Published OnlineFirst March 26, 2014; DOI: 10.1158/1535-7163.MCT-13-0634 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Nedd8-Activating Enzyme Inhibitor MLN4924 Provides Synergy with Mitomycin C through Interactions with ATR, BRCA1/BRCA2, and Chromatin Dynamics Pathways Khristofer Garcia1, Jonathan L. Blank1, David C. Bouck1, Xiaozhen J. Liu1, Darshan S. Sappal1, Greg Hather2, Katherine Cosmopoulos1, Michael P. Thomas1, Mike Kuranda1, Michael D. Pickard2, Ray Liu2, Syamala Bandi3, Peter G. Smith1, and Eric S. Lightcap1 Abstract MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standard-of-care agents and may affect the clinical development of MLN4924. As such, we studied its interaction with other DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, and gemcitabine demonstrated synergy in combi- nation with MLN4924 in vitro. The combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse xenograft model. Importantly, depletion of genes within the ataxia telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924 results in frequent repli- cation fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may provide useful information for the clinical development of these combina- tions. Mol Cancer Ther; 13(6); 1625–35. Ó2014 AACR. Introduction and regulation of p21, p27, p53, Cdc25A, Wee1, claspin, and MLN4924 is being developed as an inhibitor of the FANCM (6–10). Many standards of care in cancer chemo- Nedd8-activating enzyme (1) and is currently in phase I therapy are DNA-damaging agents and mechanistic inter- clinical trials. Nedd8 is a small ubiquitin-like protein in actions between MLN4924 and these agents may influence which the best characterized role is the activation of a class the clinical development of MLN4924. of E3 ubiquitin ligases known as cullin RING ligases (CRL; Although the mechanism engaged by MLN4924 to ref. 2). Multiple studies have demonstrated the import- induce DNA damage seemingly differs from other ance of the induction of rereplication, resulting in DNA DNA-damaging agents, the pathways involved in the damage, to the mechanism of cell death by MLN4924 (3–5). repair of the DNA damage may overlap. A previous In addition, CRLs are known to be involved in the regu- siRNA screen that evaluated the genetics of sensitivity of lation of multiple DNA replication and repair pathways, A375 and HCT-116 cell lines to MLN4924 demonstrated including nucleotide excision repair, histone modification, an engagement of a p21-dependent intra–S-phase check- point in A375 cells and an Emi1-dependent G2–M check- point in HCT-116 cells (5). In addition, roles for p53, Authors' Affiliations: Departments of 1Discovery, 2Clinical Biostatistics, BRCA1/BRCA2, and nucleotide excision repair were and 3Information Technology, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts identified. Oncogene-induced replication stress seems to play a central role in tumor progression (11, 12), suggest- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ing that rereplication may directly challenge tumor vul- nerabilities. However, it is not clear that rereplication will Current address for K. Cosmopoulos: Agios Pharmaceuticals, Cambridge, be the dominant mechanism of DNA damage in combi- Massachusetts; and current address for M.P. Thomas and P.G. Smith, H3 Biomedicine, Inc., Cambridge, Massachusetts. nation with other DNA-damaging agents and, thus, it is difficult to predict the best combination partners. Corresponding Author: Eric S. Lightcap, Discovery Oncology Biology, Takeda Pharmaceuticals International Co., 40 Landsdowne Street, Cam- We have conducted a systematic evaluation of the bridge, MA 02139. Phone: 617-551-3717; Fax: 617-551-8906; E-mail: effectiveness of combining MLN4924 with DNA-damag- [email protected] ing agents across four different cancer cell types in vitro doi: 10.1158/1535-7163.MCT-13-0634 and demonstrate synergy with mitomycin C, cytara- Ó2014 American Association for Cancer Research. bine, cisplatin, UV, SN-38, melphalan, etoposide, and www.aacrjournals.org 1625 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 26, 2014; DOI: 10.1158/1535-7163.MCT-13-0634 Garcia et al. gemcitabine. Mitomycin C gave the most consistent syn- CCL-185, HCT-116 ATCC CCL-247, and U-2 OS ATCC ergistic interaction with MLN4924 across cell lines. Much HTB-96). Consistent regions of amplification and dele- work has been done to understand the mechanism of tions were found between the cell lines of the same origin, DNA repair following mitomycin C treatment (13, 14), and correlation on genotypes was greater than 0.995. Cell providing a background to understand these results. lines were passaged for less than 8 weeks following Mitomycin C alkylates DNA with about 10% of DNA resuscitation. modifications resulting in interstrand crosslinks, the remainder being monoadduct and intrastrand crosslinks Evaluation of cell line sensitivity to DNA-damaging (14). The mechanism by which the cell repairs this DNA agents and evaluation of combination effects damage is determined by how the adduct is detected, Cells were grown in their respective growth media, which is significantly influenced by the stage of the cell supplemented with 10% FBS: A375, Dulbecco’s Modified cycle in which the damage occurs. During G1, adduct can Eagle Medium; A549, Ham’s F-12K (Kaighn’s) medium; be detected by components of either global genome nucle- HCT-116 and U-2 OS, McCoy’s 5A medium with 1% otide excision repair or by transcription-coupled nucleo- glutamine. tide excision repair (TC-NER) if the interstrand crosslink A375 melanoma (800 cells/well), HCT-116 colorectal blocks RNA polymerase. During S-phase, encounter of carcinoma (800 cells/well), A549 lung carcinoma (1,000 the interstrand crosslink by the replication fork becomes a cells/well), and U-2 OS osteosarcoma (800 cells/well) significant mechanism for detection of the crosslink and were seeded on 384-well poly-D-lysine (PDL)-coated the repair mechanism is believed to be substantially black, clear-bottom plates (BD BioCoat) and allowed to different from that experienced during G1 (14). Notably, adhere for 24 hours at 37 C, 6% CO2. Cells were then during S and G2 phases of the cell cycle, sister chromatids treated with compounds, either alone or in combination are available for exchange by homologous recombination, with MLN4924, at various doses for 48 hours (HCT-116 providing a high-fidelity mechanism of repair. and A375, doubling times of 18 and 16 hours, respectively) To gain a more complete understanding of the synergy or 72 hours (A549 and U-2 OS, doubling times of approx- between MLN4924 and mitomycin C, we used an siRNA imately 24 hours). Viability was assessed with CellTiter- screen to evaluate the effect of gene depletion on this Glo Cell Viability reagent according to the manufacturer’s synergy. Many of the genes identified in the siRNA screen instructions (Promega). Luminescence was measured underwent posttranslational modifications to their pro- using a LEADseeker imaging system (GE Healthcare). tein products upon treatment with the combination of MLN4924 will be made available to qualified researchers MLN4924 and mitomycin C, confirming their likely once a standard Material Transfer Agreement has been involvement. Comet assay experiments demonstrated executed. early induction of DNA strand breaks by the combination of MLN4924 and mitomycin C. Our results suggest that Calculation of combination metrics mitomycin C increases the frequency of replication fork The relationship between the normalized viability collision induced by MLN4924 and that the BRCA1–clas- and drug concentrations was fit with a nine parameter pin–ATR–Chk1 pathway plays an important role in the response surface model (15). To quantify the synergy, response to these collisions. These studies may provide a the combination index (16) or nonlinear blending (17) way of identifying promising drug combinations and was computed. Additional details can be found in understanding the mechanistic details of their synergy. Supplementary Materials and Methods. Such knowledge may more efficiently guide clinical development of chemotherapeutic combinations. The effect of depletion of DNA damage response genes on the synergy between MLN4924 and mitomycin C Materials and Methods A375 cells were cultured and reverse transfected with Genomic characterization and authentication of cell siRNA SMARTpools or duplexes targeting DNA damage lines response genes using DharmaFect4 transfection lipid A375, A549, HCT-116, and U-2 OS were received from (Dharmacon, GE Healthcare), as previously described the American Type culture collection (ATCC) in July 2006, (5). After 48 hours knockdown, cells