Assessment of chk1 phosphorylation as a pharmacodynamic biomarker of chk1 inhibition - PubMed (original) (raw)

. 2011 Jun 1;17(11):3706-15.

doi: 10.1158/1078-0432.CCR-10-3082. Epub 2011 Apr 11.

Yushen Qian, Daria M Tanska, Marisa Gross, Lili Zhao, Maria C Hassan, Sankari Arumugarajah, Joshua D Parsels, Linda Hylander-Gans, Diane M Simeone, Deborah Morosini, Jeffrey L Brown, Sonya D Zabludoff, Jonathan Maybaum, Theodore S Lawrence, Meredith A Morgan

Affiliations

Assessment of chk1 phosphorylation as a pharmacodynamic biomarker of chk1 inhibition

Leslie A Parsels et al. Clin Cancer Res. 2011.

Abstract

Purpose: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models.

Experimental design: We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1.

Results: AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism.

Conclusions: AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1.

©2011 AACR.

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Figures

Figure 1

Figure 1. Chk1 signaling in response to replication stress and Chk1 inhibition

In response to replication stress/DNA damage, ATR phosphorylates Chk1 at S345, leading to Chk1 autophosphorylation at S296. Phosphorylation of Chk1 at S345 is counterbalanced by PP2A phosphatase activity, which is in part dependent on Chk1 kinase activity. In addition, ATM mediates phosphorylation of Chk2 and H2AX in response to DNA damage. Active Chk1 inhibits Cdc25 phosphatases, inhibits caspase-3, and induces Rad51 focus formation, ultimately resulting in cell cycle arrest, cell survival, and HRR. In response to replication stress, in the presence of a Chk1 inhibitor, such as AZD7762 (AZ), Chk1 kinase activity and autophosphorylation are inhibited. Inhibition of Chk1 results in Cdc25 phosphatase activity, caspase-3 activation (under some conditions), and inhibition of Rad51 focus formation as well as HRR, ultimately resulting in mitotic entry (marked by pS10 histone H3), apoptosis (under some conditions), and unrepaired DNA damage. As a result of Chk1 inhibition, DNA damage accumulates which in turn amplifies ATM/ATR signaling leading to an increase in phosphorylation of H2AX, Chk1 (S345), and Chk2 (T68). In addition, the increase in pS345 Chk1 is also regulated, in part, by PP2A. Gray color indicates inhibited processes, bold indicates amplified signaling.

Figure 2

Figure 2. AZD7762 sensitizes pancreatic cancer cells to gemcitabine and affects cell cycle checkpoints

A, Schedule of treatments. B, MiaPaCa-2, M-Panc96, BxPC-3, and Panc-1 cells were treated with gemcitabine (0 – 5 uM) and AZD7762 (100nM) according to the schedule illustrated (A). At the end of the treatment period cells were processed for clonogenic survival. C, MiaPaCa-2 cells were treated with gemcitabine (50 nM) and AZD7762 (100 nM) according to treatment schedule 2. Cells were collected for immunoblotting at t = 26 and 30 hours. The surviving fraction was normalized to the control plating efficiency for `Gem Alone' samples and to the AZD7762 plating efficiency for `Gem + AZD7762' samples for each schedule (B). Data are the mean of n = 3 – 6 independent experiments (B) or are representative of 3 independent experiments (C).

Figure 3

Figure 3. AZD7762 sensitizes pancreatic tumor xenografts to gemcitabine

A, Mice bearing MiaPaCa-2 xenografts were treated with gemcitabine (120 mg/kg) on days 0, 7, and 14 and AZD7762 (25 mg/kg) on days 0, 1, 7, 8, 14, and 15. B, Alternatively, Patient J-derived xenografts were treated with gemcitabine (60 mg/kg) on days 0, 3, 7, 10, 14, and 17 and AZD7762 (20 mg/kg) on days 0, 1, 3, 4, 7, 8, 10, 11, 14, 15, 17, and 18. Data are expressed as the portion of tumors doubled in volume (A–B) or the median time required for tumor volume doubling (P<0.05) (C). Each treatment group contained 6–8 (A) or 10–20 (B) tumors.

Figure 4

Figure 4. AZD7762 increases pS345 Chk1 in response to gemcitabine

MiaPaCa-2 xenografts were treated with gemcitabine (120 mg/kg) on day 0 and AZD7762 (25 mg/kg) on days 0 and 1 (A, C). Alternatively, Patient-J-derived xenografts were treated with gemcitabine (60 mg/kg) on day 0 and AZD7762 (20 mg/kg) on days 0 and 1 (B, D). Tumors were harvested on day 1, following the last dose of AZD7762 and prepared for immunoblotting (A–B) or immunohistochemistry (C–D). Data are from a single experiments representative of 3 – 4 tumors per treatment group.

Figure 5

Figure 5. Induction of pS345 Chk1 in hair follicles and colon by gemcitabine and AZD7762

Mice were treated with gemcitabine (30 – 60 mg/kg) and three hours later with AZD7762 (5 – 40 mg/kg). Three hours post-AZD7762, tissues were harvested and fixed for immunohistochemistry. Images are from single experiment with 60 mg/kg gemcitabine and 20 mg/kg AZD7762, as indicated in hair follicles (A) or colon (B). The mean H-score ± standard error in hair follicles for 2 – 4 animals per treatment condition is shown (C). Statistically significant differences from control* or gemcitabine‡ are indicated (P<0.05) and were determined by one-way ANOVA.

Figure 6

Figure 6. pS345 Chk1 induction occurs independent of PP2A and correlates with increased DNA damage response

MiaPaCa-2 cells were treated according to Schedule 2 (Fig. 1A). At t = 24 hours cells were treated with AZD7762 and/or okadaic acid (OA, 1uM) for 1 hour. At the end of treatment, cells were prepared for immunoblotting (A, B) or FACS analysis (C). Data are from a single experiment (A) or are the mean of 3 – 4 independent experiments (B–C). Statistically significant differences (P<0.05) were determined by a paired t-test comparing control versus 7762*, Gem versus Gem 7762‡, OA versus 7762 OA or Gem OA†, versus Gem 7762 OAπ.

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