Optimising treatment with combinations of novel anti-kinase drugs and cytotoxic therapy

5395 Background There is considerable clinical interest in combining kinase inhibitors with cytotoxic drugs. The cyclin dependent kinase 2 (CDK2) inhibitor O 6 - cyclohexylmethylguanine (NU2058) has been shown to potentiate cisplatin cytotoxicity in vitro (Fishel et al. J Pharmacol Exp Ther., 312 :206-13, 2005), and the aim of this study was to investigate the mechanism of potentiation. Methods Human SQ20b head and neck or LoVo colorectal tumour cells were exposed to 100 μM NU2058 for 2 hr and then a further 2 hr in the presence of the cytotoxic drug. Survival was measured by clonogenic assay. Platinum-DNA adducts and cellular platinum levels were analysed by inductively coupled plasma mass spectrometry (ICP-MS). Melphalan DNA adducts were measured by an ELISA, using an antibody specific for the N7 guanine adduct. Results While NU2058 alone was non-toxic to SQ20b cells, it increased the cytotoxicity of cisplatin with a LC 50 dose modification factor (DMF) of 3.1. Similarly, NU2058 (100 μM) increased the cytotoxicity of carboplatin (DMF 3.9), oxaliplatin (DMF 2.0), and melphalan (DMF 2.3), but not temozolamide or ionising radiation. Similar results were observed with LoVo cells. Treating cells with NU2058 immediately prior to or after, but not during, cisplatin treatment produced no potentiation of cytotoxicity. Platinum-DNA adduct levels immediately after cisplatin exposure were linearly related to cisplatin concentration, and NU2058 increased adduct levels 2-fold. NU2058 had no effect on the rate of loss of platinum-DNA adducts following drug exposure, when cells were incubated in drug-free media. NU2058 increased total intracellular platinum levels by 1.5-fold immediately after cisplatin treatment. Uptake studies suggested that NU2058 increased platinum influx, but there was no clear effect on the loss of platinum from cells upon incubation with NU2058 following cisplatin treatment. In contrast, NU2058 did not increase the formation of melphalan-DNA adducts despite causing a 2.3 fold increase in cytotoxicity. 200 structurally related CDK2 inhibitors were screened in combination with cisplatin in SQ20b cells. 60 compounds increased cisplatin cytotoxicity; however, no relationship between CDK2 inhibition and cisplatin potentiation was observed. Conclusions This study has shown that the potentiation of cisplatin by NU2058 in SQ20b and LoVo tumour cells is caused partly by enhanced drug-target interaction (i.e. increased platinum-DNA adduct levels), which is associated with an increased cisplatin influx, and partly by an altered response of cells to the DNA damage. Since NU2058 had no effect on melphalan DNA-adduct formation, we suggest that potentiation of melphalan by NU2058 was caused solely by an altered cellular response to DNA damage. The cellular target responsible for the effects of NU2058 has not been defined; however, it is unlikely to be CDK2.