The kinase inhibitor O6-cyclohexylmethylguanine (NU2058) potentiates the cytotoxicity of cisplatin by mechanisms that are independent of its effect upon CDK2

Abstract

O(6)-Cyclohexylmethylguanine (NU2058) was developed as an inhibitor of CDK2 and was previously shown to potentiate cisplatin cytotoxicity in vitro. The aim of this study was to investigate the mechanism of cisplatin potentiation by NU2058. SQ20b, head and neck cancer cells were treated for 2h with NU2058 (100 microM) and then for a further 2h with cisplatin and NU2058. NU2058 increased cisplatin cytotoxicity, by clonogenic assay, with a dose modification factor (DMF) of 3.1. NU2058 increased total intracellular platinum levels 1.5-fold, and platinum-DNA adduct levels twofold. Furthermore, the cisplatin-DNA adducts formed were more toxic in the presence of NU2058. To investigate whether the effects of NU2058 on cisplatin adduct levels and toxicity were dependent on CDK2 activity, additional CDK2 inhibitors were tested. NU6230 (CDK2 IC(50) 18 microM) was equipotent to NU2058 (CDK2 IC(50) 17 microM) as a CDK2 inhibitor in cell-free and cell-based assays, yet did not potentiate cisplatin cytotoxicity. Furthermore, NU6102 was >1000-fold more potent than NU2058 as a CDK2 inhibitor (CDK2 IC(50) 5 nM) yet was no more active than NU2058 in potentiating cisplatin. NU2058 also potentiated melphalan (DMF 2.3), and monohydroxymelphalan (1.7), but not temozolomide or ionising radiation. Whilst NU2058 increased melphalan cytotoxicity, it did not increase melphalan-DNA adduct formation. These studies demonstrate that NU2058 alters the transport of cisplatin, causing more Pt-DNA adducts, as well as sensitizing cells to cisplatin- and melphalan-induced DNA damage. However, the effects of NU2058 are independent of CDK2 inhibition.

Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines

“…This compound, VE-821, also blocked phosphorylation of CHK1 at ser345 (Reaper et al , 2011). We have taken a different approach, based on the observation that NU2058 and NU6027 sensitised cells to cisplatin independently of their CDK2 inhibitory activity (Harrison, 2009; Harrison et al , 2009). We developed a cell-based assay using ATR kinase-dead cells, and used it to demonstrate that NU6027 is, in fact, a more potent inhibitor of ATR than CDK2 in living cells.…”

“…The CDK2 inhibitor, NU2058, (Arris et al , 2000), profoundly sensitises cells to cisplatin cytotoxicity independently of CDK2 inhibition (Harrison et al , 2009) and the analogous pyrimidine, NU6027 (2,6-diamino-4-cyclohexyl-methyloxy-5-nitroso-pyrimidine; Supplementary Figure 1) has similar effects (Harrison, 2009). These observations, coupled with the known cisplatin sensitisation by ATR knockdown led us to investigate whether NU6027 inhibited ATR.…”

Chemical inhibition of DNA repair kinases as a promising tool in oncology

“…NU6027 (2,6-diamino-4-cyclohexyl-methyloxy-5-nitroso-pyrimidine) was identified as a CDK2 inhibitor 78 . Peasland et al found out that NU6027 was a more potent inhibitor of ATR (IC 50 :2.8-2.9 μM) than of CDK2 (IC 50 : 10 μM).…”

Blockade of Nuclear β‐Catenin Signaling via Direct Targeting of RanBP3 with NU2058 Induces Cell Senescence to Suppress Colorectal Tumorigenesis

“…[ 6 ] Interestingly, NU2058 was also reported to exert a CDK2‐independent effect on cancer chemoresistance. [ 7 ] To date, the anticancer bioactivity of NU2058 in CRC and the underlying mechanism have not been reported.…”