The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of P53-Mutated Multiple Myeloma Cells

Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-11-0949

Molecular Cancer Preclinical Development Therapeutics

The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of p53-Mutated Multiple Myeloma Cells

Heather J. Landau1, Samuel C. McNeely1,4, Jayasree S. Nair4, Raymond L. Comenzo5, Takashi Asai3, Hillel Friedman3, Suresh C. Jhanwar2, Stephen D. Nimer1,3, and Gary K. Schwartz1,4

Abstract DNA cross-linking agents are frequently used in the treatment of multiple myeloma–generating lesions, which activate checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response. Chk1 activation promotes cell survival by regulating cell-cycle arrest and DNA repair following genotoxic stress. The ability of AZD7762, an ATP-competitive Chk1/2 inhibitor to increase the efﬁcacy of the DNA-damaging agents bendamustine, melphalan, and doxorubicin was examined using four human myeloma cell lines, KMS-12- BM, KMS-12-PE, RPMI-8226, and U266B1. The in vitro activity of AZD7762 as monotherapy and combined with alkylating agents and the "novel" drug bortezomib was evaluated by studying its effects on cytotoxicity, signaling, and apoptotic pathways. The Chk1/2 inhibitor AZD7762 potentiated the antiproliferative effects of bendamustine, melphalan, and doxorubicin but not bortezomib in multiple myeloma cell lines that were p53- deﬁcient. Increased gH2AX staining in cells treated with bendamustine or melphalan plus AZD7762 indicates a

greater degree of DNA damage with combined therapy. Abrogation of the G2–M checkpoint by AZD7762 resulted in mitotic catastrophe with ensuing apoptosis evidenced by PARP and caspase-3 cleavage. In summary, the cytotoxic effects of bendamustine, melphalan and doxorubicin on p53-deﬁcient multiple myeloma cell lines were enhanced by the coadministration of AZD7762. These data provide a rationale for testing these combinations in patients with relapsed and/or refractory multiple myeloma. Mol Cancer Ther; 11(8); 1781–8. 2012 AACR.

Introduction FGFR3 and loss of expression or mutation in TP53 play a Multiple myeloma is an incurable hematologic malig- key role in determining tumor progression and drug nancy that arises from the dysregulated proliferation of resistance in multiple myeloma (2). The introduction of plasma cells. While traditional chemotherapy regimens novel drugs such as thalidomide, bortezomib, and lena- including alkylating agents and anthracyclines have mod- lidomide has dramatically improved patient outcomes est benefits in this disease, high-dose melphalan sup- both during the initial therapy and at relapse. In some ported by autologous stem cell transplantation has been studies these drugs overcome the poor prognosis associ- shown to increase the incidence of complete and near ated with specific chromosomal lesions (3). However, complete remissions in approximately 50% of patients (1). there are no reliable data showing improved survival However, almost all patients relapse and respond to when patients with p53 abnormalities are treated with subsequent therapy only temporarily. Dysregulation of these agents or even allogeneic stem cell transplant (4). Therefore, new therapeutic strategies are needed. Chemotherapy, commonly used to treat multiple mye- Authors' Affiliations: Departments of 1Medicine and 2Pathology, 3Molec- loma, includes several DNA-damaging agents such as the ular Pharmacology and Chemistry Program, Sloan Kettering Institute, 4Laboratory of New Drug Development, Memorial Sloan-Kettering Cancer nitrogen mustards, bendamustine and melphalan, and Center, New York; and 5Department of Medicine, Tufts Medical Center, doxorubicin that is an anthracycline antibiotic. All 3 Boston, Massachusetts agents cause DNA cross-links and strand breaks thereby Note: Supplementary data for this article are available at Molecular Cancer eliciting the DNA damage response (DDR), which ensures Therapeutics Online (http://mct.aacrjournals.org/). genomic stability by activating cell-cycle checkpoints and H.J. Landau and S.C. McNeely are co-first authors. promoting DNA repair. Checkpoint kinase 1 (Chk1) is a critical component of the DDR to replicative stress. Once Corresponding Author: Heather J. Landau, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, activated by ATR, Chk1 promotes cell-cycle arrest via NY 10065. Phone: 212-639-8808; Fax: 212-717-3394; E-mail: phosphorylation of Cdc25 phosphatases resulting in their [email protected] destruction or sequestration, preventing the reversal of doi: 10.1158/1535-7163.MCT-11-0949 inhibitory phosphorylation of the cyclin-dependent 2012 American Association for Cancer Research. kinases (5). Chk1 also inhibits firing of replication origins

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Figure 1. Chemical structures of AZD7762 (A), melphalan (B), bendamustine (C), and doxorubicin (D).

(6), stabilizes stalled replication forks (7), and regulates from the ATCC Technical Bulletin no. 8; Manassas: proteins involved with DNA repair (8, 9). A structurally ATCC; 2007. The cells lines were not authenticated at dissimilar checkpoint kinase, Chk2 is activated by ataxia our institution. All cells were maintained in RPMI with telangiectasia mutated (ATM) in response to double 10% FBS (HyClone) and 1% penicillin/streptomycin. strand breaks; it promotes both Cdc25 inactivation and Cells were grown at 37 C, 90% humidity, and 5% CO2. p53-dependent cell-cycle arrest. For all experiments, multiple myeloma cells were seeded Chk1 inhibitors have been shown to potentiate conven- at 4 105 to 6 105 cells/mL in fresh media. tional DNA-targeted chemotherapeutics both in vitro and in vivo (10, 11). AZD7762, an ATP-competitive Chk1/2 Drugs inhibitor that has been studied clinically, increases the AZD7762 (12) was provided by AstraZeneca. Benda- level of DNA damage and abrogates cell-cycle arrest in mustine, melphalan, and doxorubicin were purchased cells treated with gemcitabine or irinotecan (12, 13). Given from Sigma. Bortezomib was commercially obtained from the known clinical activity of alkylating agents in multiple Millenium Pharmaceuticals, Inc. The concentrations of myeloma, we examined whether combining AZD7762 melphalan, bendamustine, doxorubicin, and bortezomib with bendamustine, melphalan, and doxorubicin could used in these studies were selected on the basis of in vitro potentiate the cytotoxic effects of these agents on myeloma combination studies in multiple myeloma (15, 16), and the cell lines. We also investigated AZD7762 in combination concentrations and conditions selected for AZD7762 have with the proteasome inhibitor bortezomib, an active agent been previously reported (12). in multiple myeloma that is not known to elicit the DDR. Proliferation assays Materials and Methods Cells were seeded at 4 105 to 6 105 cells/mL in fresh Cells media in 96-well plates. After 24 hours, cells were treated The KMS-12-BM and KMS-12-PE cell lines were pur- with drug combinations concurrently for 72 hours and chased from the German Collection of Microorganisms assayed with WST-8 (Dojindo Molecular Technologies, and Cell Cultures and were characterized as per pub- Inc.) as per manufacturer’s directions. lished guidelines (14). RPMI-8226, U266B1, and HCT116 colorectal cancer cell lines were purchased from the Immunoblotting American Type Culture Collection (ATCC) and were Immunoblotting was conducted as previously veriﬁed according to established recommendations described (17). Mouse monoclonal antibodies were

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KMS-12-BM KMS-12-PE ABKMS-12-BM KMS-12-PE 100 100 100 100

10 10 10 10 Proliferation (% control) Proliferation (% control) Proliferation Proliferation (% control) Proliferation (% control) Proliferation 0 25 50 75 100 0 25 50 75 100 0 5 10 15 20 25 0 5 10 15 20 25 Bendamustine (µmol/L) Bendamustine (µmol/L) Melphalan (µmol/L) Melphalan (µmol/L)

RPMI-8226 U266B1 RPMI-8226 U266B1 100 100 100 100

10 10 Control 10 10 Control

Proliferation (% control) Proliferation (% control) Proliferation 100 nmol/L AZD7762

Proliferation (% control) Proliferation (% control) Proliferation 100 nmol/L AZD7762 0 255075100 02550 75 100 0 5 10 15 20 25 0 5 10 15 20 25 Bendamustine (µmol/L) Bendamustine (µmol/L) Melphalan (µmol/L) Melphalan (µmol/L) CD KMS-12-BM KMS-12-PE KMS-12-BM KMS-12-PE

100 100 100 100

10 10 10 10 Proliferation (% control) Proliferation (% control) Proliferation Proliferation (% control) Proliferation (% control) Proliferation 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0 Doxorubicin (µmol/L) Doxorubicin (µmol/L) Bortezomib (nmol/L) Bortezomib (nmol/L)

RPMI-8226 U266B1 RPMI-8226 U266B1

100 100 100 100

10 10 10 10 Control Control 100 nmol/L AZD7762 100 nmol/L AZD7762 (% control) Proliferation (% control) Proliferation Proliferation (% control) Proliferation (% control) Proliferation 0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 Bortezomib (nmol/L) Bortezomib (nmol/L) Doxorubicin (µmol/L) Doxorubicin (µmol/L)

Figure 2. Bendamustine, melphalan, and doxorubicin are potentiated by Chk1/2 inhibitor AZD7762. KMS-12-BM, KMS-12-PE, RMPI-8226, and U266B1 cells were treated with the indicated concentrations of bendamustine (A), melphalan (B), doxorubicin (C), or bortezomib (D) along with vehicle or 100 nmol/L AZD7762 for 72 hours before assay with WST-8 reagent.

Cdc25A (F-6), Chk1 (G-4), Chk2 (A-11), cyclin A (H-432), apoptotic on the basis of the presence of condensed p53 (DO-1; Santa Cruz Biotechnology, Inc.); p21 (Ab-1; fragmented chromatin. Micronucleated cells were Calbiochem/Merck KGaA); PARP (19F4; Cell Signaling); defined as cells containing multiple (3) small decon- and g-H2AX (JBW301; Millipore). Rabbit polyclonal anti- densed interphase nuclei. At least 500 cells were counted bodies were phospho-Ser317 Chk1, phospho-Ser345 Chk1 per sample. The percentage of apoptotic and micronu- (133D3), phospho-Thr68 Chk2, phospho-Ser15 p53, cleated cells was measured using quantitative fluorescent a-tubulin (11H10), phospho-Ser10 histone H3, cleaved microscopy (QFM; ref. 17). caspase-3 (Asp175), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 14C10; Cell Signaling). Statistical considerations All studies were done in at least triplicate unless oth- Flow cytometry erwise indicated. Mean and SD were calculated and t Cell-cycle distribution was analyzed using flow cyto- statistical significance was calculated by the Student test. metry (fluorescence-activated cell-sorting analysis; FACS) for DNA content and mitotic marker MPM-2 (18). Results In vitro cytotoxicity of AZD7762 combinations on Measurement of apoptosis and micronucleation multiple myeloma cell lines Cells were harvested, fixed in 3% paraformaldehyde, The cytotoxicity of AZD7762 alone (100 nmol/L) and in stained with 40,6-diamidino-2-phenylindole, and exam- combination with doxorubicin, melphalan, and benda- ined with fluorescence microscopy. Cells were scored as mustine (Fig. 1) was investigated using KMS-12-BM,

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Figure 3. AZD7762 in combination with bendamustine and melphalan increases apoptosis and micronucleation. Nuclear morphology of KMS-12-PE cells was scored after 48-hour treatment with 50 or 100 mmol/L bendamustine (A) and 10 or 25 mmol/L melphalan (B) with and without 100 nmol/L AZD7762. C, representative apoptotic and micronucleated KMS-12-PE cells following treatment with AZD7762 and 50 mmol/L bendamustine.

KMS-12-PE, RPMI-8226, and U266B1 cells. As a single apoptosis (19, 20). Representative apoptotic and micro- agent, exposure to AZD7762 did not result in growth nucleated cells are shown in Fig. 3C. inhibition. However, as shown in Fig. 2, a fixed concen- tration of AZD7762 (100 nmol/L) potentiated growth Signaling and cell-cycle analyses inhibition induced by increasing concentrations of bend- Immunoblot analysis of KMS-12-PE cells treated with amustine (25–100 mmol/L; Fig. 2A), melphalan (5–25 bendamustine for 24 and 48 hours showed that histone mmol/L; Fig. 2B), or doxorubicin (0.5–1.5 mmol/L; Fig. H2A.X was phosphorylated at Ser139 forming gH2AX, a 2C) on KMS-12-BM, KMS-12-PE, and RPMI-8226 cells marker of DNA damage (Fig. 4A). Consistent with acti- with modest effects on U266B1 cells. Enhanced growth vation by DNA damage, Chk1 and Chk2 underwent inhibition was not observed following treatment with the phosphorylation at Ser317/Ser345 and Thr68, respective- combination of AZD7762 (100 nmol/L) and bortezomib ly. Chk1 activation was accompanied by a diminution of (at concentrations from 2.5to 10 nmol/L; Fig. 2D). both Cdc25A levels. In addition, bendamustine caused an accumulation of cyclin A and a decrease in the mitotic Induction of apoptosis and micronucleation marker phosho-Ser10 histone H3 indicative of S-phase The induction of apoptosis and micronucleation was cell-cycle arrest. Analysis via flow cytometry confirmed evaluated in KMS-12-PE cells treated with bendamustine that cells were arresting in S-phase at 24 hours, with DNA (50 or 100 mmol/L) or melphalan (10 or 25 mmol/L) content between 2N and 4N (Fig. 4B), accompanied by a together with AZD7762 (100 nmol/L) for 48 hours (Fig. loss of staining for mitotic marker MPM2. By 48 hours, 3). This caused a dose-dependent increase in apoptosis cells accumulated in G2 consistent with S-phase arrest from 3% (with 50 mmol/L bendamustine alone) to 25% were more transient than arrest in either G1 or G2 (21). with the combination, and from 5% (with 100 mmol/L Addition of 100 nmol/L AZD7762 to bendamustine bendamustine alone) to 35% with the combination (Fig. treatment caused a significant induction of gH2AX at 24 3A). An increase in micronucleation was also seen at both hours and increased Chk2 Thr68 phosphorylation, sug- concentrations of bendamustine with the greatest increase gesting Chk1 inhibition resulted in double strand breaks, occurring when 50 mmol/L bendamustine was used (1%– which increased ATM activity. AZD7762 also abrogated 50%; Fig. 3A). AZD7762 also increased apoptosis from 5% cell-cycle arrest as indicated by stabilization of Cdc25A with 10 mmol/L melphalan alone to 25% with the combi- and decreased cyclin A. Flow cytometry confirmed S- nation and from 15% with 25 mmol/L melphalan alone to phase checkpoint abrogation with the majority of cells 45% with the combination (Fig. 3B). Micronucleation also having 4N DNA content. AZD7762 increased the percent- increased with combination therapy, especially when 10 age of MPM2-positive cells from 0.2% to 5.75%. There was mmol/L melphalan was used (going from 1% to 35%). also an increase in p-histone H3 at serine 10, indicative of These results suggest a mitotic catastrophe with ensuing cells entering mitosis and undergoing mitotic catastrophe,

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Figure 4. AZD7762 abrogates cell-cycle arrest and induces mitotic catastrophe. A–C, immunoblot analysis in KMS-12-PE cells after 24 and 48 hours of treatment as indicated. B, flow cytometry for DNA content and MPM2 staining in KMS-12-PE cells after 24 and 48 hours of treatment with no drug control (100 nmol/L AZD7762, 50 mmol/L bendamustine, or 50 mmol/L bendamustine combined with 100 nmol/L AZD7762). Top right, histogram of DNA content by propidium iodide staining. Top left, percentage of MPM2 within boxed area. D, flow cytometric analysis as described above after treatment with no drug control, 100 nmol/L AZD7762, 10 mmol/L melphalan, or 10 mmol/L melphalan combined with 100 nmol/L AZD7762. FITC, fluorescein isothiocyanate. consistent with the increase in micronucleated cells ogous effects (Fig. 4C and D). Similar results were observed with the combination therapy (Fig. 3). Despite observed when RPMI-8226 cells were treated with bend- an increase in mitotic cells at 24 hours, a commensurate amustine or melphalan in combination with AZD7762 increase in G1 cells was not observed. Rather, at 48 hours, (Supplementary Data S1). In contrast, when KMS-12-PE cells accumulated with sub-G1 DNA content. PARP and cells were treated with bortezomib and AZD7762, no caspase-3 cleavage were also observed consistent with the increase in phospho-gH2AX was seen (Supplementary increase in apoptosis noted with theQFM. The disappear- Data S2). Although PARP and caspase-3 cleavage can be ance of Chk1 at 48 hours in the cells with combined induced with increased concentrations of bortezomib in treatment is probably a result of cleavage by active cas- the KMS-12-PE cells, this is not appreciably enhanced pases, as the cells become highly apoptotic. This is con- with the addition of AZD7762, especially with prolonged sistent with the studies of Matsuura and colleagues, who exposure of the drug (48 hours). showed that Chk1 is cleaved by caspase during apoptosis induced by genotoxic stress (22). Induction of p53 The benzimidazole ring system of bendamustine (Fig. The induction of p21 in multiple myeloma cell lines was 1C) endows it with antimetabolite properties and distin- studied following exposure to 10 Gy irradiation as a guishes it from most conventional alkylating agents (23). marker of p53 functionality. Relative to the induction of However, FACS and immunoblot analysis of KMS-12-PE p21 in p53-proficient HCT116 colorectal cancer cells, cells treated with melphalan and AZD7762 showed anal- U266B1 cells were the only multiple myeloma cells with

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decreases with each salvage regimen. p53 inactivation is associated with resistance to therapy in multiple myeloma, even with proteasome inhibitors, which are arguably the most active class of drugs for multiple myeloma (3, 28). In this report, we show in vitro data on the molecular pharmacology of a potent Chk1 inhibitor (AZD7762) administered in combination with commonly used chemotherapy to treat human myeloma cells that contain mutated p53. AZD7762 (100 nmol/L) not only potentiated the growth inhibitory effects of bendamustine, melphalan, and doxorubicin on human multiple myeloma cell lines, but it also initiated apoptosis and led to micronucleation when combined with bendamustine and melphalan (17). Chk1 inhibitors are known to predominantly sensitize p53-deficient cancer cells to DNA damage (12, 29, 30). KMS-12-BM and KMS-12-PE cells harbor only one copy of p53, and RPMI- 8226 and U266B1 cells each have distinct p53 mutations (data not shown). However, U266B1 cells can activate p21 and Gadd45 following genotoxic stress (31), and in our experiments, an amplified DNA damage response Figure 5. Induction of p21 in U266B1 cells following DNA damage. and intact p53 axis, characterized by induction of p21, Induction of p21 was determined in multiple myeloma cells 2, 4, and 8 hours after 10 Gy irradiation. U, untreated. Equal amounts of protein were was detected in U266B1 cells. This may correlate with the loaded onto gels for immunoblotting. Blots were incubated with identical reduced sensitivity of U266B1 cells to the combination of antibody dilutions and exposed on the same film. Data are representative AZD7762 and alkylating agent compared with the other of 2 independent experiments with similar results. cell lines tested. The staurosporine derivative UCN-01, a multityrosine kinase inhibitor, is also a potent Chk1 p21 induction indicative of an intact p53 axis (Fig. 5). inhibitor. Interestingly, interfering with the NF-kB path- RPMI-8226 cells showed attenuated p21 induction, way with Bay 11-7082 enhanced the apoptotic effects of thought to be p53-independent (24), given the distinct UCN-01 on multiple myeloma cell lines (32). Although difference with respect to HCT116 cells. the activity of bortezomib on multiple myeloma cells is, at least in part, mediated through NF-kB inhibition (33), we did not observe increased potency when we com- Discussion bined bortezomib and AZD7762. Thus, the previous Dysregulation of cell-cycle checkpoints is a salient fea- reported effect of UCN-01 may not have been Chk1- ture of malignant transformation; however, this abnor- dependent (32). mality in checkpoint control may selectively sensitize All data generated were with concurrent treatment. The tumor cells to genotoxic stress. Indeed, this is the rationale importance of sequence has been shown in vitro, especial- for evaluating therapeutic strategies that combine con- ly with the sequential treatment with the topoisomerase I ventional cancer treatments with inhibitors of cell-cycle inhibitor irinotecan, followed by a Chk1 inhibitor, such as checkpoints. Chk1 is a serine/threonine kinase involved CHIR-124 (17). Although, concomitant therapy shows a in the induction of cell-cycle checkpoints in response to very strong proapoptotic effect, our data do not address DNA damage and replicative stress and is essential for whether sequential treatment would have an even greater both S and G2–M phase checkpoints. Chk1 is a potential effect target for combination approaches to enhance antitumor In summary, the current study shows that pharmaco- activity of DNA-damaging agents, especially in tumors logic disruption of checkpoint function sensitizes multi- that are p53-deficient (17). ple myeloma cells with intrinsic checkpoint defects to Mutation or deletion of the TP53 gene is rarely detected genotoxic stress imparted by standard chemotherapy. at diagnosis in patients with multiple myeloma, but When combined with bendamustine or melphalan, such abnormalities become more frequent in advanced AZD7762 induced apoptosis and mitotic catastrophe. disease (25). The survival of patients with multiple mye- Drugs like the Chk1/2 inhibitor AZD7762 represent novel loma has improved over the last decade due to active new therapeutic agents for patients with multiple myeloma, agents (thalidomide, bortezomib, and lenalidomide), as perhaps even for those with p53 mutations, a population well as the optimal use of stem cell transplantation and with limited and suboptimal treatment options. Studies to improvements in supportive care (26). However, most examine the clinical benefit of adding a checkpoint kinase patients eventually relapse and die from their disease or inhibitor to standard DNA-damaging agents should be its complications (27). Although options for treating conducted in patients with relapsed and refractory mul- relapsed disease have increased, the remission duration tiple myeloma.

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Disclosure of Potential Conﬂicts of Interest Writing, review, and/or revision of the manuscript: H.J. Landau, S.C. R.L. Comenzo and H.J. Landau served on the advisory board for McNeely, R.L. Comenzo, S.C. Jhanwar, S.D. Nimer, G.K. Schwartz Millenium Pharmaceuticals. R.L. Comenzo is a consultant/advisory board Administrative, technical, or material support (i.e., reporting or orga- member for Millenium Pharmaceuticals. No potential conﬂicts of interest nizing data, constructing databases): H.J. Landau, S.C. McNeely, T. Asai, were disclosed by the other authors. G.K. Schwartz Study supervision: H.J. Landau, G.K. Schwartz Authors' Contributions Conception and design: H.J. Landau, S.C. McNeely, R.L. Comenzo, S.D. Grant Support Nimer, G.K. Schwartz This work was supported by Geoffrey Beene Cancer Research Center Development of methodology: H.J. Landau, S.C. McNeely, S.D. Nimer, G. Grant (S.D. Nimer). K. Schwartz The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Acquisition of data (provided animals, acquired and managed patients, advertisement provided facilities, etc.): H.J. Landau, S.C. McNeely, T. Asai, H. Friedman, in accordance with 18 U.S.C. Section 1734 solely to indicate S.C. Jhanwar, G.K. Schwartz this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): H.J. Landau, S.C. McNeely, J.S. Nair, T. Received November 29, 2011; revised May 4, 2012; accepted May 7, 2012; Asai, H. Friedman, S.D. Nimer, G.K. Schwartz published OnlineFirst May 31, 2012.

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1788 Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics

The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of p53-Mutated Multiple Myeloma Cells

Heather J. Landau, Samuel C. McNeely, Jayasree S. Nair, et al.

Mol Cancer Ther 2012;11:1781-1788. Published OnlineFirst May 31, 2012.

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