Abstract 5048: Targeting regulators of Chk1 to enhance cytotoxic efficacy of HDAC inhibitors
Authors: Kreahling Jenny M. , Gemmer Jennifer Y. , Ma Yihong , Cress Douglas , Johnson Jackie , Chellappan Srikumar P. , Altiok Soner 
Published: June 2012

Introduction: Predicting and overcoming drug resistance is a major difficulty facing the efficacy of drugs in clinical trials. Histone deacetylase inhibitors (HDACis) are known to induce cell death and have been used in clinic trials as anticancer drugs, however, the cytotoxic effects of HDACis remains to be limited with no clear mechanism of resistance has been elucidated to explain this limited efficacy. In this study we showed that downregulation of the G2 checkpoint kinase 1 (Chk1) plays an essential role in inducing cell death by HDACis. We identified E2F as a main regulator of Chk1 expression in cancer cells, suggesting that inhibition of Chk1 through E2F may enhance the cytotoxic effects of HDACis in clinical studies.

Methods: A549, H1299, PC9, HCT116, HT1080, MNNG, H2172, Saos-2, MG63, U2OS, p21-/- and p21+/+ MEF cell lines were grown in appropriate medium and treated with the HDAC inhibitors, SaHa, LBH589 or MS275. Western blot and qRT-PCR experiments were performed to assess the effect of HDACis on Chk1/G2-M checkpoint pathways and on E2F-mediated gene expression profiles. Additionally, knock out and gain of function experiments were done to better understand the interaction between Chk1, E2F and p21Waf1 in HDACi mediated cell death. The results of the in vitro cell lines studies were corroborated in the ex vivo experiments with patient tumor tissue.

Results: We showed that HDACi treatment leads to inhibition of Chk1 expression and consequently to increased activity of CDC25 and CDC2 (CDK1) enzymes causing premature mitotic entry and cell death. In the knock-down experiments we found that inhibition of E2F1 or E2F3 leads to reduction of Chk1 expression and potentiation of cell death by HDACis. Similar results were observed using patient-derived tumor samples treated with SaHa and an E2F inhibitor (HLM006474). Additionally, over-expression of E2F1 or E2F3 dramatically decreased HDACi-mediated cell death. In time course experiments, Chk1 as well as other E2F-responsive genes were down regulated after HDACi treatment accompanied by an expected p21Waf1 induction. In p21Waf1 +/+ and -/- MEFs we showed that lack of p21Waf1 results in increased apoptosis, suggesting that early induction of p21Waf1 by HDACis negatively regulates HDACi-induced cell death, which likely contributes to HDACi resistance in tumor cells by preventing cells to undergo mitotic cell death.

Conclusions: Our results demonstrate that E2F-mediated downregulation of Chk1 plays a key role in HDACi-induced cell. We provide evidence that lack of Chk1 downregulation and activation of CDC2 is associated with resistance to HDACis indicating that Chk1 may represent a clinically relevant biomarker to assess the efficacy of HDACis in patient tumor samples. Taken together our data lends insight into the molecular mechanism of sensitivity and resistance to HDACi-induced cell death and will hopefully provide a stronger foundation for future clinical translation with HDACis.

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 5048. doi:10.1158/1538-7445.AM2011-5048