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
Ovarian cancer (OC) is a molecularly and histologically heterogeneous disease; however, standard treatment is the same for all subtypes. Highgrade serous OC initially responds to chemotherapy; however, low-grade serous and clear cell OC are relatively chemo-resistant. Limited treatment options are available upon recurrence. p53 mutations are found in over 90% of high-grade serous OC. Low-grade serous OC harbor wild type p53, but contain other mutations. During later stages of OC, tumors are a heterogeneous population of mutant cells; thus development of a novel drug that addresses these molecular differences is highly desirable. Previously, we showed that Kevetrin stabilized wild type p53 and induced transcriptional targets in human lung carcinoma. We sought to validate Kevetrin as a potential treatment for OC with varied p53 status. Endometrioid carcinoma (A2780, wt p53), high-grade serous (OVCAR-3, mutant p53; OV-90, mutant p53) and atypical non-serous clear cell (SKOV-3, partially deleted p53) OC cell lines and tumors were treated with Kevetrin for our studies.
Our studies showed that Kevetrin™, a small molecule currently under development, has potent antitumor activity in several wild type and mutant p53 human tumor xenografts e.g. A549, PC-3, MDA-MB-231, HT-29, NCI-H1975, HCT-15, K-562, LNCaP. To investigate the mechanism of action of its antitumor activity in different xenograft models, we assessed Kevetrin's effects on apoptosis and cell cycle progression. Rb-E2F and MDM2-p53 pathways are crucial regulators of cell cycle progression. Remarkably, both Rb-E2F and MDM2-p53 pathways are defective in most, if not all, human tumors. There is extensive crosstalk between the Rb-E2F and MDM2-p53 pathways, especially between the transcription factors E2F1 and p53, which influence vital cellular decisions. In wild type p53 human lung carcinoma (A549), Kevetrin showed a concentration dependent increase in activation of p53 by Western blot analysis. Activated p53 triggered apoptosis by inducing the expression of PUMA. Kevetrin also increased the expression of the target gene p21, an inhibitor of cell cycle progression. Kevetrin induced apoptosis in a transcriptional independent way by altering the E3 ligase processivity of MDM2. Kevetrin strongly induced apoptosis in multiple tumor cell lines characterized by activation of caspase3 and PARP. E2F1 is a pivotal transcription factor that integrates signals from a variety of G1/S phase regulators and modulates diverse functions, such as DNA synthesis, mitosis, apoptosis and senescence. E2F1 overexpression, observed in most tumors, is associated with tumor growth. Cells which overexpress E2F1 proved to be immune to induction of senescence. A recent study showed that depletion of E2F1 hinders cell cycle progression and induces senescence. Kevetrin down regulated E2F1 expression in various p53 wild type and mutant cell lines. Kevetrin also down regulated the E2F1 target gene thymidylate synthase (TS) in various tumor cell lines. Several reports have shown E2F1 overexpression correlates with TS expression. Effective inhibition of TS by Kevetrin could play a crucial role in tackling the problem of resistance. Kevetrin acting through both major pathways of tumor suppression, Rb-E2F and MDM2-p53, has far reaching consequences. Due to the complexity of proapoptotic and antiapoptotic pathways with multiple players involved and redundant signaling networks, blocking only one antiapoptotic factor may not result in robust antitumor activity. However, Kevetrin acts via multiple targets to produce potent efficacy in various xenograft tumor models. We have also demonstrated that Kevetrin is non-genotoxic. DNA damaging drugs result in rapid phosphorylation of H2A.X at Ser 139 by PI3K-like kinases; however, Kevetrin did not induce phosphorylation of H2A.X. Since Kevetrin was well-tolerated in GLP safety pharmacology and toxicity studies, we have submitted an IND application for a Phase I clinical trial at Dana Farber / Harvard Cancer Center for 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2874. doi:1538-7445.AM2012-2874
TPS2627 Background: Thioureidobutyronitrile, kevetrin, has demonstrated anti-tumor activity in several wild type and mutant p53 human tumor xenografts without evidence of genotoxicity. In wild type p53 models, kevetrin induces cell cycle arrest and apoptosis through activation and stabilization of wild type p53, resulting in increased expression of p53 target genes p21 and PUMA. Kevetrin also alters processivity of MDM2, and induces monoubiquitination of wild type p53, enhancing its stability (AACR 2011 abstract 4470). Mutant p53 is a complex target since it is an array of mutant proteins with oncogenic properties. Kevetrin induces degradation of oncogenic mutant p53 and induces apoptosis (AACR 2012 abstract 2874). Kevetrin therefore has the unique ability to target both wild type and mutant p53 tumors thereby controlling tumor growth in a wide range of preclinical tumor models. Methods: Adults with refractory locally advanced or metastatic solid tumors, acceptable liver and kidney function, and hematological status were eligible. Objectives include determination of DLT, MTD, recommended phase 2 dose, pharmacokinetics (PK), pharmacodynamics (PD), and evaluating preliminary evidence of antitumor activity. Kevetrin is given as a 1-hour intravenous infusion once weekly for 3 weeks in 28-day cycles. The starting dose was 10 mg/m2. In a 3+3 design, groups of 3-6 patients are evaluated for toxicity at each dose level. Dose escalation is based upon the number and intensity of adverse events in cycle 1. Kevetrin PK is characterized for the first and last doses given in cycle 1. Kevetrin induced expression of p21 in lymphocytes in preclinical studies; therefore p21 expression in peripheral blood mononuclear cells will be measured as a PD biomarker. Antitumor activity by RECIST 1.1 criteria and serum tumor markers will be assessed. The p53 status of tumors of selected patients will be determined. The second cohort is currently under evaluation. Conclusion: In preclinical models, Kevetrin has activity against tumors harboring both wild type and mutant p53 by diverse mechanisms. A first-in-human dose escalation trial is underway. Clinical trial information: NCT01664000.
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