This could be a concern since MDM2 antagonism can alleviate drug-induced DNA damage based on data above. which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations. gene are uncommon. This includes melanoma, which harbors a relatively low (~?15%) rate of mutations (Hodis et al., 2012). However, the p53 pathway is suppressed in most melanomas via mutations, deletions or promoter methylation of the gene (Freedberg et al., 2008; Goldstein et al., 2007; Hodis et al., 2012). p14Arf, a product of this gene, negatively regulates MDM2, which is a main ubiquitin kinase that targets p53 for degradation (Kubbutat et al., 1998). In addition to inactivation, gene is amplified in a subset of melanoma tumors (about 5% of cases) (Hodis et al., 2012). These alterations can diminish p53 activity in malignant cells. Therefore, Ambroxol targeting the MDM2-p53 interaction with specific small molecule antagonists may benefit melanoma patients with wild type loss or SIR2L4 amplifications. Early phase clinical trials of MDM2 antagonists showed evidence of anti-tumor activity in patients with leukemia and liposarcoma (Burgess et al., 2016). For instance, in the phase 1 study of RG7112 in liposarcoma, partial response was achieved in 1 out of 20 patients and 14 had stable disease (Vu et al., 2013). In a phase 1 leukemia trial, complete or partial response to RG7112 was seen in 5 out of 30 patients with AML (Andreeff et al., 2016). More promising results were obtained using the next generation MDM2 antagonist, RG7388 (idasanutlin), which induced complete responses in about a quarter of enrolled AML patients (Yee et al., 2014). However, clinical trials of MDM2 antagonists also reported serious on-target adverse events including GI toxicities and prolonged myelosuppression. These data suggest that using MDM2 antagonists at a lower dose and in combination with other therapies may be more effective than single agent therapy. Finding rational and effective combination partners for MDM2 inhibitors in melanoma which avoid excessive toxicity was a goal of the study discussed here. We have recently reported that the combination of MDM2 antagonist with a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) has a potent anti-melanoma activity (Vilgelm and Richmond, 2015; Vilgelm et al., 2015). In mouse Ambroxol studies this drug combination induced senescence and immune clearance of cancer cells by antitumor leukocytes that were recruited into the tumor via NF-B-dependent induction of CCL5, CCL1, and CXCL9. As a result, prominent responses were detected Ambroxol in vivo in several melanoma models. In addition, the AURKA and MDM2 combination therapy showed adequate bioavailability and low toxicity to the host (Vilgelm et al., 2015). Notably, we found that melanoma cells treated with AURKAi had high levels of DNA damage (Liu et al., 2013). The p53 protein is the master regulator of Ambroxol DNA damage responses. Therefore here we investigated whether activation of p53 using MDM2 antagonists can affect melanoma response to AURKAi-induced DNA damage. 2.?Materials and Methods 2.1. Chemical Reagents, Cell Culture and Cell Transfection Protocols Nutlin-3a was synthesized Ambroxol as described previously (Davis and Johnston, 2011; Davis et al., 2013). MLN8237 was kindly provided by Takeda Pharmaceuticals, Inc. Idasanutlin was provided by Roche Pharmaceuticals. Chemotherapeutic drugs were purchased from Selleck (Houston, TX). Stock solutions of drugs for in vitro studies were prepared in DMSO. Stock solutions of dNTPs were prepared as follows: adenosine (Sigma (St. Louis, MO), A4036; resuspended in sterile water to 10?mM), guanosine (Sigma, G6264; resuspended in sterile DMSO to 10?mM), thymidine (Sigma, T1895; resuspended in sterile water to 10?mM), cytosine (Sigma, C4654; resuspended in sterile water to 10?mM) in accordance with previously published literature (Aird et.