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

Open AccessPublished:February 01, 2019DOI:https://doi.org/10.1074/jbc.RL119.007419
      Koh (
      • Koh S.-B.
      Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent.
      ) correctly notes that gemcitabine elicits two mechanisms of action, but proposes that chain termination predominates. We believe its ability to inhibit ribonucleotide reductase and deplete dNTP is more critical (
      • Warren N.J.H.
      • Eastman A.
      Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.
      ). We have previously discussed our rationale for this position (
      • Montano R.
      • Khan N.
      • Hou H.
      • Seigne J.
      • Ernstoff M.S.
      • Lewis L.D.
      • Eastman A.
      Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.
      ). Both mechanisms result in S phase arrest and then replication catastrophe upon addition of a CHK1 inhibitor. Koh agrees with this conclusion (
      • Koh S.-B.
      • Wallez Y.
      • Dunlop C.R.
      • Bernaldo de Quirós Fernández S.
      • Bapiro T.E.
      • Richards F.M.
      • Jodrell D.I.
      Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine.
      ). His primary concern is that we use high concentrations of gemcitabine.
      Our experiments are designed to reflect clinically relevant concentrations and schedules. Patients receive gemcitabine by a short infusion, and it is cleared rapidly from plasma. Accordingly, we incubate cells briefly with gemcitabine. After removal, cells continue to accumulate in S phase as observed in vitro, xenografts, and human tumors (
      • Montano R.
      • Khan N.
      • Hou H.
      • Seigne J.
      • Ernstoff M.S.
      • Lewis L.D.
      • Eastman A.
      Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.
      ). The CHK1 inhibitor is most effective when administered after cells arrest in S phase (
      • Montano R.
      • Thompson R.
      • Chung I.
      • Hou H.
      • Khan N.
      • Eastman A.
      Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration of schedule in vitro and in vivo.
      ). In contrast, Koh incubated cells with gemcitabine and CHK1 inhibitor continuously for 72 h (
      • Koh S.-B.
      • Wallez Y.
      • Dunlop C.R.
      • Bernaldo de Quirós Fernández S.
      • Bapiro T.E.
      • Richards F.M.
      • Jodrell D.I.
      Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine.
      ), neither of which is a clinically relevant schedule. Koh notes his low concentrations of gemcitabine permit continued proliferation for 72 h (
      • Koh S.-B.
      • Wallez Y.
      • Dunlop C.R.
      • Bernaldo de Quirós Fernández S.
      • Bapiro T.E.
      • Richards F.M.
      • Jodrell D.I.
      Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine.
      ). These low concentrations are not clinically relevant as the standard dose (1000 mg/m2) causes durable S phase arrest in the tumor cells (
      • Montano R.
      • Khan N.
      • Hou H.
      • Seigne J.
      • Ernstoff M.S.
      • Lewis L.D.
      • Eastman A.
      Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.
      ). We also showed that considerably lower concentrations of gemcitabine in xenograft models exhibited durable S phase arrest (
      • Montano R.
      • Khan N.
      • Hou H.
      • Seigne J.
      • Ernstoff M.S.
      • Lewis L.D.
      • Eastman A.
      Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.
      ). Consequently, low concentrations of gemcitabine that permit ongoing replication have little relevance to the clinical administration of these drugs. Consequently, we believe our experimental design is far more relevant to human patients.

      References

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        Signaling dynamics of DNA damage response invoked by combination therapy are dose-dependent.
        J. Biol. Chem. 2019; 294: 2191
        • Warren N.J.H.
        • Eastman A.
        Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.
        J. Biol. Chem. 2019; 294 (30573684): 1763-1778
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        • Khan N.
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        Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.
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        Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine.
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        • Thompson R.
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