Synergistic antitumor interactions between MK-1775 and panobinostat in preclinical models of pancreatic cancer

Abstract

Pancreatic cancer remains a clinical challenge, thus new therapies are urgently needed. The selective Wee1 inhibitor MK-1775 has demonstrated promising results when combined with DNA damaging agents, and more recently with CHK1 inhibitors in various malignancies. We have previously demonstrated that treatment with the pan-histone deacetylase inhibitor panobinostat (LBH589) can cause down-regulation of CHK1. Accordingly, we investigated using panobinostat to down-regulate CHK1 in combination with MK-1775 to enhance cell death in preclinical pancreatic cancer models. We demonstrate that MK-1775 treatment results in increased H2AX phosphorylation, indicating increased DNA double-strand breaks, and activation of CHK1, which are both dependent on CDK activity. Combination of MK-1775 and panobinostat resulted in synergistic antitumor activity in six pancreatic cancer cell lines. Finally, our in vivo study using a pancreatic xenograft model reveals promising cooperative antitumor activity between MK-1775 and panobinostat. Our study provides compelling evidence that the combination of MK-1775 and panobinostat has antitumor activity in preclinical models of pancreatic cancer and supports the clinical development of panobinostat in combination with MK-1775 for the treatment of this deadly disease.

Introduction

Pancreatic cancer remains a very difficult disease to treat. The fourth most lethal cancer in the United States, pancreatic cancer has a grim prognosis, with a 5-year survival rate of 6% [1]. Standard chemotherapeutic care for pancreatic cancer involves treatment with gemcitabine, a nucleoside analogue, but offers only modest benefit [2]. Furthermore, combinations of other cytotoxic agents with gemcitabine have, in general, offered little improvement [3]. Thus, there is an obvious need to develop more effective therapy for this disease.

Cell cycle progression is a tightly regulated process. The cell cycle is driven by the activation of various cyclin dependent kinases (CDKs), which are regulated by a combination of cyclin expression and inhibitory phosphorylation. The Wee1 kinase is capable of phosphorylating CDK1 and CDK2 on Tyr-15 (Y15), preventing progression through G2/M and S phase, respectively [4], [5]. This phosphorylation can be removed by the CDC25 phosphatases, which are in turn inactivated when phosphorylated by CHK1 [6], [7]. Finally, CHK1 activity is controlled primarily by ATR kinase, which activates CHK1 by phosphorylation upon sensing replication stress or DNA damage [8], [9]. Thus, both the CHK1 and the Wee1 pathways contribute to maintaining inhibitory phosphorylation of CDKs.

MK-1775, the first potent and selective inhibitor of Wee1, has been investigated primarily as an agent that can target the G2/M checkpoint to exert toxicity specifically in p53-mutant cells [10]. It has been shown that, when combined with DNA damaging agents, MK-1775 is able to abrogate the G2/M checkpoint and enhance apoptosis, although recent studies have questioned the p53-dependence of these effects [10], [11], [12], [13], [14], [15], [16], [17], [18]. Importantly, it has been shown that Wee1 inhibition alone can induce DNA damage, likely through the induction of replication stress secondary to overactive CDKs and inhibition of DNA repair [19].

Histone deacetylase (HDAC) inhibitors (HDACIs) have been shown in vitro to have promising anti-cancer activity, but their single-agent effectiveness in the clinic has been only modest [20], [21], [22], [23], [24], [25], [26], [27]. However, there are many clinical trials investigating the role of HDACIs in combination therapies (NCT01242774, NCT01742793, NCT02061449, and NCT02145715, clinicaltrials.gov). Previous work from this lab has demonstrated the ability of HDACIs to synergize with standard chemotherapeutic agents, at least in part by enhancing DNA damage [28], [29], [30], [31]. Importantly, we recently demonstrated that treatment with the pan-HDACI panobinostat (LBH589) was able to down-regulate CHK1 [28], [29]. It has been reported that combined inhibition of Wee1 and CHK1 is effective at inducing cancer cell death [13], [32], [33], [34], leading us to consider the combination of MK-1775 and panobinostat for the treatment of pancreatic cancer.

In this work, we use pre-clinical pancreatic cancer models to investigate the effects of the combination of MK-1775 and panobinostat, and the mechanism by which panobinostat enhances MK-1775-induced apoptosis. We demonstrate that MK-1775 alone is able to induce DNA damage and activate CHK1 in a CDK-dependent fashion. Panobinostat treatment down-regulates CHK1 and synergizes with MK-1775 to enhance apoptosis and cell growth inhibition. Importantly, we demonstrate that in some cell lines, the CHK1 pathway is able to overcome single agent Wee1 inhibition and maintain phosphorylation of CDK1. This demonstrates a potential mechanism of resistance to treatment with MK-1775 and emphasizes the importance of combinations with agents such as panobinostat.