Abstract 4470: Kevetrin™, a novel small molecule, activates p53, enhances expression of p21, induces cell cycle arrest and apoptosis in a human cancer cell line

Abstract

We have previously shown that Kevetrin, a small molecule currently under development, has potent antitumor activity in human multi-drug resistant carcinoma xenograft models while being well tolerated. To investigate the mechanism of action for its potent antitumor activity, we assessed Kevetrin's effect on apoptosis, cell cycle progression, including underlying molecular mechanisms. Here we report that Kevetrin strongly induced cell cycle arrest and apoptosis in a human lung adenocarcinoma cell line (A549) using the TUNEL assay and FACS analysis; similar results were observed using a human breast carcinoma cell line (MDA-MB-231). Treatment of A549 cells with 400 µM of Kevetrin for 48 hours resulted in G2/M phase cell cycle arrest that was associated with a marked decline in levels of G2/M regulatory proteins, including CDK1 and cdc25B, and increased expression of Wee1. Further, Kevetrin-mediated growth inhibition of A549 correlated with apoptosis induction that was characterized by cleavage of procaspase-3 and poly (ADP-ribose) polymerase (PARP). The p53 tumor suppressor is a well characterized transcription factor controlling cell growth and apoptosis during times of cellular stress. The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. In cancer development, the resistance of cells to apoptosis is one of the crucial steps. The reactivation of p53 in tumor cells should trigger massive apoptosis and eliminate the tumor. Interestingly, Kevetrin treatment caused enhanced activated p53 levels in A549 cells. Activation of p53 can lead to cell cycle arrest and apoptosis. Western blotting revealed a concentration dependent increase in phosphorylation of p53 at serine 15. The phosphorylation of p53 at serine 15 leads to reduced interaction between p53 and its negative regulator, the oncoprotein MDM2. We also found that Kevetrin increased expression of p53 target genes such as p21 (Waf1). The tumor suppressor protein p21 (Waf1) acts as inhibitor of cell cycle progression. Finally, we also found that p53 induced apoptosis by inducing the expression of PUMA. Our study shows that Kevetrin activates p53 functions in tumor cells. Thus Kevetrin is a strong candidate as an anticancer drug that targets p53. Based on our studies we plan to initiate a Phase I clinical trial in 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2011-4470

Kevetrin induces apoptosis in TP53 wild‑type and mutant acute myeloid leukemia cells

“…The p53 pathway was modulated by a 48-h kevetrin treatment, with transcriptional upregulation of several p53 targets in TP53-wt and -mutant models. In the wt cell lines, kevetrin induced a dose-dependent upregulation of the p21 protein, in line with the trend observed in TP53-wt solid tumor cells (18,20). In the TP53-mutant models, p21 upregulation was observed at an intermediate kevetrin dose, with increased p53 nuclear localization (also detected at the highest dose), together with a high fraction of p53-positive cells with nuclear fragmentation.…”

“…We did not observe cell cycle alterations in KASUMI-1 or MOLM-13 models, whereas a significant accumulation of cells in the G0/G1 phase was observed in OCI-AML3 and NOMO-1 cells. Only TP53-wt OCI-AML3 cells displayed an increase in the percentage of G2/M cells after treatment, as previously reported in the TP53-wt A549 lung carcinoma cell line (18). The difference observed between KASUMI-1 and NOMO-1 mutated models may be attributable to the predominant apoptotic effect induced by kevetrin on KASUMI-1 cells and to the different TP53 mutational status.…”

“…Under stress conditions, p53 is phosphorylated by various sensor kinases, promoting the dissociation of the p53-MDM2 complex and p53 stabilization and activation (16,17). Kevetrin (thioureidobutyronitrile or 3-cyanopropyl carbamimidothioate hydrochloride, C5H10ClN3S) is a small-molecule compound that exhibits p53-dependent and -independent activity in solid tumors, including lung, breast, colon and ovarian cancer cell and xenograft models (18)(19)(20). In TP53-wt models, kevetrin induces cell cycle arrest and apoptosis through the alteration of the E3 ligase processivity of MDM2 and activation and stabilization of p53, with increased expression of its targets, including protein 21 (p21) and p53-upregulated modulator of apoptosis (18,19).…”

“…Kevetrin (thioureidobutyronitrile or 3-cyanopropyl carbamimidothioate hydrochloride, C5H10ClN3S) is a small-molecule compound that exhibits p53-dependent and -independent activity in solid tumors, including lung, breast, colon and ovarian cancer cell and xenograft models ( 18 – 20 ). In TP53 -wt models, kevetrin induces cell cycle arrest and apoptosis through the alteration of the E3 ligase processivity of MDM2 and activation and stabilization of p53, with increased expression of its targets, including protein 21 (p21) and p53-upregulated modulator of apoptosis ( 18 , 19 ). A p53-independent upregulation of p21 expression was observed in TP53 -mutant ovarian cancer cell lines ( 20 ).…”

Restoration of conformation of mutant p53

“…Kevetrin (thiobutyronitrile) has several effects, one of which is HDAC inhibition. It might also have direct effect on mutant p53 and has been shown to lead to phosphorylation of p53 at serine 15 and G2/M cell division arrest with a decline in Wee1, p21 and PUMA, and tumor cell death [40,41]. A possible mechanism is induction of mutant p53 degradation by downregulation of HSP90 through inhibition of HDAC6.…”