunofluorescence images of gH2AX (red), RAD51 (green), nuclear DAPI (blue). Scalebar, 2.5 mm. Representative images of three independent experiments. I, Quantification of gH2AX, RAD51 staining. Representative images of three differentexperiments. Data are mean� SD of biological replicates. unpaired two-sided t tests. J,Quantification of average gH2AX foci per cell. A minimum of 250 cells werecounted. K, RT-PCR analysis of DNA repair genes in MB231 cells treated with DMSO, niraparib (1 mmol/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.






Fig. S6B). As an alternative objective to identify a secondary targetin patients with BRCAmut breast cancer that can be used as abiomarker for PARPi response, we mined TCGA databases forBRCAmut breast (n ¼ 27) and ovarian (n ¼ 69) cancer patients,revealing that MYC was the most frequently amplified gene inthese aggressive cancers (Supplementary Fig. S6C and 6D).

Pearson correlation analysis demonstrated that RAD51was thethird most significant DNA repair gene associated with MYCexpression in TNBC breast tumor samples (SupplementaryFig. S6E, Pearson correlation ¼ 1.022). Analysis of both TNBCand ovarian cancer tumors supported an upregulation of DNArepair genes including RAD21, RAD54L, and RAD51, in bothbreast and ovarian cancers (Supplementary Fig. S6F–I).Moreover,gene expression analysis of 817 human breast tumor samplesdemonstrated a significant correlation between MYC and RAD51expression (R2 ¼ 0.2294, P � 0.001; Fig. 2D, R2 ¼ 0.2294, P �0.001), more so thanMYC target gene EIF4E (Fig. 2E, R2¼ 0.124,P � 0.011). Collectively, these analyses point to MYC as amediator of DNA repair via HR in TNBC.

To directly test this hypothesis in patient samples, we interro-gated whether the correlation between c-myc and RAD51 persistsat the protein level in human TNBC samples obtained before andafter neoadjuvant chemotherapy using IHC (Fig. 2F). Fifty-threeTNBCpatients with stage II and III TNBC enrolled in a prospectivestudy at MD Anderson Cancer Center received neoadjuvant che-motherapy with anthracyclins, taxanes, or a combination. Pre-treatment core biopsy samples from 40 patients and postche-motherapy surgical samples from 37 of those patients wereavailable for immunohistochemical staining for c-myc andRAD51 (Supplementary Fig. S7A). We recorded their clinical andpathologic treatment responses then evaluated and compared thepatient, tumor, and treatment characteristics in the twogroups as afunction 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 ofboth markers indicated favorable response (Fig. 2G and H, P ¼0.0001 and 0.0002, respectively). Although, c-myc and RAD51expression individually predicted poor overall response, dualexpression predicted the strongest correlation in both the pre-and posttreatment cohorts (Supplementary Fig. S7B–D, P ¼0.0001). Of the 53 patients, 24 had residual disease followingchemotherapy allowing for comparison of RAD51 and c-mycexpression in matched pre-/post-chemotherapy samples.Although c-myc expression was rarely altered in response totreatment (Supplementary Fig. S7e), RAD51 expression wasaltered (in both directions; Supplementary Fig. S7F). However,all patients with c-myc�/RAD51� tumors retained the phenotypein response to chemotherapy, whereas only 35% of c-mycþ/RAD51þ tumors reverted to a c-myc�/RAD51� phenotype(Supplementary Fig. S7G). Furthermore, assessment ofpathologic complete response (pCR) in pretreatment tumorsamples supported previous findings that the c-myc�/RAD51�

phenotype predicted the highest chemotherapy response rate(71%),whereas the c-mycþ/RAD51þphenotypepredicted a lowerpCR response (Supplementary Fig. S7H). These observationssuggested that low c-myc/RAD51 expression predicts responseof TNBC patients to standard-of-care therapy, whereas highexpression can be used to identify tumors likely responsive toc-myc–targeted therapy.

Wenext investigatedwhetherMYC regulates RAD51 expressionand the DNA repair pathway in breast cancer. MYC siRNA

downregulated RAD51 mRNA and protein expression in MB231cells (Fig. 2I and J) consistent with previous observations oftranscriptional regulation of RAD51 via MYC (2). Consistently,MYC overexpression in normal breast cells (MCF10A) increasedRAD51 protein andmRNA expression (Fig. 2K and I). To examineif downregulation of MYC alters the sensitivity of cells to PARPis,we treated BRCAwt MDA231, and BRCAmut SUM149 cells withsiRNAs for MYC and RAD51 for 48 hours and then cultured themwith the PARPi niraparib for 72 hours followed by 72 hours ofrecovery. MYC and RAD51 siRNA in combination with PARPiincreased apoptosis (Fig. 2M), decreased clonogenicity (Fig. 2N),and decreased cell proliferation (Fig. 2O) ofMB231 and SUM149cells. In addition, treatment of TNBC BRCAmut (SUM149,HCC1937) and BRCAwt (MDA231) with MYC siRNA in combi-nation with either platinum DNA damaging agent, cisplatin or ataxane, docetaxel, revealed only increased efficacy of cisplatin incombination with MYC siRNA, but not with docetaxel. Further-more, the immortalized MCF-10A cells were resistant to thecombination treatments with MYC siRNA as revealed in minimalincreased cell death (Supplementary Fig. S8A). Cell-cycle changesas a result of MYC siRNA treatments had no effect on PARPiresponse (Supplementary Fig. S8B). However, MYC siRNAmarkedly impaired RAD51 foci formation in PARPi-treated cellsas compared with control (Fig. 2P and Q; Supplementary Fig.S8C–D). Finally, MYC downregulation impaired HR activity inMB231 and HCC1937 cells as measured using a DR-GFP reporterassay (Fig. 2R). Collectively, these results suggested that MYCinhibition of HR is comparable with RAD51 loss in both MB231and HCC1937 cells, providing further evidence of the HR-regu-latory role of MYC in TNBCs.

Pharmacologic inhibition of MYC expression induces PARPisensitivity

The downstream oncogenic function of MYC is primarilydependent on its heterodimerization with basic helix-loop-helixprotein MAX, resulting in the activation of transcriptional targets(22). The small-molecule 10058-F4 inhibitedMYC-MAX binding(23), which resulted in the downregulation of RAD51 protein(Fig. 3A) and RNA (Fig. 3B) expression in MB231 and SUM149cells. Treatment with 10058-F4 also resulted in transcriptionaldownregulation 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 adecreased RAD51-gH2AX ratio (Fig. 3F–H; Supplementary Fig.S9C). Collectively, these results suggest the following criteria foridentifying agents that when combined with PARPi could lead tosynergistic activity in TNBC cells: (i) downregulates MYC/RAD51expression, (ii) induces synergistic growth inhibition in HTSAs,and (iii) increases apoptosis in combination with PARPi(Fig. 3I–M; Supplementary Fig. S10). We tested several com-pounds including JQ1, a BRD4 inhibitor, and known MYCexpression inhibitor (24), dinaciclib, a pan cyclin-dependentkinase (CDK) 1,2,5,9 inhibitor with potent antiproliferativeactivity (25), THZ1, a CDK7 inhibitor that transcriptionallyregulates MYC expression (26) and nitazoxanide, an antiparasiticagent that inhibits c-myc expression (27). Of all these agents, theonly one that met all three criteria—reducing the expression ofboth MYC and RAD51 in both MB231 and SUM149 cells (Fig. 3Iand J), exhibiting synergism with niraparib HTSAs (Fig. 3K), and


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Figure 5.

Dinaciclib þ niraparib treatment in vivo. A–C, SUM149 cells were injected into immunocompromised mice and allowed to grow to approximately200 mm3; xenografts were treated with either vehicle, dinaciclib 25 mg/kg three times weekly, niraparib 5 mg/kg five times weekly, or dinaciclib þ niraparib(combo) therapy for 4 weeks. Tumor volume measurements (A) and Kaplan–Meier survival analysis (B) of mice treated with indicated drug treatment armsfor 4 weeks (28 days), followed by release without treatment until mice reached maximum allowed tumor size. C, Percent change in tumor volume normalizedby 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 cellswere injected into nudemice and cells were allowed to grow to >500mm3 before treatment was initiated. Mice were treated with vehicle, dinaciclib 25 mg/kg threetimes weekly, niraparib 5 mg/kg five times weekly, or dinaciclib þ niraparib (combo) therapy for 3 weeks. Upon completion, tumors were extracted andanalyzed 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 formultiple comparison. G–K Tumors from mice treated with each of the treatment arms were extracted and weighed (G and H) and subjected to IHC analysisof treated tumors for hematoxylin and eosin (H&E), Ki67, c-myc, RAD51, and gH2AX (I), resulting in a comparison of the H-score of c-myc with RAD51 expressionin MB231T–treated xenografts (J), and immunoblot analysis for c-myc, RAD51, gH2AX, Zeb1, Axl, E-cadherin, N-cadherin, and actin (K).






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significantly inducing apoptosis (Fig. 3L and M) in combinationtherapy was dinaciclib. Dinaciclib has an extensive clinical profilein several types of cancer including breast cancer (28).

Dinaciclib induces TNBC sensitivity to PARPisTreatment of a panel of TNBC BRCAmut and BRCAwt TNBC cell

lines with dinaciclib and niraparib revealed that the synergism ofthis combination therapy (assessed via HTSA) increased propor-tionally with the dinaciclib concentration (Fig. 4A and B) thatresulted 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 olaparibandvelaparib exhibited similar synergism inTNBCcell lineswhencombined with dinaciclib (Supplementary Fig. S11A–C). Thecombination therapy was not synthetically lethal in the immor-talized mammary epithelial cell line MCF10A, suggesting thatinduction of apoptosis by this combination strategy is tumorspecific and dependent upon MYC expression (Fig. 4D). Dinaci-clib þ niraparib combination therapy consistently inhibitedgrowth in both BRCA wild-type (MB231, MB157) and BRCAmutant (SUM149, HCC1937; Supplementary Fig. S11D).Induced MYC expression in MB231 cells partially reversed theeffect of dinaciclib and niraparib combination therapy (Supple-mentary Fig. S11E and S11F). Protein expression for severalproapoptotic and anti-apoptoticmarkers including cleavedPARP,caspase-3, caspase-7,Mcl-1, and Bcl-2 did not delineate activationof a conventional apoptotic pathway in the cells treated withmonotherapy versus combination therapy (Supplementary Fig.S11G). However, the DR-GFP assay demonstrated impaired HRactivity in the dinaciclib-treated cells (Fig. 4G). Dinaciclib inhib-ited RAD51 foci formation in MB231, HCC1937, and SUM149cells (Fig. 4G–J; Supplementary Fig. S12A–S12D). Combinationtherapy with dinaciclib and niraparib further increased the num-ber of gH2AX-positive cells (Fig. 4H and I; SupplementaryFig. S12A–S12G). Therefore, themechanism of synthetic lethalityof dinaciclib and niraparib is manifested through increasedgH2AX DNA damage concomitant with downregulation ofRAD51 foci formation. BRCA1 foci formation was also impairedby dinaciclib and niraparib in MB231 cells combination (Sup-plementary Fig. S12H). Therefore, dinaciclib can impair the HRpathway by inhibiting BRCA1 and RAD51 activity via c-myc inBRCAwt cells. However, in cells with retainedRAD51 functionalityin the absence of BRCA1, dinaciclib and niraparib also inducesynthetic lethality via RAD51 inhibition.Downregulation ofMYCtarget gene expression in response to treatment with dinaciclibalone or combinedwith niraparib supports the effect of dinaciclib

on c-myc and transcriptional regulation of the DNA repair path-way (Fig. 4K).

Wenext examined the in vivo efficacy of dinaciclib andniraparibagainst 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 celltypes, combination therapy produced superior growth inhibitionversus either agent alone (Fig. 5A–F).We also determinedwhethergrowth inhibition during this combination therapy (four cycles)was sustained in BRCAmut SUM149 xenografts after treatment(Fig. 5B; Supplementary Fig. S13A). Only the dinaciclib þniraparib combination therapy arm showed significant increasesin median survival duration of 43 days as compared with vehicle,niraparib, and dinaciclib arms with 15-, 26-, and 23-day mediansurvival times, respectively (P ¼ 0.0115; Fig. 5B).

We injected BRCAwt MB231T cells into nude mice and allowedthe resulting tumors to grow >500 mm3 before initiating treat-ment with dinaciclib and niraparib to determine whether theinitial tumor size had any bearing on therapeutic efficacy(Fig. 5D). Treatment with dinaciclib and niraparib resulted insignificant tumor growth inhibition (Fig. 5D; SupplementaryFig. S13D), increased overall survival (Fig. 5E), decreased percentchange in tumor volume (Fig. 5F; Supplementary Fig. S13F), and areduced tumor weight (Fig. 5G and H). IHC analysis demonstrat-ed 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 combina-tion treatment armalso demonstrated the greatest upregulation ofgH2AX expression (Fig. 5I; Supplementary Fig. S13J). Down-regulation of c-myc expression correlated with RAD51 and Ki67downregulation in treated xenografts (Fig. 5J; Supplementary Fig.S13G and S13K). Western blot analysis revealed downregulationof c-myc and RAD51 in both dinaciclib and combination treat-ment arms (Fig. 5K). In addition, we observed loss of pro-grammed cell death ligand1 (PD-L1) expression in treated tumors(Supplementary Fig. S13L). Consistent with downregulation ofEMT markers, dinaciclib given either alone or in combinationtherapy 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 reductionof mammosphere formation of MB231 cells treated with siMYCand dinaciclib compared with control, supporting a role of MYCon the CSC phenotype in TNBC (Supplementary Fig. S13N).Although MYC itself is a marker of the CSC phenotype, severalothers (e.g., Vimentin, ALDH) were downregulated at the proteinlevel. Reverse phase protein array (RPPA) analysis demonstrated

Figure 6.Dinaciclib induces synthetic lethality in niraparib acquired resistant and high MYC cancers. A, Seventy-two hour HTSA for niraparib was performedon 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 mmol/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, andHCC1806 (R) treated with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSyn software (<0.9 ¼ synergism;0.9–1.1 ¼ additive; >1.1 antagonistic). D, Clonogenic assay of MB436, MB436 (R), HCC1806, and HCC1806 (R) cells treated for 72 hours with DMSO (ctrl), dinaciclib10 nmol/L, niraparib 1mmol/L, and combo, followedby 72-hour drug release.E,TCGAanalysis ofMYCamplification across several cancer types.F andG, Seventy-twohour HTSA was performed on c-myc–high cancers with dinaciclib 10 nmol/L in combination with niraparib and synergism was determined using CalcuSynsoftware. H, Clonogenic assay of MYC-high cancer cell lines treated for 72 hours with DMSO (ctrl), dinaciclib 10 nmol/L, niraparib 1 mmol/L, and combo, followedby 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.






upregulation of the proapoptotic pathway but downregulationof the DNA repair, antiapoptotic, and epithelial–mesenchymaltransition proteins (Supplementary Fig. S13O–S13Q). Down-regulation of the EMT pathway was confirmed by proteindownregulation of Axl, Vimentin, Zeb1, and N-cadherin proteinexpression and upregulation of E-cadherin expression (Fig. 5K).These alterations correlated with downregulation of c-myc andRAD51 expression (Fig. 5J and I). Collectively, our in vivo analy-sis suggested that c-myc is a useful predictive biomarker fortreatment with the combination of dinaciclib and niraparibhaving synthetic lethality via HR repair and dinaciclib inhibitsthe EMT and CSC pathways in TNBC xenografts.

Dinaciclib resensitizes PARPi-resistant cells to PARP inhibitionNext, we sought to determine whether therapy with dinaciclib

and niraparib is also effective against TNBC cells with acquiredresistance to PARP inhibition. We generated PARPi-sensitiveBRCAmut, MB436 and BRCAwt, HCC1806 TNBC cell lines(Fig. 1A), resistant to increasing concentrations of the PARPiniraparib in a step-wise fashion, resulting in 5- to 18-foldincreased resistance according to IC50 values (Fig. 6A). Dinaciclibalone reversed the resistance of these cells to PARP inhibition,while inducing a synthetically lethal increase in apoptosis inci-dence (Fig. 6B). Also, the combination therapy induced synergis-tic growth inhibition (Fig. 6C and D). To further analyze theefficacy of combination therapy of dinaciclib and niraparib, weinduced PARPi resistance via 53BP1 loss. Knockdown of 53BP1expression in parental HCC1806 cells induced PARPi resistanceby two-fold (Supplementary Fig. S14A and S14b). The combina-tion treatment also induced synergistic growth inhibitionresponse in 53BP1 knockdown cells (Supplementary Fig. S14Cand S14D). Conversely, 53BP1 knockdown by short hairpin RNAdid not induce PARP inhibitor resistance inMB436 cells (data notshown). Thus, treatment with dinaciclib and niraparib can over-come both acquired and induced resistance to PARPi.

Dinaciclib and PARP inhibition are effective against MYC-driven cancers

MYC is a potent oncogene that is amplified in several cancertypes. Analysis of several TCGApatient cohorts revealedhighMYCgene expression in uveal, colorectal, head and neck, lung, andovarian tumors (Fig. 6E; Supplementary Fig. S15). Often, patientswith these high MYC-expressing cancers have very poor responserates to standard of care therapies, indicating a need for noveltherapeutic strategies (13). Therefore, we sought to determine theapplicability of c-myc and PARP inhibition for other aggressivecancer types besides TNBC. We selected a panel of high MYC-expressing ovarian, prostate, pancreatic, lung, and coloncancer cell lines (Fig. 6F). In vitro analysis of dinaciclib andniraparib revealed synergistic growth inhibition (i.e., combina-tion index < 1) in all cell lines examined (Fig. 6F–H). In addition,the combination therapy significantly increased apoptosisincidence, as compared with either single agent, in several celllines tested irrespective of cancer type (Fig. 6I). These resultssuggested that the combination of dinaciclib and niraparib is aneffective treatment strategy extending to many tumors with highMYC expression.

DiscussionClinical 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-chang-ing modality versus conventional chemotherapy. However, mostcancer patients do not benefit from PARPi use, as the BRCAmut

population represents only a small percentage of cancer patientsoverall. De novo and acquired resistancemechanisms further limitPARPi efficacy (29), demonstrating the need for increased clinicalPARPi use in combination therapy. We developed and tested apromising treatment strategy based on MYC oncogene addictionand DNA repair. Oncogene MYC induction induces replicationstress, causing double-strand DNA breaks, which require theactivation of HR pathway to repair lesions (3). Our study demon-strates that MYC expression directly invokes HR activation inresponse to DNA damage and can potentially be used as apredictor of response of TNBC to therapy with PARPicombination.

The connection between MYC and RAD51 (as an indicator ofHR activity) is based upon (i) correlation between TNBC andovarian cancer tumor tissue gene expression of MYC and severalDNA repair genes including RAD51 in the TCGA cohort(Supplementray Fig. 6E–I); (ii) IHC analysis of MYC and RAD51in TNBC patient cohorts accrued at MD Anderson Cancer Center(Fig. 2G and H; Supplementary Fig. S7A–S7D); (iii) downregula-tion of RAD51 in response to MYC knockdown in MDA231 cells(Fig. 2I and J); (iv) MYC regulation of RAD51 in knock-inexperiments (Fig. 2K and L). Collectively these results, which areconsistent with published reports showing MYC is a transcrip-tional regulator of RAD51 (30–33), provide experimental evi-dence for the functional relationship between MYC and RAD51.

Although, oncogenic MYC transformation embodies severalaspects of the "Hallmarks of Cancer," it is designated as anundruggable target (1). Therefore, targeting of MYC-driven can-cers through a synthetically lethal approach has therapeuticpotential. MYC expression has long been associated with BRCAmutation frequency in breast cancer cases (34, 35). Studies oftransgenic mousemodels have demonstrated that MYC facilitatesBRCAmut progression of ovarian cancer (36). The frequency ofMYC upregulation in BRCAmut tumors and correlation with HRrepair genes suggests that MYC compensates for BRCA loss viaupregulation of a compensatory HR pathway, via increasedRAD51 expression (37). The correlation ofMYC and RAD51 geneexpression in sporadic TNBCs (Fig. 2D) supports MYC depen-dence on HR repair as a function of MYC-driven tumors. RAD51also regulates TNBC resistance to PARPis via the CSC resistancephenotype in TNBC (38). Inhibition of RAD51 expression incombination with PARPi-based treatment abrogates this CSCresistance phenotype, supporting our use of RAD51 as a markerof response to PARPis. Although RAD51 is classified as a tumorsuppressor, high RAD51 expression compensates for BRCA loss intumors, causing decreased sensitivity to irradiation and chemo-therapy (37). Independent of MYC, increased RAD51 expressionpromotes TNBC metastasis, supporting high MYC, and RAD51individual expression as negative predictors of poor outcome ofTNBC (7).

The clinical use of CDK inhibitors to treat cancer has yieldedminimal success until recently with the approval of the CDK4/6inhibitors palbociclib and riibociclib (39). Dinaciclib, a panCDK 1/2/5/9 inhibitor, has undergone several rounds of clinicalinvestigation for several types of cancer including breast cancer(28, 40). However, the lack of a suitable biomarker for patientselection has plagued the advancement of dinaciclib beyond

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phase II clinical trials. In addition, dinaciclib combined withconventional chemotherapy has compounded patient toxicity,leading to treatment discontinuation, which further supports theuse of dinaciclib in combination with targeted therapy to min-imize patient toxicity profiles (28). Although dinaciclib was notdeveloped as an agent targeted against MYC, several studies havecorroborated our observation of dinaciclib's regulation of MYCexpression (41, 42). Ideally, MYC downregulation would inducesynthetic lethality in MYC-driven tumors, however studies havedemonstrated that MYC loss alone is insufficiently lethal (31–33)further strengthening the need for combination therapy.

The precise mechanism of dinaciclib-induced MYC downregu-lation remains unclear. CDK1 and CDK9 inhibition are bothsynthetically lethal in high MYC-expressing cancer cells (43).Several CDKs (CDK2, CDK1, CDK5, CDK12) have been linkedtoDNA repair regulation,withmanyCDKsphosphorylatingDNArepair genes directly (BRCA1, BRCA2; refs. 44–48). CDK5 hasbeen directly linked to regulation of DNA repair in cancer (49).Transcriptional downregulation of MYC in response to dinaciclibexposure points to a transcriptional CDK-regulating MYC expres-sion. Recently, Johnson and colleagues have suggested thatCDK12 mediates dinaciclib-induced sensitivity to PARP inhibi-tion in TNBC (50). In support of these findings, loss of CDK12expression is synthetic lethal in c-myc–overexpressing cells, thuscharacterizing CDK12 as an upstream effector of MYC expressionvia RNA polymerase phosphorylation (50). Loss of CDK12expression also predicts sensitivity of ovarian cancer to PARPis(51). Traditionally, the degree of PARP trapping by PARPis plays asignificant role in the efficacy of PARPis against HR-deficient cells(52). Our study demonstrates a synergistic growth inhibitoryeffect of dinaciclib on TNBC cells when given with several differ-ent PARPis, suggesting that PARP trapping does not dictatesensitivity to this combination therapy.

We have also presented a novel mechanism of stratifyingTNBC patients for therapy with PARPis based upon MYC

expression (Supplementary Fig. S16). This model corrects anumber of deficiencies in treatment options for TNBC patientsby making MYC druggable, enhancing the therapeutic effect ofPARPis in non-BRCAmut TNBC patients while targeting severalcancer hallmarks including DNA repair, EMT, and the CSCphenotype, which lie at the crux of conventional chemother-apeutic efficacy (Fig. 7).

Predictive IHC analysis of c-myc/RAD51 expression points to aless favorable response to chemotherapy of tumors that expressboth c-myc and RAD51 (Fig. 2f–h). By harnessing this knowledgewe seek to improve patient outcomes in both c-myc-/RAD51- (viachemotherapy) and c-mycþ/RAD51þ (via treatment with dinaci-clib and niraparib) patient cohorts. In addition, implementationof dinaciclib andniraparib as aneoadjuvant therapeutic treatmentthat downregulates both c-myc and RAD51 expression inferssubsequent response to chemotherapy and/or radiation therapy,if necessary. As a proto-oncogene, upregulation of MYC can drivetherapeutic resistance to other targeted therapeutic agents (53,54). Therefore, the combination strategy with dinaciclib andniraparib may be a secondary treatment option for patients whohave developed MYC-dependent resistance to therapy.

Disclosure of Potential Conflicts of InterestJ.K. Litton reports receiving a commercial research grant from Pfizer and

Astra-Zeneca and also is a consultant/advisory board member for Pfizer andAstra-Zeneca. K.K. Hunt reports receiving a commercial research grant fromEndomagnetics and Lumicell and is also a consultant/advisory board memberfor Lumicell and Armada Health. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: J.P.W. Carey, K. Mikule, K.K. Hunt, K. KeyomarsiDevelopment of methodology: J.P.W. Carey, Y. Zhao, K. KeyomarsiAcquisition 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

Figure 7.

A graphical representation oftargeting both MYC and PARP inMYC-high TNBC could elicitantitumor effects.






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. KeyomarsiWriting, review, and/or revision of the manuscript: J.P.W. Carey, Y. Zhao,K. Mikule, J.K. Litton, K.K. Hunt, K. KeyomarsiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.P.W. Carey,Study supervision: J.P.W. Carey, K.K. Hunt, K. Keyomarsi

AcknowledgmentsWe thank Dr. Debu Tripathy, Dr. Banu Arun, and Dr. Gabriel Hortobagyi

for their insightful comments during the course of this study. Researchreported in this publication was supported by the National Cancer Instituteof the NIH under award (P30CA016672) to The University of Texas

MD Anderson Cancer Center, R01 grants CA87548 and CA1522218 andCancer Prevention and Research Institute of Texas (CPRIT) RP170079grants (to K. Keyomarsi), the Susan G. Komen for the Cure grant KG100521(to K.K. Hunt), the Susan G. Komen postdoctoral fellowship grantPDF14302675 (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 thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 31, 2017; revised September 29, 2017; accepted November 7,2017; published OnlineFirst November 27, 2017.

References1. Stine ZE,WaltonZE,AltmanBJ,HsiehAL,DangCV.MYC,metabolism, and

cancer. Cancer Discov 2015;5:1024–39.2. Walz S, Lorenzin F, Morton J, Wiese KE, von Eyss B, Herold S, et al.

Activation and repression by oncogenic MYC shape tumour-specific geneexpression profiles. Nature 2014;511:483–7.

3. Cottini F, Hideshima T, Suzuki R, Tai YT, Bianchini G, Richardson PG, et al.Synthetic lethal approaches exploiting DNA damage in aggressive myelo-ma. Cancer Discov 2015;5:972–87.

4. AlHilli MM, BeckerMA,Weroha SJ, Flatten KS, Hurley RM,Harrell MI, et al.In vivo anti-tumor activity of the PARP inhibitor niraparib in homologousrecombination deficient and proficient ovarian carcinoma. Gynecol Oncol2016;143:379–88.

5. RING-less BRCA1 induces PARP inhibitor and platinum resistance. CancerDiscov 2016;6:OF6.

6. Wang Y, Bernhardy AJ, Cruz C, Krais JJ, Nacson J, Nicolas E, et al. TheBRCA1-Delta11q alternative splice isoform bypasses germline mutationsand promotes therapeutic resistance to PARP inhibition and cisplatin.Cancer Res 2016;76:2778–90.

7. Wiegmans AP, Al-Ejeh F, Chee N, Yap PY, Gorski JJ, Da Silva L, et al. Rad51supports triple negative breast cancer metastasis. Oncotarget 2014;5:3261–72.

8. Balaji K, Vijayaraghavan S, Diao L, Tong P, Fan Y, Carey JP, et al. AXLInhibition suppresses the DNA damage response and sensitizes cells toPARP inhibition in multiple cancers. Mol Cancer Res 2017;15:45–58.

9. Nanos-WebbA, JabbourNA,Multani AS,WingateH,OumataN,GalonsH,et al. Targeting low molecular weight cyclin E (LMW-E) in breast cancer.Breast Cancer Res Treat 2012;132:575–88.

10. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.

11. Aravalli RN, Talbot NC, Steer CJ. Gene expression profiling of MYC-driventumor signatures in porcine liver stem cells by transcriptome sequencing.World J Gastroenterol 2015;21:2011–29.

12. Nanos-Webb A, Bui T, Karakas C, Zhang D, Carey JP, Mills GB, et al.PKCiota promotes ovarian tumor progression through deregulation ofcyclin E. Oncogene 2016;35:2428–40.

13. Jung M, Russell AJ, Liu B, George J, Liu PY, Liu T, et al. A Myc activitysignature predicts poor clinical outcomes in Myc-associated cancers. Can-cer Res 2017;77:971–81.

14. Hartman AR, Kaldate RR, Sailer LM, Painter L, Grier CE, Endsley RR, et al.Prevalence of BRCA mutations in an unselected population of triple-negative breast cancer. Cancer 2012;118:2787–95.

15. Kwon JS, Gutierrez-Barrera AM, YoungD, SunCC,DanielsMS, LuKH, et al.Expanding the criteria for BRCAmutation testing in breast cancer survivors.J Clin Oncol 2010;28:4214–20.

16. Gonzalez-Angulo AM, Timms KM, Liu S, Chen H, Litton JK, Potter J,et al. Incidence and outcome of BRCA mutations in unselected patientswith triple receptor-negative breast cancer. Clin Cancer Res 2011;17:1082–9.

17. Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, Gonzalez-AnguloAM, et al. Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol 2008;26:4282–8.

18. Bayraktar S, Gluck S. Systemic therapy options in BRCA mutation-associ-ated breast cancer. Breast Cancer Res Treat 2012;135:355–66.

19. Sorlie T, Tibshirani R, Parker J, Hastie T,Marron JS, Nobel A, et al. Repeatedobservation of breast tumor subtypes in independent gene expression datasets. Proc Natl Acad Sci U S A 2003;100:8418–23.

20. Peshkin BN, Alabek ML, Isaacs C. BRCA1/2 mutations and triple negativebreast cancers. Breast Dis 2010;32:25–33.

21. Buisson R, Dion-Cote AM, Coulombe Y, Launay H, Cai H, Stasiak AZ, et al.Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 instimulating homologous recombination. Nat Struct Mol Biol 2010;17:1247–54.

22. Hurlin PJ, Dezfouli S. Functions of myc:max in the control of cell prolif-eration and tumorigenesis. Int Rev Cytol 2004;238:183–226.

23. Follis AV, Hammoudeh DI, Daab AT, Metallo SJ. Small-molecule pertur-bation of competing interactions between c-Myc and Max. Bioorg MedChem Lett 2009;19:807–10.

24. ShaoQ, Kannan A, Lin Z, Stack BC Jr., Suen JY, Gao L. BET protein inhibitorJQ1 attenuates Myc-amplified MCC tumor growth in vivo. Cancer Res2014;74:7090–102.

25. Parry D, Guzi T, Shanahan F, Davis N, Prabhavalkar D, Wiswell D, et al.Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinaseinhibitor. Mol Cancer Ther 2010;9:2344–53.

26. Kwiatkowski N, Zhang T, Rahl PB, Abraham BJ, Reddy J, Ficarro SB, et al.Targeting transcription regulation in cancer with a covalent CDK7 inhib-itor. Nature 2014;511:616–20.

27. Chipumuro E, Marco E, Christensen CL, Kwiatkowski N, Zhang T, Hathe-way CM, et al. CDK7 inhibition suppresses super-enhancer-linked onco-genic transcription in MYCN-driven cancer. Cell 2014;159:1126–39.

28. Mitri Z, Karakas C,Wei C, Briones B, Simmons H, IbrahimN, et al. A phase1 studywithdose expansionof theCDK inhibitor dinaciclib (SCH727965)in combination with epirubicin in patients with metastatic triple negativebreast cancer. Invest New Drugs 2015;33:890–4.

29. Kaufman B, Shapira-Frommer R, Schmutzler RK, Audeh MW, FriedlanderM, Balmana J, et al. Olaparib monotherapy in patients with advancedcancer and a germline BRCA1/2 mutation. J Clin Oncol 2015;33:244–50.

30. Middleton FK, Patterson MJ, Elstob CJ, Fordham S, Herriott A, Wade MA,et al. Common cancer-associated imbalances in theDNAdamage responseconfer sensitivity to single agent ATR inhibition. Oncotarget 2015;6:32396–409.

31. Lee JY, Kim DK, Ko JJ, Kim KP, Park KS. Rad51 regulates reprogrammingefficiency through DNA repair pathway. Dev Reprod 2016;20:163–9.

32. Gravina GL, Festuccia C, Popov VM, Di Rocco A, Colapietro A, Sanita P,et al. c-Myc sustains transformed phenotype and promotes radioresistanceof embryonal rhabdomyosarcoma cell lines. Radiat Res 2016;185:411–22.

33. Ambrosio S, Amente S, Napolitano G, Di Palo G, Lania L, Majello B. MYCimpairs resolution of site-specific DNA double-strand breaks repair. MutatRes 2015;774:6–13.

34. Grushko TA, Dignam JJ, Das S, Blackwood AM, Perou CM, Ridderstrale KK,et al.MYC is amplified in BRCA1-associated breast cancers. ClinCancer Res2004;10:499–507.

35. Rhei E, Bogomolniy F, Federici MG,Maresco DL, Offit K, RobsonME, et al.Molecular genetic characterization of BRCA1- and BRCA2-linked heredi-tary ovarian cancers. Cancer Res 1998;58:3193–6.

Carey et al.





36. Xing D, Orsulic S. A mouse model for the molecular characterization ofbrca1-associated ovarian carcinoma. Cancer Res 2006;66:8949–53.

37. Martin RW, Orelli BJ, Yamazoe M, Minn AJ, Takeda S, Bishop DK. RAD51up-regulation bypasses BRCA1 function and is a common feature ofBRCA1-deficient breast tumors. Cancer Res 2007;67:9658–65.

38. Liu Y, BurnessML,Martin-Trevino R,Guy J, Bai S,Harouaka R, et al. RAD51mediates resistance of cancer stem cells to PARP inhibition in triple-negative breast cancer. Clin Cancer Res 2017;23:514–22.

39. Hortobagyi GN. Ribociclib forHR-positive, advanced breast cancer. N EnglJ Med 2017;376:289.

40. Chen XX, Xie FF, Zhu XJ, Lin F, Pan SS, Gong LH, et al. Cyclin-dependent kinase inhibitor dinaciclib potently synergizes with cis-platin in preclinical models of ovarian cancer. Oncotarget 2015;6:14926–39.

41. Gregory GP, Hogg SJ, Kats LM, Vidacs E, Baker AJ, Gilan O, et al. CDK9inhibition by dinaciclib potently suppresses Mcl-1 to induce durableapoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo.Leukemia 2015;29:1437–41.

42. Horiuchi D, Kusdra L, Huskey NE, Chandriani S, Lenburg ME, Gonza-lez-Angulo AM, et al. MYC pathway activation in triple-negative breastcancer is synthetic lethal with CDK inhibition. J Exp Med 2012;209:679–96.

43. Huang CH, Lujambio A, Zuber J, Tschaharganeh DF, Doran MG,Evans MJ, et al. CDK9-mediated transcription elongation is requiredfor MYC addiction in hepatocellular carcinoma. Genes Dev 2014;28:1800–14.

44. Liu W, Li J, Song YS, Li Y, Jia YH, Zhao HD. Cdk5 links with DNA damage`response and cancer. Mol Cancer 2017;16:60.

45. Wang H, Kim NH. CDK2 is required for the DNA damage response duringporcine early embryonic development. Biol Reprod 2016;95:31.

46. Trovesi C, Manfrini N, Falcettoni M, Longhese MP. Regulation of the DNAdamage response by cyclin-dependent kinases. J Mol Biol 2013;425:4756–66.

47. Cerqueira A, Santamaria D, Martinez-Pastor B, Cuadrado M, Fernandez-Capetillo O, Barbacid M. Overall Cdk activity modulates the DNA damageresponse in mammalian cells. J Cell Biol 2009;187:773–80.

48. Johnson N, Shapiro GI. Cyclin-dependent kinases (cdks) and the DNAdamage response: rationale for cdk inhibitor-chemotherapy combinationsas an anticancer strategy for solid tumors. Expert Opin Ther Targets 2010;14:1199–212.

49. Shupp A, Casimiro MC, Pestell RG. Biological functions of CDK5and potential CDK5 targeted clinical treatments. Oncotarget 2017;8:17373–82.

50. JohnsonSF,CruzC,Greifenberg AK,Dust S, StoverDG,ChiD, et al. CDK12inhibition reverses De novo and acquired PARP inhibitor resistance inBRCA wild-type and mutated models of triple-negative breast cancer. CellRep 2016;17:2367–81.

51. Bajrami I, Frankum JR, Konde A, Miller RE, Rehman FL, Brough R, et al.Genome-wide profiling of genetic synthetic lethality identifies CDK12 as anovel determinant of PARP1/2 inhibitor sensitivity. Cancer Res 2014;74:287–97.

52. Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, et al.Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res2012;72:5588–99.

53. Xiao D, Yue M, Su H, Ren P, Jiang J, Li F, et al. Polo-like Kinase-1 RegulatesMyc stabilization and activates a feedforward circuit promoting tumor cellsurvival. Mol Cell 2016;64:493–506.

54. Liu H, Ai J, Shen A, Chen Y, Wang X, Peng X, et al. c-Myc alterationdetermines the therapeutic response to FGFR inhibitors. Clin Cancer Res2017;23:974–84.






2018;78:742-757. Published OnlineFirst November 27, 2017.Cancer Res Jason. P.W. Carey, Cansu Karakas, Tuyen Bui, et al. CancerBlockade Is Independent of BRCA Status in Triple-Negative Breast Synthetic Lethality of PARP Inhibitors in Combination with MYC

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