Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine

Koh, Siang-Boon; Wallez, Yann; Dunlop, Charles R.; Fernández, Sandra Bernaldo de Quirós; Bapiro, Tashinga E.; Richards, Frances M.; Jodrell, Duncan I.

doi:10.1158/0008-5472.can-17-3932

Cited by 36 publications

(48 citation statements)

References 49 publications

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“…Both mechanisms result in S phase arrest and then replication catastrophe upon addition of a CHK1 inhibitor. Koh agrees with this conclusion (4). His primary concern is that we use high concentrations of gemcitabine.…”

supporting

confidence: 56%

Reply to Koh: Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent

Warren

Eastman

2019

Journal of Biological Chemistry

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supporting

confidence: 56%

Reply to Koh: Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent

Warren

Eastman

2019

Journal of Biological Chemistry

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“…The ability of S phase-arrested cells to undergo premature mitosis before completing DNA synthesis is well-recognized (30). More recently, it was demonstrated that CHK1i can force some gemcitabine-arrested cell lines to prematurely enter mitosis (15)(16)(17). Mitotic catastrophe is a well-characterized cytotoxic insult that could explain CHK1i-mediated sensitization to gemcitabine (31).…”

Section: Premature Mitotic Entry Is Not Required For Chk1i-mediated Dmentioning

confidence: 99%

“…It has also been reported that inhibiting CHK1 in the absence of dNTPs may aberrantly activate endonucleases, such as MUS81 (14), albeit this could also be a consequence of dissociating RAD51 foci. CHK1i can also induce premature activation of cyclin-dependent kinase 1 (CDK1) in S phase-arrested cells leading to premature mitosis (15)(16)(17). Another area of investigation has been the restart of stalled replication following recovery from the genotoxic insult (18).…”

mentioning

confidence: 99%

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Warren

Eastman

2019

Journal of Biological Chemistry

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Edited by Patrick SungCombining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)-and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive singlestrand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells. . 2 The abbreviations used are: CHK1i, checkpoint kinase 1 inhibitor; ssDNA, single-strand DNA; TBS, Tris-buffered saline; FBS, fetal bovine serum; HRP, horseradish peroxidase; RPA, replication protein A; ␥H2AX, pSer-139 histone 2AX; PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase; ATR, ATM and Rad3-related; DAPI, 4Ј,6-diamidino-2-phenylindole.Figure 11. Proposed mechanism of replication catastrophe from gemcitabine plus delayed CHK1i. The normal firing of early replication origins involves CDC7-dependent phosphorylation of the MCM2-7 DNA helicase core and CDK2-dependent loading of the helicase co-factor, CDC45. Gemcitabine arrests replication by depleting dNTPs. Accumulation of RPA at stalled replication forks activates the DNA-damage response through ATR and CHK1. Over time, replication machinery dissociates from stalled replication forks, preventing their restart. CHK1 prevents firing of dormant origins. Inhibition of CHK1 activates CDK2 which, in concert with CDC7, leads to CDC45 loading and dormant origin firing. Active helicases unwind DNA, but in the continued absence of dNTPs, extensive single-strand DNA is produced that exceeds the ability of replication protein A to p...

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“…Many anticancer compounds have low potency, poor therapeutic index or suffer from development of resistance [34]. Monotherapy is rarely efficient and instead drug cocktails are widely used in the clinic [23, 26]. Establishing these combinations can enhance the scope of preclinical studies and inform the design of future clinical trials.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, combination anticancer therapies have been used clinically for over 50 years to improve the responses achieved by monotherapies alone. Cancer cell line-based models for these combination therapies are easy and inexpensive to perform using high-throughput drug screening protocols (HTS) to identify the most effective drug combination [25, 26]. HTS helps to explore the relation between the cell line characteristics and drug specific dose responses [25].…”

Section: Introductionmentioning

confidence: 99%

Chemogenomic screening Identifies the Hsp70 Co-chaperone HDJ2 as a Hub for Anticancer Drug Resistance

Nitika

Blackman

Knighton

et al. 2019

Preprint

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24Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates 25 oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone 26 drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 27 is regulated by a suite of co-chaperone molecules that bring "clients" to the primary 28 chaperone for efficient folding. Therefore, rather than targeting Hsp70 itself, here we have 29 examined the feasibility of inhibiting the co-chaperone HDJ2, a member of the J domain 30 protein family, as a novel anticancer strategy. We found HDJ2 to be upregulated in a 31 variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the 32 NIH Approved Oncology collection for chemical-genetic interactions with loss of HDJ2 in 33 cancer. 41 compounds showed strong synergy with HDJ2 loss, whereas 18 dramatically 34 lost potency. Several of these hits were validated using a HDJ2 inhibitor (116-9e) in 35 castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, 36 these results confirmed that HDJ2 is a hub for anticancer drug resistance and that HDJ2 37 inhibition may be a potent strategy to sensitize cancer cells to current and future 38 therapeutics. 39 40 41 42 104 Materials and Methods 105 Cell culture. The HAP1 Chronic Myelogenous Leukemia cancer cell line and HDJ2 106 Knockout cell line was purchased from Horizon Discovery and were cultured in Iscove's 107 Modified Eagle Medium (Invitrogen) with 10% fetal bovine serum (Gibco), 100 units/ml 108 penicillin, and 100 μg/ml streptomycin at 5% CO2 and 37° C. The LNCaP cancer cell line 109 was purchased from ATCC and were cultured in RPMI-1640 medium (Invitrogen) with 110 10% fetal bovine serum (FBS, Clontech), 100 units/ml penicillin, and 100 μg/ml 111 streptomycin at 5% CO2 and 37° C. 112 6 Drug Screening. Approved Oncology Drug plates consisting of the most current FDA 113 approved anticancer drugs were obtained from National Cancer Institute (NCI). For 114 experiments delineating the synergy between the loss of HDJ2 and approved anticancer 115 drug, HAP1 cells and HAP1 (HDJ2 KO) cells were plated in growth media at 20% 116 confluency 1 day prior to drug treatment. On Day 1 of treatment, cells were treated with 117 DMSO (control), Approved oncology anticancer drugs at 50 µM for 72 hours. Following 118 drug treatments, Cell Titer-Glo reagent was added directly to the wells according to 119 manufacturer's instructions. The luminescence was measured on Bio-Tek Plate reader. 120 Luminescence reading was normalized to and expressed as a relative percentage of the 121 plate averaged DMSO control. The data shown are the mean and SEM of three 122 independent biological replicates. 123 Combination index (CI) calculations. For IC50 calculations, LNCaP cells were seeded 124 in triplicates in 96-well white bottom Nunc plates in growth media at 20% confluency 1 day 125 prior to initiation of drug treatment. On Day 1 of treatment, cells were treated with DMSO 126 (control) and ten ...

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Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine

Cited by 36 publications

References 49 publications

Reply to Koh: Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent

Reply to Koh: Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Chemogenomic screening Identifies the Hsp70 Co-chaperone HDJ2 as a Hub for Anticancer Drug Resistance

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