Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in P53-Mutant Triple-Negative Breast Cancer Natalie A

Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0519

Small Molecule Therapeutics Molecular Cancer Therapeutics Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in p53-Mutant Triple-Negative Breast Cancer Natalie A. Jabbour-Leung1, Xian Chen1, Tuyen Bui1, Yufeng Jiang1, Dong Yang1, Smruthi Vijayaraghavan1, Mark J. McArthur2, Kelly K. Hunt3, and Khandan Keyomarsi1

Abstract

Triple-negative breast cancer (TNBC) is an aggressive malig- Roscovitine treatment arrests TNBC cells in the G2–M cell-cycle nancy in which the tumors lack expression of estrogen receptor, phase, priming them for DNA damage. Combination treatment progesterone receptor, and HER2. Hence, TNBC patients cannot increased frequency of DNA double-strand breaks, while simul- benefit from clinically available targeted therapies and rely on taneously reducing recruitment of homologous recombination chemotherapy and surgery for treatment. While initially respond- proteins compared with doxorubicin treatment alone. Further- ing to chemotherapy, TNBC patients are at increased risk of more, this combination therapy significantly reduced tumor developing distant metastasis and have decreased overall survival volume and increased overall survival compared with single drug compared with non-TNBC patients. A majority of TNBC tumors or concomitant treatment in xenograft studies. Examination of carry p53 mutations, enabling them to bypass the G1 checkpoint isogenic immortalized human mammary epithelial cells and and complete the cell cycle even in the presence of DNA damage. isogenic tumor cell lines found that abolishment of the p53 Therefore, we hypothesized that TNBC cells are sensitive to cell- pathway is required for combination-induced cytotoxicity, mak- cycle–targeted combination therapy, which leaves nontrans- ing p53 a putative predictor of response to therapy. By exploiting formed cells unharmed. Our findings demonstrate that sequential thespecificbiologicandmolecularcharacteristicsofTNBCtumors, administration of the pan-CDK inhibitor roscovitine before doxo- thisinnovativetherapycangreatlyimpactthetreatmentandcareof rubicin treatment is synthetically lethal explicitly in TNBC cells. TNBC patients. Mol Cancer Ther; 15(4); 593–607. 2016 AACR.

Introduction resulting from BRCA1 mutations, may contribute to TNBC patients initially responding well to chemotherapy; however, Triple-negative breast cancer (TNBC) is a clinical diagnosis many patients' tumors recur (6). While there are several targeted that afflicts 10% to 20% of breast cancer patients (1). TNBC therapies being developed in clinical trials, including PARP and tumors are characterized by lacking expression of estrogen EGFR inhibitors, there are currently no clinically available and receptor (ER), progesterone receptor (PR) and the growth factor effective targeted therapies for TNBC patients (6–8). receptor HER2. TNBC patients are more likely to be premeno- The majority (54%–82%) of TNBC tumors harbor p53 muta- pausal, of African descent, have a poor prognosis and have tions, enabling them to bypass the G checkpoint and complete greater risk of recurrence within the first 5 years of diagnosis (2). 1 the cell cycle even with unrepaired DNA damage (6, 9, 10). In Less than one-third of metastatic TNBC patients survive 5 years comparison, only 13% of hormone receptor-positive luminal A (3). About 70% of TNBC are classified as basal-like breast cancer tumors have p53 mutations (11). Moreover, 50% of these breast (BLBC), and share many of the same characteristics, including cancers overexpress cyclin D1, inhibiting retinoblastoma (Rb) more likely occurring in patients with BRCA1/BRCA2 gene regulation of E2F (12). Notably, overexpression of cyclin E serves mutations (4, 5). Dysfunction in the DNA repair pathway, as a poor prognostic marker in breast cancer and correlates to negative ER and PR status (13, 14). Owing to deregulation of the cell cycle in cancer cells, cyclin-dependent kinase (CDK) inhibi- 1Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2Veterinary Med- tors were developed to inhibit tumor cell proliferation and icine and Surgery,The University of Texas MDAnderson Cancer Center, induce apoptosis (15). However, CDK inhibitors have not been 3 Houston,Texas. Breast Surgical Oncology,The University of Texas MD effective clinically, despite having promising results both in vitro Anderson Cancer Center, Houston, Texas. and in vivo (16, 17). Roscovitine, a pan CDK inhibitor with activity Note: Supplementary data for this article are available at Molecular Cancer against CDK1, 2, 5, 7, and 9 (18, 19), became the first orally Therapeutics Online (http://mct.aacrjournals.org/). bioavailable drug from this class to go into clinical trials based on N.A. Jabbour-Leung and X. Chen contributed equally to this article. the preclinical data showing induction of apoptosis in tumor cells. Corresponding Author: Khandan Keyomarsi, Department of Experimental However, of the 77 solid tumor patients treated with single-agent Radiation Oncology, The University of Texas MD Anderson Cancer Center, roscovitine, one partial response was seen in hepatocellular 6565 MD Anderson Blvd, Box 1052, Houston, TX 77030. Phone: 713-792- carcinoma, 2 prolonged stable disease observed in non–small 4845; Fax: 713-794-5369; E-mail: [email protected] cell lung cancer (14 and >18 months), while stable disease was the doi: 10.1158/1535-7163.MCT-15-0519 best response seen in the remaining solid tumors (20–22). One of 2016 American Association for Cancer Research. the reasons that these CDK inhibitors have not been more effective

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clinically is that they are either being used as single agents or when 10A, 76NE6, and 76NF2V, the breast cancer cell lines MDA-MB- they are used in combination therapy, both agents were delivered 157, MDA-MB-231, HCC1806, MCF-7, ZR75-1, T47D, MDA- concomitantly to the patient (23). In addition, there was no MB-468 (Supplementary Table S1A), and the isogenic colorectal þ þ attempt to identify those patients most likely to respond to these cancer cells HCT116 p53 wild-type (p53 / ) and p53 knockout agents based on their biology. In fact, very few patients with breast (p53 / ) were previously described (29–31). HEK-293T cells cancer of any subtype were accrued to these trials. for lentiviral packaging were maintained in DMEM supplemen- CDK1 participates in the DNA double-strand break (DSB) ted with 10% FCS. All cells were free of mycoplasma contam- repair pathway homologous recombination (HR). HR faithful- ination, and their identities were authenticated by karyotype ly repairs DNA DSBs that occur in late S, G2,andM(24).CDK and short tandem repeat analysis using the MD Anderson's activity is required for the recruitment of the endonucleases Characterized Cell Line Core Facility (http://www.mdander- Sae2 or CtIP that excise the DNA DSB to generate single strands son.org/characterized-cell-line-core-facility/index.html) on a during HR in both yeast and mammalian cells, respectively (25, routine basis (every 6 months). 26). Moreover, CDK activity is required for the recruitment and association of BRCA1 to the MRN (Mre11-Rad50-Nbs1) com- Immunofluorescence to detect DNA repair foci plex during HR (27). Concordantly, CDK inhibition with Following treatment of cells with roscovitine (IC50) for 24 roscovitine reduced the recruitment of HR downstream protein hours, doxorubicin (IC50) for 48 hours, or both drugs sequentially RPA34 in irradiated sarcoma cells due to an inability to gen- as indicated, they were fixed in 4% paraformaldehyde for 20 min- erate single strands (28). Thus, compromising HR via CDK utes followed by permeabilization with a 0.3% triton solution (20 inhibition may provide a strategy to augment TNBC cell sen- mol/L Hepes, 50 mmol/L NaCl, 3 mmol/L MgCl2, 300 mmol/L sitivity to chemotherapy. sucrose, and TritonX-100) for 20 minutes. Cells were then blocked No clinically available treatment strategies target the TNBC- with 10% BSA and 2% horse serum PBS for one hour. Antibodies deregulated cell cycle to exploit TNBC-cell sensitivity to DNA- were diluted 1:500 and 1:1000 for anti-g-H2AX (EMD Millipore) damaging agents (e.g., chemotherapeutics). Because TNBC cells and anti-Rad51 (32), respectively, and incubated at 4C over- have a deregulated G1 checkpoint, enabling them to re-enter the night. Secondary goat anti-mouse or goat anti-rabbit antibodies cell cycle while harboring DNA damage, we hypothesized that (Alexa Fluor 594 and 488, respectively, EMD Millipore) were – TNBC cells are sensitive to cell-cycle targeted combination diluted at 1:750 and incubated at room temperature at 1 hour. therapy, which leaves nontransformed cells unharmed. Ideally, Nuclei were stained with DAPI (Life Technology) at 1mg/mL for 5 this therapeutic strategy will be synthetically lethal against minutes room temperature. Cells were mounted with Dako TNBC cells by inhibiting pathways that can compensate for fluorescent mounting medium. Images were captured using the one another during tumorigenesis. We therefore examined the Olympus FV1000 Laser Confocal Microscope. Cells with 5 foci effect of combining the pan-CDK inhibitor roscovitine and the were considered g-H2AX positive. chemotherapeutic doxorubicin both sequentially and concomitantly. Our data suggest that sequential treatment of roscovitine before doxorubicin is synthetically lethal explicitly in Neutral comet assay TNBC cells due to p53 pathway ablation. A neutral comet assay was performed according to Trevigen protocols to measure DNA DSBs in HMEC and TNBC cells in response to single and combination drug treatment. Briefly, Materials and Methods following treatment, cells were harvested and combined with Cell lines and culture conditions low-melting agarose, and spread onto CometSlides (Trevigen). The source of cell lines and culture conditions of the immor- Following cell adherence to the slides, cells were lysed with talized human mammary epithelial (HMEC) cell lines MCF- Trevigen lysis solution. The slides were then placed in an

Figure 1. Sequential administration of roscovitine and doxorubicin is synergistic only in TNBC cells. A, a panel of p53 wild-type, p53 heterozygous, and p53-mutant breast cells, including immortalized human mammary epithelial cells (HMEC cells; MCF-10A, 76NF2V, and 76NE6), estrogen receptor–positive (ER-positive cells; T47D, MCF- 7, ZR-75-1) and triple-negative breast cancer (TNBC cells; MDA-MB-468, HCC1806, MDA-MB-157, and MDA-MB-231) cells was treated with roscovitine (R)at 20 mmol/L for 24 hours followed by flow cytometry for cell-cycle analysis. Untreated cells harvested at the same time served as a control (C). B, average percent change in cell-cycle phase following treatment of roscovitine as compared with untreated cells according to p53 status. The p53 wild-type cell lines included were MCF-10A, 76NF2V, MCF-7, and ZR75-1; p53-mutant/nonfunctional cell lines included were 76NE6, MDA-MB-468, HCC1806, MDA-MB-157, and MDA-MB-231 (C), HMEC (MCF-10A), ER-positive (ZR75-1) and TNBC (MDA-MB-157, MDA-MB-231, and MDA-MB-468) cells were subjected roscovitine and doxorubicin (D) combination treatment in 96-well plates as described in the Materials and Methods. Drugs were administered both simultaneously (RþD) for 72 hours and sequentially in both directions (D!RorR!D). During sequential treatment the first drug, or drug A, was administered at IC10,IC25,andIC50 concentrations for 24 hours. Following removal of drug A, drug B was administered at IC10 to IC50 concentrations for 48 hours. The IC values for all treatment conditions are listed in Supplementary Fig. S1B. Following 72 hours of drug treatment, cells were cultured in drug-free medium for an additional 9 days where drug-medium was replaced every 48 hours. Cells were subjected to MTT assay on day 12. Isobolograms and combination index values (CI) were generated using CalcuSyn, in which eachpoint refers to a specific combination of drug A to drug B. CI values <1.0 are synergistic, CI values ¼ 1.0 are additive, and CI values >1.0 are antagonistic. Average CI values per combination treatment are shown in the bar graphs. D, MCF-10A and MDA-MB-468 cells were transiently transfected with siRNA against CDK1, CDK2, or both. Nontargeting siRNA (siControl) served as a control. Western blot analysis with antibodies against CDK1 and CDK2 was used to confirm efficiency of downregulation. Next, transfected cells were subjected to HTSA either in the presence of absence of doxorubicin at IC50 values (30 nmol/L for MCF-10A and 25 nmol/L for MDA-MB-468 cells), Supplementary Fig. S1B) for 48 hours. After 48 hours, doxorubicin-containing medium was replaced with drug-freemedium every 48 hours until day 12 when cell viability was assessed with MTT. Percent viability for each condition was calculated as compared with siControl plotted in the bar graphs. For all panels, experiments were repeated three times; error bars, 95% confidence intervals. The Student t test (two-tailed, equal variance) was used to derive the P values.

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electrophoresis chamber with neutral electrophoresis buffer. A different receptor and p53 status, we treated 10 different cell current of 21 V was used for 21 minutes. After drying, samples lines, including three HMEC, three ER/PR-positive cell lines, were stained with SYBR Green I and allowed to dry at room and four triple-negative cell lines, (Supplementary Table S1A) temperature in the dark. Images of nuclei were captured using an with 20 mmol/L roscovitine for 24 hours followed by flow- Eclipse 90i microscope equipped with the NIS-Elements Br 3.10 cytometric analysis (Fig. 1A). Roscovitine induced only a 10% software program (Nikon). The tail moment was measured using or less increase in G2–M phase in the p53 wild-type HMEC the CometScore software program (TriTek). cells (MCF-10A and 76NF2V), p53 wild-type ER/PR-positive cells (ZR75-1 and MCF-7), and p53 heterozygous ER/PR-pos- Xenograft studies itive cells (T47D; 34). In contrast, treatment of the p53-mutat- 6 A total of 4.5 10 MDA-MB-468 cells in a 1:2 ratio with ed TNBC cell lines (MDA-MB-468, MDA-MB-157, HCC1806) Matrigel (BD Bioscience) were injected into the mammary fat and p53 inactivated HMECs (76NE6) resulted in a 30% and pad of 4- to 6-week-old female Balb/c Nu/nu mice (Taconic). 20% increase of G –M phase, respectively, demonstrating a fl 2 Mice received injections on both the right and left anks. Once significantly greater change (P < 0.05) in the G –Mphase 2 3 2 the tumors reached a volume (L x W /2) of 100 to 150 mm , compared with p53 wild-type cells (Fig. 1A and 1B; Supple- þ mice were treated with vehicle vehicle, roscovitine (50 mg/kg) mentary Fig. S1A). 4 days on/3 days off, doxorubicin (2mg/kg) once a week, These results raised the hypothesis that sequential treat- sequentially with 4 days of roscovitine followed by 1 day of ment of TNBC cells by roscovitine would accumulate them in doxorubicin, or concomitantly with 4 days of roscovitine and 1 the G2–M phase, and when followed by a DNA-damaging day of doxorubicin administered on day 1 for four cycles. agent, such as doxorubicin, would lead to synergistic cell All drugs were administered i.p. Roscovitine was diluted at killing. We tested this hypothesis by examining the schedule 100 mg/mL in DMSO and then further diluted to 10 mg/mL and duration of combination therapy with roscovitine in a in a carrier solution consisting of 10% Tween 80 (Sigma- panel of five cell lines, including HMEC MCF-10A cells, ER- Aldrich), 20% N-N-dimethylacetamide (Acros Organics), and positive ZR75-1 and TNBC MDA-MB-157, MDA-MB-231 and 70% polyethylene glycol 400 (Sigma-Aldrich; 33). The tumor MDA-B-468 cell lines (Fig. 1C). A high throughput survival volume and weight of the mice were measured twice a week. assay (HTSA) was used as a means for determining cell Mice were euthanized when the total tumor burden (sum of survival. This assay evaluates in vitro survival of cells in 3 right and left tumors) was equal to 1,500 mm .Micewere response to different treatment strategies, either as single fi housed ve per cage in sterilized micro-isolator cages (Lab agents or in combination (35, 36). As depicted in the diagram Products) furnished with corncob bedding. Mice received care (Supplementary Figs. 1B and C), each cell line was treated in accordance with the Animal Welfare Act, the NIH "Guide for with three different combination strategies: (i) concomitant the Care and Use of Laboratory Animals," and the institutional treatment with roscovitine and doxorubicin; (ii) sequential guidelines of MD Anderson Cancer Center (Houston, TX). treatment with doxorubicin followed by roscovitine; and (iii) sequential treatment with roscovitine followed by doxorubi- Statistical analysis cin. At the conclusion of the experiment (12 days), cell Each experiment was carried out at least three times. Contin- viability was assessed based upon a combinational index (CI) uous outcomes were summarized with mean and SD. Compar- algorithmic that the statistical program CalcuSyn provides. CI isons among groups were conducted using GraphPad Prism values of 0–0.9 are indicative of synergistic response, whereas software using either unpaired or paired Student t test with a CI values of 0.9–1.1 or >1.1 indicate an additive or antago- 95% confidence interval. Overall survival curves were generated nistic response, respectively (37). The CI values demonstrated with the Kaplan–Meir method and compared using the Mantel– that concomitant treatment induced an antagonistic or addi- Cox test (GraphPad Prism Software). tive response in all cell lines (Fig. 1C, R þ D). Sequential administration of doxorubicin treatment before roscovitine Results also induced antagonism or additivity in all cell lines (Fig. 1C Sequential combination treatment with roscovitine followed and D ! R). However, administration of roscovitine before by doxorubicin synergistically inhibits TNBC cells doxorubicin induced a synergistic response only in TNBC cell To interrogate whether treatment of breast cancer cells with lines, but an antagonistic response in the p53 wild-type roscovitine would alter their cell-cycle profiles based on their HMEC and ER-positive cell lines (Fig. 1C, R ! D) with a

Figure 2. Sequential combination treatment of roscovitine anddoxorubicininducesapoptosisinTNBCcells.A,HMEC(MCF-10A)andTNBC(MDA-MB-157andMDA-MB- 436) cells were treated with roscovitine (R) at IC50 concentrations (13 mmol/L MCF-10A and 32 mmol/L and 24 mmol/L for MDA-MB-157 and MDA- MB-468, respectively), for 24 hours, doxorubicin (D; 20 nmol/L for MCF-10A, 15 nmol/L for MDA-MB-157, and 21 nmol/L for MDA-MB-468) for 48 hours or roscovitine followed doxorubicin (R!D), each at the aforementioned IC50 values for each cell lines. Cell death was assessed directly following drug treatment and after (24 hours, 72 hours, 120 hours, or 9 days) release into drug-free medium using a dye-exclusion assay with propidium iodide (PI) on unﬁxed cells using ﬂow cytometry. Staurosporine (S) served as a positive control for each cell line. B, HMEC and TNBC cells were treated with R, D, and R!D(atIC50 values, Supplementary Fig. S1B) and harvested immediately following drug treatment or at indicated times after release. Western blot analysis was used to detect full-length and cleaved PARP-1. Staurosporine (S) was used as a positive control. Densitometry analysis was performed usingImageJ.Theratioofcleavedtofull-lengthPARPwasdeterminedandplottedasbargraphs.CandD,MCF-10AandMDA-MB-468cellswere transfected with siControl, siCDK1, siCDK2, or both siCDK1/siCDK2 with or without the sequential addition of doxorubicin (at IC50 concentrations of 20 nmol/L for MCF-10A and 21 nmol/L for MDA-MB-468 cells) treatment for 48 hours. Cells were harvested following drug treatment (top) or 48 hours after release from drug treatment (bottom). Western blot analysis was used to detect full-length and cleaved PARP1 in each condition. Densitometry analysis was performed using ImageJ. The ratio of cleaved to full-length PARP was determined and plotted as bar graphs.

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significantly lower (P <0.05) CI value in all TNBC cell lines after treatment (Fig. 2A, left). Doxorubicin treatment steadily (Fig. 1C). Furthermore, when TNBC cells were treated with reduced cell viability over time; with MDA-MB-157 cells show- roscovitine first for 24 hours followed by another 48 hours ing over 30% PI positivity at 9 days after drug removal (Fig. 2A, concomitantly with doxorubicin, we measured synergism and middle). Combination-sequential treatment of cells with ros- additivity (Supplementary Fig. S2A). To examine the speci- covitine followed by doxorubicin induced only a 15% PI ficity of the synergistic response to anthracyclines, we also positivity in MCF-10A cells, while the combination treatment performed sequential treatment of TNBC cells with roscov- induced 45% and 30% PI positivity in MDA-MB-157 and MDA- itine followed by the antimicrotubule chemotherapeutic tax- MB-468 cells, respectively. Moreover, only TNBC cells contin- ol, which induced an additive and antagonistic response in ued to exhibit decreased cell viability, or PI positivity, following MDA-MB-157 and MDA-MB-231 cells, respectively (Supple- release from treatment with maximum PI positivity at 72 hours mentary Fig. S2B). Therefore, only sequential combination after treatment in MDA-MB-157 and 120 hours after treatment treatment of roscovitine followed by doxorubicin can specif- in MDA-MB-468 cells (Fig. 2A, right). ically inhibit TNBC cells, and not HMEC or ER-positive cells. PARP-1 cleavage was not observed in MCF-10A cells follow- Next, we examined whether inhibition of CDK1, CDK2, or ing any of the treatment arms, or following drug removal both CDKs simultaneously was required for synergism with (Fig. 2B and Supplementary Fig. S2E). However, both TNBC doxorubicin treatment. MCF-10A, ZR75-1, and MDA-MB-468 cell lines showed sustained levels of PARP-1 cleavage both cells were transiently transfected with siRNA against either CDK during and following release from combination treatment or both, with nontargeting siRNA used as a control (Fig. 1D; (Fig. 2B). For example, MDA-MB-468 cells had the highest Supplementary Fig. S2C and 2D). Cell viability was assessed by ratio of PARP-1 cleavage at 24 hours during the combination HTSA using siCDK1or siCDK2 as single agents and in combi- treatment, which was sustained up to 72 hours after release nation with doxorubicin. Although siCDK1 or siCDK1/siCDK2 (Fig. 2B) suggesting a persistent apoptotic signal only in TNBC reduced cell viability in MCF-10A by 50% to 60%, there was no cells and not in MCF-10A cells (Supplementary Fig. S2E). further reduction in viability in these cells with the addition of We also examined whether the synergism observed between doxorubicin. Knockdown of CDK2 did not decrease percent CDK1 siRNA þ doxorubicin (Fig 1C–1D) also resulted in higher viability; however, the addition of doxorubicin treatment again PARP-1 cleavage. Western blot analysis revealed that MCF-10A reduced viability by 50% to 60% in MCF-10A cells (Fig. 1E). cells, showed no change (compared with siControl) in their Similar results were seen with ZR75-1 cells (Supplementary Fig. ability to cleave PARP-1 following either CDK1/2 siRNA or S2D). Knockdown of CDK1 or both CDKs simultaneously treatment with doxorubicin (Fig. 2C, top). In contrast, knock- reduced viability by about 50% in MDA-MB-468 cells, whereas down of CDK1, CDK2, or both increased PARP-1 cleavage in siCDK2 caused only 20% cell inhibition. However, the addition MDA-MB-468 cells compared with siControl transfected cells. of doxorubicin to either siCDK1 or siCDK1/siCDK2 knock- Moreover, the addition of doxorubicin to CDK knockdown cells down cells caused 90% cell inhibition only in the TNBC MDA- increased cleaved PARP-1 expression (Fig. 2D, top). Forty-eight MB-468 cells, but not in the MCF-10A (Fig. 1E) or ZR75-1 hours after treatment, MCF-10A knockdown and combination cells (Supplementary Fig. S2D). These results suggest that treated cells showed complete recovery, expressing only full- inhibition of CDK1 combined with doxorubicin treatment is length PARP-1 (Fig. 2C, bottom). However, MDA-MB-468 cells sufficient to induce synergistic cell inhibition in TNBC but not had persistent cleaved PARP-1 expression in both CDK transiently in non-TNBC cells. knockdown cells in the presence or absence of doxorubicin 48 hours after treatment (Fig. 2D, bottom). These findings suggest Combination treatment increases apoptosis specifically in that TNBC cells do not readily recover from pharmacologic TNBC cells inhibition or molecular knockdown of CDK combined with To examine cell death in TNBC and HMEC cells in response doxorubicin, leading to an enduring apoptotic signal. to single and combination treatments both during and following drug treatment, we used both dye-exclusion assay with TNBC cells bypass the G1 checkpoint in response to propidium iodide (PI) and PARP-1 cleavage as readouts for combination treatment apoptosis. PI positivity is detected when the cell membrane is The sequential combination treatment of roscovitine fol- compromised, a characteristic of apoptosis (38) as depicted by lowed by doxorubicin resulted in an accumulation of cells in the use of staurosporine as a positive control in our assays G2–M and generated a polyploid population only in TNBC cell, (Fig. 2A). Roscovitine-induced PI positivity peaked at 24 hours with 55% of MDA-MB-231 cells accumulating in G2–M(Fig.3A after drug exposure with no further increase up to 9 days and Supplementary Fig. S3A). The TNBC polyploid population

Figure 3. Sequential combination treatment of roscovitine and doxorubicin induces a G2–M arrest and accumulation of polyploidy population. A, MCF-10A and MDA-MB-231 cells were treated with roscovitine (24 hours), doxorubicin (48 hours), or sequential combination treatment followed by cell-cycle analysis with ﬂow cytometry. Drugs were administered at IC50 concentrations (see Supplementary Fig. S1B). B, HMEC (MCF-10A) and TNBC (MDA-MB-231 and MDA- MB-468) cells were transfected with siControl, siCDK1, siCDK1, or both siCDK1/siCDK2. Western blot analysis was used to conﬁrm knockdown of CDK1 and CDK2. C, cell-cycle analysis was performed on HMEC (MCF-10A) and TNBC (MDA-MB-231 and MDA-MB-468) siRNA-transfected cells in the absence or presence of doxorubicin (48 hours at IC50 concentration; see Supplementary Fig. S1B). D, MCF-10A and MDA-MB-157 cells were treated with roscovitine (R), doxorubicin (D), or combination treatment (R!D) at IC50 concentrations, harvested (using RIPA buffer), and subjected to Western blot analysis with the indicated antibodies. Actin was used for equal loading determination. E, ImageJ was used to measure relative protein expression of each antigen. Protein expression was normalized to actin.

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increased by 20% following the sequential combination ther- and HCT116 p53 / cell lines. 76NE6 cells were immortalized apy, compared with untreated cells, whereas HMEC cells did from 76N primary HMEC cells via transfection with the viral not accumulate in G2–M or increase their polyploid population oncoprotein HPV16-E6, which binds to and degrades p53; following any of the treatments (Fig. 3A). The G2–Mand while 76NF2V cells were immortalized with the F2V-mutant polyploidy population induced by concomitant treatment HPV-16E6 gene, which renders cells immortalized but with a (45%) was less than sequential (55%) treatment for TNBC functional and wild-type p53 (40, 41). Sequential administra- cells (Supplementary Fig. S3B). tion of roscovitine followed by doxorubicin resulted in an TransientknockdownofCDK1,CDK2ofbothCDKs(Fig. additive response in p53 wild-type cells 76NF2V, while it 3B) revealed that HMEC cells only had about a 10% increase in induced synergism in the p53 inactive 76NE6 cells (Fig. 4A), G2–M upon CDK knockdown, with the addition of doxorubi- with twice as many cells with polyploid nuclei in 76NE6 cin either reverting cells to a G1 accumulation or causing no compared with 76NF2V cells (Fig. 4B). Levels of phospho-and change in their cell-cycle profiles (Fig. 3C). Conversely, in total Rb were reduced following single and combination ther- TNBC cells, knockdown of CDK1 or CDK1/CDK2 lead to a apy in 76NF2V cells compared with 76NE6 cells. In addition, G2–M accumulation that was augmented with the addition of p21 levels were induced only in 76NF2V cells, in response to doxorubicin. In MDA-MB-468 cells, 80% of the cells accumu- combination treatments (Fig. 4C). lated in the G2–M when the knockdown of CDK1 or CDK1/ Because drug treatment reduced phospho-Rb expression in CDK2 was combined with doxorubicin treatment as compared both MCF-10A (Fig. 3D) and 76NF2V (Fig. 4C) cells, while with single drug treatments. There was no additional gain in the remaining elevated in MDA-MB-157 cells, we interrogated G2–M phase when both CDKs were knocked down in the whether loss of Rb would render 76NF2V cells more synergistic presence of doxorubicin compared with CDK1 knockdown to the combination treatment. Rb was stably knocked down in plus doxorubicin in TNBC cells (Fig. 3C). 76NF2V cells (Fig. 4D) and subjected to HTSA followed by In MCF-10A cells, the expression of both total and phospho- CalcuSyn analysis (Fig. 4D). Roscovitine–doxorubicin combi- Rb was reduced, whereas the expression of p21 and p27 were nation treatment induced only additivity or antagonism in the both increased following single and combination treatment Rb knocked down cells (Fig. 4D). Moreover, ablation of the Rb (Fig. 3D and 3E). In contrast, treatment of the TNBC MDA-MB- pathway did not cause 76NF2V cells to accumulate more in the 157 cells with single or combination treatment increased G2–M phase compared with shScramble cells (Supplementary both total and phospho Rb with minimal changes in p21 or Fig. S3B). Therefore, Rb inactivation is not sufficient to cause p27 expression (Fig. 3D and 3E). These results suggest that the roscovitine-doxorubicin-induced synthetic lethality in HMEC G1 checkpoint activation is intact only in the HMEC cells. In cells. þ þ addition, p21 was also transcriptionally activated upon com- When HCT116 p53 / and HCT116 p53 / cells were bination treatment only in MCF-10A cells (data not shown), subjected to HTSA and CalcuSyn analysis, p53 wild-type cells suggesting that wild-type p53 induced a G1 arrest that may responded antagonistically to combination treatment, while protect MCF-10A cells against the toxic effects of combination deletion of p53 induced synergism in HCT116 cells (Fig. therapy. 4E,4F).Sequentialroscovitine–doxorubicin treatment reduced phospho-Rb expression in p53 wild-type cells. More- Loss of wild-type p53 activity is required for roscovitine/ over, both single and combination drug treatment caused doxorubicin synergistic response increased expression of p27 and p21 only in the p53 wild- The results from the cell line panel study (Fig. 1) suggested type cells (Fig. 4G). Finally, combination treatment increased that absence of a wild-type p53 could predict for a synergistic G2–M accumulation and polyploidy in both p53 wild-type response of roscovitine followed by doxorubicin treatment. and knockout cells. However, combination-treated p53 Since the majority of TNBC cell lines identified to date harbor knockout cells had twice the amount of sub-G1 cells compared p53 mutations (39), we utilized two different isogenic cell with p53 wild-type cells both during and after treatment systems to test this hypothesis: breast epithelial cells (HMECs) (Fig. `4H) suggesting that the loss of p53 is required for þ þ 76NF2V and 76NE6 and colorectal cancer cells HCT116 p53 / synergism in tumors cells.

Figure 4. Absence of p53 is required for synergism between roscovitine and doxorubicin. A, isogenic HMEC cells, 76NF2V and 76NE6, were subjected to sequential roscovitine-doxorubicin treatment via HTSA followed by CalcuSyn analysis. The concentrations used for each agent are listed in Supplementary Fig. S1B. Average combination index (CI) is shown for each cell line. B, 76NF2V and 76NE6 cells were treated with roscovitine (R) for 24 hours, doxorubicin (D) for 48 hours, or sequential combination treatment (R!D) at IC50 values (Supplementary Fig. S1B). Following fixation, samples were subjected to cell-cycle analysis via flow cytometry. Untreated (C) samples served as a control. C, cells were treated with R, D, or R!DatIC50 values (Supplementary Fig. S1B) followed by Western blot analysis of G1 checkpoint proteins. D, lentivirus was used to generate stable Rb knockdown 76NF2V cells. Western blot analysis of total Rb was used to confirm knockdown compared with parental and stably expressing nontargeting shScamble cells. Rb knockdown and shScramble cells were treated with sequential roscovitine-doxorubicin treatment via HTSA followed by CalcuSyn analysis. Average combination index (CI) is shown for the shSramble, the two shRB variants of 76NF2V. E, Western blot analysis of p53 expression in HCT116 p53 / þ þ cells compared with wild-type HCT116 p53 / and MCF-10A cells. F, HCT116 wild-type and knockout cells were subjected to roscovitine–doxorubicin combination treatment via HTSA followed by CalcuSyn analysis. The concentrations used for each agent are listed in Supplementary Fig. S1B. Average combination index (CI) is shown for each cell line. G, Western blot analysis was performed to examine the effect of single and sequential combination drug treatment on G1 checkpoint proteins of HCT116 p53 wild-type and knockout cells. Actin was used as a loading control. H, HCT116 wild-type and knockout cells treated with R!DatIC50 concentration of each drug and cells were removed either immediately after treatment or 24 hours after drug treatment (RþDþ24) was subjected to cell-cycle analysis.

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Combination-based treatment increased DNA double-strand Deletion of p53 allows combination treatment to increase DNA breaks and decreased DNA repair DSBs þ þ Because CDK1 activity is required for HR, we next hypoth- HCT116 p53 / and HCT116 p53 / were used to determine esized that this combination treatment strategy mediates its the effect of p53 on DNA damage and repair. Presence or absence activity through the alteration of DNA damage response. To this of p53 had little effect on doxorubicin-induced DNA damage, end, we initially examined a neutral comet assay to quantitate with 41% and 48% of p53 wild-type and p53 knockout cells DNA DSB in our panel of HMEC and TNBC cells following having g-H2AX positivity, respectively (Supplementary Fig. S4A, single and combination treatments (Fig. 5A). The read-out of B, and C). However, combination treatment significantly the neutral comet assay is the tail moment, or the amount of increased (P <0 .01) g-H2AX–positive cells only in p53 knockout DNA in the distance traveled, which measures the extent of cells compared with doxorubicin-treated cells. Combination DNA DSBs. As expected, doxorubicin treatment induced DNA treatment did not augment g-H2AX positivity in p53 wild-type DSBs, indicated by an increased tail moment, in all cell lines cells. Moreover, combination-treated p53 knockout cells had examined (Fig. 5A). However, administering roscovitine before significantly more (P <0 .01) g-H2AX–positive cells (by 20%) doxorubicin caused a significant (P < 0.05) increase in the compared with combination-treated p53 wild-type cells (Supple- amount of DNA DSBs only in TNBC cells. Furthermore, doxo- mentary Fig. S4C). rubicin treatment induced g-H2AX foci, a marker of DNA DSBs, Furthermore, upon doxorubicin treatment, p53 wild-type in MCF-10A cells and both TNBC cell lines compared with and p53 knockout cells recruited Rad51 to g-H2AX sites in untreated control cells (Fig. 5B–D). However, treatment with 63% and 59% of cells, respectively (Supplementary Fig. S4A, B, roscovitine before doxorubicin significantly increased (P < and D). Combination treatment had no effect on Rad51 0.05) the percentage of g-H2AX–positive cells by 20% explicitly recruitment in p53 wild-type cells. However, p53 knockout in TNBC cells with no increase in MCF-10A cells (Fig. 5B–D). cells had a significant (P <0 .05) decrease in the percent of Moreover, Western blot analysis revealed that only combina- g-H2AX–positive cells with Rad51 foci compared with doxo- tion treatment induced an increase in phospho-H2AX rubicin-treated cells (Supplementary Fig. S4A, S4B, and S4D). (g-H2AX) in TNBC MDA-MB-157 cells, but not in HMEC Despite having more G2–M cells, p53 knockout cells had less MCF-10A cells (Fig. 5B). recruitment of Rad51 foci that p53 wild-type cells during Quantification of g-H2AX–positive cells with Rad51 foci was combination treatment (Fig. 4G; Supplementary Fig. S4A, used next to examine the recruitment of downstream HR proteins. S4B, and S4D). Collectively, these results suggest that deletion Untreated control HMEC and TNBC cells had limited DNA of p53 causes tumor cells to have increased DNA damage and damage, but had over 60% or over 80% cells with Rad51 positivity reduces the ability of tumor cells to recruit downstream HR when g-H2AX was present, respectively (Fig. 5C–E). Roscovitine proteins in response to roscovitine–doxorubicin combination treatment decreased the recruitment of Rad51 in both MCF-10A treatment. and MDA-MB-157 cells. In addition, roscovitine treatment reduced Rad51 protein expression in both MCF-10A and MDA- Combination treatment increases overall survival and MB-157 cells (Fig. 5B). However, combination treatment signif- decreases proliferation in human breast cancer xenograft icantly reduced (P < 0.05) the formation of Rad51 foci compared MDA-MB-468 cells were used to establish human xenograft with doxorubicin treatment only in TNBC cells even though these tumors in the right and left mammary fat pads of nude mice cells had increased g-H2AX positivity (Fig. 5C–5F). Combination (Fig.6A).Therewerefive treatment arms: vehicle, roscovitine and doxorubicin only treated MCF-10A cells were able to recruit for 4 days on/3 days off, doxorubicin once a week, concom- Rad51 foci at a similar percentage (Fig. 5C and 5F). In parallel, itant treatment of roscovitine with doxorubicin administered although Rad51 expression decreased in combination-treated on day 4, or sequential treatment of roscovitine followed by MDA-MB-157 cells, it remained elevated in MCF-10A combina- doxorubicin for four cycles. Sequential combination-treated tion-treated cells (Fig. 5B). These results suggest that treating tumors were the only arm of the study that did not increase 3 TNBC cells with roscovitine before doxorubicin can enhance DNA in volume while on treatment. Averaging at 125 mm ,sequen- damage while impairing their ability to repair DNA DSBs, regard- tial combination-treated tumors were significantly smaller less of increased DNA damage. (P <0.01), than vehicle-treated tumors that averaged to be

Figure 5. Sequential combination treatment of roscovitine and doxorubicin causes more DNA double-strand breaks while simultaneously reducing downstream homologous recombination proteins. A, neutral comet assay was performed on HMEC (MCF-10A) and TNBC (MDA-MB-157 and MDA-MB-468) cells that were treated with roscovitine (R) for 24 hours, doxorubicin (D) for 48 hours, or sequential combination treatment (R!D) at IC50 concentrations (Supplementary Fig. S2B). Samples were subjected to a current of 21V for 21 minutes and subsequently stained with SYBR green for visualization (right). Images were captured at 10 magnification with an Eclipse 90i microscope equipped with the NIS-Elements Br 3.10 software program. The tail moment (the amount of DNA in the distance traveled) was measured using Comet Score software and plotted as bar graphs (left). B, (A) MDA-MB-157 and MCF-10A cells were treated with roscovitine (R), doxorubicin (D), or sequential combination treatment (R!D). Untreated cells were used as a control (C). Following treatment, cells were harvested and Western blot analysis was performed to detect expression of phospho-H2AX and Rad51. C and D, MCF-10A and MDA-MB-231 cells were treated with single and combination drug treatment as in A and B. DNA repair foci g-H2AX and Rad51 were detected with immunofluorescence. DAPI was used to detect nuclei. Images were captured at 100 with an Eclipse 90i microscope. E, percent of g-H2AX–positive was quantified in HMEC and TNBC cells in response to single and combination drug treatment. Only cells with 5 foci were considered as positive. One hundred cells per sample, per replicate were counted. F, percent of g-H2AX–positive cells with Rad51 foci recruitment was quantified. Cells with 1Rad51 foci were considered positive. Images for quantification were captured at 60 with a confocal Olympus FV100 microscope. Experiments were repeated three times; error bars, 95% confidence intervals. The Student t test (two-tailed, equal variance) was used to derive the P values.

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330 mm3 on day 26 (Fig. 6B and Supplementary Fig. S5A and Discussion S5C). Indeed, concomitantly treatedmicehada3-foldincrease Here, we describe that sequential administration of roscov- in tumor size on day 26 (Supplementary Fig. S5C). Moreover, itine followed by doxorubicin is a well-tolerated, synthetic sequential combination-treated tumors were significantly lethal combination treatment strategy that explicitly targets smaller (P <0.05) than roscovitine, doxorubicin, and concom- TNBC cells, while leaving nontransformed cells unharmed. itant-treated tumors during and following treatment (Fig. 6B Hallmarks of cancer include limitless proliferation and and Supplementary Fig. S5A and S5C). Notably, no measur- genome instability (42, 43). By targeting CDK1, which has able difference was observed between vehicle and roscovitine- aroleinbothcell-cyclepromotionandDNADSBrepair, treated mice; supporting clinical findings that roscovitine is followed by treatment with a chemotherapeutic, we have inefficient as a single agent (Fig. 6A). developed a novel synthetic lethal combination. Simultaneous In addition, none of the sequential combination-treated drug administration or treating cells with doxorubicin prior to mice suffered from increased toxicity or tumor burden during roscovitine was antagonistic in TNBC cells and toxic in vivo. the 60-day experiment (Fig. 6D and Supplementary Table S2). Furthermore, roscovitine followed by doxorubicin treatment As such, sequential combination therapy significantly increased inhibited tumor growth in vivo and significantly increased overall survival (P < 0.05) compared with vehicle, single-agent, overall survival compared with mice receiving concomitant and concomitant-treated mice (Fig. 6C). Overall toxicity was treatment of the two drugs or either of the drugs as single assessed by weight loss during and following treatment. Nei- agents. ther the roscovitine alone arm nor the combination arm ani- Because TNBC cells have lost their G –S regulation, treatment mals suffered any weight loss, while 80% and 50% of doxo- 1 of these cells with roscovitine resulted in their accumulation in rubicin and concomitant-treated mice had to be sacrificed G –M phase, while non-TNBC or HMEC cells with a regulated due to >20% weight loss, respectively, again revealing the 2 G –S transition, accumulated in the G phase of the cell cycle. In limitations of doxorubicin as monotherapy and the importance 1 1 addition, combination treatment maintained a G –M arrest and/ of drug delivery scheduling (Fig. 6C; Supplementary Fig. S5E 2 or induced polyploid nuclei specifically in TNBC cells. Moreover, and Supplementary Table S2). knockdown of CDK1, a G –M-specific CDK, was sufficient to These results suggested that treatment with roscovitine could 2 augment the percentage of G –M accumulation in the presence of limit doxorubicin-induced toxicities. To test this, a dose–esca- 2 doxorubicin only in TNBC cells. Collectively these results suggest lation study examining weight loss and blood count was that a sequential treatment strategy that differentially targets the performed on nontumor-bearing nude mice that were treated cell cycle in TNBC versus HMEC cells is more likely to produce a with vehicle, roscovitine (50 mg/kg), doxorubicin (5 mg/kg or synergistic response only in those cells with a deregulated cell 10 mg/kg), or sequential therapy. At 1 week into treatment, cycle (i.e., TNBC cells). sequential-treated mice maintained normal weights compared TNBC cells with p53 mutations had a diminished capacity to with doxorubicin treatment at 5 mg/kg. However, as the induce p21 transcription compared with p53 wild-type HMEC amount of doxorubicin accumulated,theprotectiveeffect cells. Knockdown of Rb was insufficient to cause a synergistic of roscovitine against weight loss diminished (Supplementary response in HMEC cells. However, combination treatment was Fig. S6A). Following the completion of treatments, blood was synthetically lethal only in p53-compromised HEMC and collected from all mice. Treatment of roscovitine before doxo- tumors cells, whereas the paired isogenic p53 wild-type cells rubicin was able to rescue the reduction in the white blood were additive or antagonistic to the combination treatment. cell (WBC) count caused by doxorubicin alone (Supplementary Moreover, the cell-cycle profile of HMEC and tumor cells Fig. S6B). In addition, doxorubicin significantly (P < .05) lacking p53 activity closely mimicked the cell-cycle profile of reduced the platelet count in mice. However, sequentially TNBC cells. Thus, p53 inactivation could serve as a predictor of treated mice had twice as many platelets (Supplementary Fig. synergistic response to sequential roscovitine-doxorubicin S6C). None of the treatments affected the red blood cell count combination treatment. (RBC; Supplementary Fig. S6D). Therefore, sequential combi- Mechanistically, administration of roscovitine before doxo- nation therapy rescues doxorubicin-induced neutropenia and rubicin caused increased DNA DSBs while simultaneously platelet loss. inhibiting recruitment of downstream HR proteins in TNBC Toassessproliferationoftumorsineachtreatmentarm, cells. Combination treatment did not subject HMEC cells bromodeoxyuridine (BrdUrd) incorporation was measured to increased DNA damage. HR is the preferred method of at the end of treatment (day 26) and revealed that both DNA DSB repair during S and G –M phase, and CDK1 activity doxorubicin and combination-treated tumors had a significant 2 is required for the excision of DNA DSBs during HR (25). (P < 0.05) decrease in proliferation compared with vehicle- Therefore, inhibiting CDK1 activity (with roscovitine) treated tumors (Fig. 6D). Western blot analysis of PARP-1 primes and sensitizes TNBC cells to doxorubicin. Nontrans- on tumors resected on day 26 revealed that combination formed cells and tumor cells with an intact p53 pathway were therapy significantly increased cleaved PARP-1 expression less sensitive to combination treatment because they did (P <0.05) compared with doxorubicin-treated tumors, indic- not accumulate at the G –M phase before doxorubicin ative of apoptosis (Fig. 6E). On the basis of hematoxylin 2 administration. and eosin staining, tumors from all four treatment arms The requirement of p53 inactivity for the sequential-combi- had similar histology, with combination-treated tumors hav- nation–induced synergism provides a putative predictor of ing marked fibrous (Supplementary Fig. S5D). Overall, response. TNBC tumors are heterogeneous, with genome anal- these data suggest that roscovitine–doxorubicin combination ysis to identifying four or six subgroups within TNBC tumors therapy is both well tolerated and efficacious against (44, 45). Thus, identifying patients that would benefitthemost TNBC.

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Figure 6. Sequential roscovitine–doxorubicin combination treatment is effective and well tolerated in vivo. A, a schematic illustrating that 4.5106 MDA-MB-468 cells in a 1:2 ratio with Matrigel were injected into the right and left mammary fat pads of the four groups of 4- to 6-week-old female Balb/c Nu/nu mice. B, once tumors reached 100 to 150 mm3, they were treated with vehicle, roscovitine (50 mg/kg) 4 days on/3days off, doxorubicin (2 mg/kg) once a week, or combination treatment for four cycles. Weekly tumor volume measurements shown. C, overall survival graphed as Kaplan–Meier curve. Mice treated with concomitant combination drug treatment (4 days of roscovitine with doxorubicin administered on day 4) included in survival analysis. Mice were euthanized if total tumor burden was 1,500 mm3 or if mice lost 20% of initial body weight. Statistical analysis was performed using Mantel–Cox method. D, percent BrdUrd-positive cells were averaged from three tumors resected on day 26 per each treatment arm. E, Western blot analysis performed on tumors resected on day 26. Densitometry analysis performed using ImageJ.

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from combination treatment, while sparring nonresponsive underwent more apoptosis, indicating increased cytotoxicity. patients to treatment, is crucial to successfully implementing Furthermore, doxorubicin only treated mice suffered from more this treatment strategy. Moreover, developing a drug treatment weight loss compared with combination-treated mice. Thus, the strategy that incorporates the clinically available agent doxo- combination therapy of roscovitine followed by doxorubicin can rubicin may hasten the adoption of this combination in the decrease tumor volume while limiting toxicity. Combining che- clinic. motherapies with a CDK inhibitor could potentially lead to a Although the sequential roscovitine followed by doxorubicin reduction in the chemotherapy dose administered to patients. treatment was well tolerated in vivo (this study), a phase I Overall, roscovitine–doxorubicin sequential therapy increased clinical trial examining the MTD and efficacy of dinaciclib (the the therapeutic benefit of doxorubicin while reducing toxicity, next-generation analogue of roscovitine) and the anthracycline supporting future clinical studies. epirubicin found that this treatment was very toxic, ending the fi trial before ef cacy could be determined (46). Dose-limiting Disclosure of Potential Conflicts of Interest toxicities included grade 3 neutropenia, syncope, and vomiting No potential conflicts of interest were disclosed. (46). One solution to increase the efficacy of combination treatment while reducing toxicity is to couple the drugs with a nanocarrier delivery system. Coupling CDK inhibitors or Authors' Contributions doxorubicin with nanotechnology drug delivery system will Conception and design: N.A. Jabbour-Leung, X. Chen, K.K. Hunt, K. Keyomarsi Development of methodology: N.A. Jabbour-Leung, X. Chen, Y. Jiang, D. Yang, limit dispersal of the drug to only the site of action, protecting K.K. Hunt, K. Keyomarsi other organs and tissues from cytotoxicity (47). There is pre- Acquisition of data (provided animals, acquired and managed patients, cedence for such a strategy as anti-HER2 immunoliposomes provided facilities, etc.): Y. Jiang, D. Yang, S. Vijayaraghavan, M.J. McArthur, containing doxorubicin were effectively targeted to HER2-over- K.K. Hunt, K. Keyomarsi expressing tumors, increasing the therapeutic benefitofdoxo- Analysis and interpretation of data (e.g., statistical analysis, biosta- rubicin while reducing toxicity in a xenograft mouse model tistics, computational analysis): N.A. Jabbour-Leung, D. Yang, K.K. Hunt, K. Keyomarsi (48). Alternatively, CDK inhibitors could be directly delivered Writing, review, and/or revision of the manuscript: N.A. Jabbour-Leung, to the tumor site if it was bound to a ligand-mediated active X. Chen, K. Keyomarsi binding nanoparticle. EGFR, which is overexpressed in the Administrative, technical, or material support (i.e., reporting or organizing majority of TNBC tumors, is a cell surface receptor that provides data, constructing databases): N.A. Jabbour-Leung, X. Chen, T. Bui a putative target to deliver roscovitine to the tumor site. Indeed, Study supervision: N.A. Jabbour-Leung, K. Keyomarsi in vitro in vivo EGFR-targeted polymer nanocarriers delivered paclitaxel and Other (performed experiments; and ): N.A. Jabbour-Leung ionidamine to multidrug resistant EGFR-overexpressing tumor cells, increasing drug cytotoxicity (49). Developing EGFR-tar- Grant Support geted nanocarriers to deliver roscovitine directly to the tumor This work was supported by NIH grants CA87458 and CA1522228 (to K. Keyomarsi), by the Center for Clinical and Translational Sciences at NIH, TL1 site could increase the therapeutic benefitofCDKinhibition – RR024147 (to N.A. Jabbour-Leung), and by NCI CCSG grant CA16672 (to M.D. while reducing toxicities. Moreover, liposomal doxorubicin, Anderson Cancer Center). which accumulates at tumor sites due to leaky vasculature, is The costs of publication of this article were defrayed in part by the clinically available to treat breast cancer (50). Thus, it would be payment of page charges. This article must therefore be hereby marked clinically beneficial to consider incorporating CDK inhibitors advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate or doxorubicin combination treatment with nanoparticle deliv- this fact. ery system. Although doxorubicin and combination-treated tumors had Received June 22, 2015; revised December 31, 2015; accepted January 4, the same amount of proliferation, combination-treated tumors 2016; published OnlineFirst January 29, 2016.

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www.aacrjournals.org Mol Cancer Ther; 15(4) April 2016 607

Sequential Combination Therapy of CDK Inhibition and Doxorubicin Is Synthetically Lethal in p53-Mutant Triple-Negative Breast Cancer

Natalie A. Jabbour-Leung, Xian Chen, Tuyen Bui, et al.

Mol Cancer Ther 2016;15:593-607. Published OnlineFirst January 29, 2016.

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