Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S-and G 2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used. Mol Cancer Ther; 12(11); 2285-95. Ó2013 AACR.
Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G 2 -M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968-80. Ó2013 AACR.
Targeting cancer metabolism is currently being evaluated as a potential therapeutic target. Identification of tumor subtypes with specific metabolic requirements will enable the success of such therapies. Using cell line models of pancreatic ductal adenocarcinoma (PDAC), we conducted broad metabolite profiling and identified 3 metabolic subtypes through non-negative matrix factorization (NMF) consensus clustering. Among the most distinguishing features between the subtypes were levels of metabolites important for lipid synthesis, amino acid, and energy-related pathways, suggestive of distinct metabolic dependencies. Using RNAseq we show that these subtypes are defined at the transcriptional level and are associated with known subtypes of PDAC. In metabolic flux experiments, we also show that glucose and glutamine are differentially utilized by each subtype, and as a consequence, exhibit distinct sensitivity to inhibition of various metabolic pathways. Finally, through screening of a large panel of tumor cell lines with various metabolic inhibitors, we cross-validate our finding in PDAC and provide strong evidence that these same subtypes exist and can be identified in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity to metabolic inhibitors in cancer cells. Citation Format: Anneleen Daemen, David Peterson, Nisebita Sahu, Ron McCord, Kaska Kowanetz, Anna Hitz, Xiangnan Du, Bonnie Liu, Min Gao, John Moffat, Rebecca Hong, Deepak Sampath, Mark Merchant, Thomas O'Brien, Bob Yauch, Jeff Settleman, Jing Qing, Georgia Hatzivassiliou, Marie A. Evangelista. Identification of distinct metabolic subtypes within pancreatic ductal adenocarcinoma through broad metabolite profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2014-1430
<p>Representative data from enzyme assays evaluating the relative potency of GNE-900 against ChK1 or ChK2.</p>
<p>Representative data from a cell-based checkpoint abrogation assay.</p>
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