Abstract B20: Schedule-dependent activation of DNA helicases by Checkpoint Kinase 1 inhibition following dNTP depletion causes CDK2-independent replication catastrophe
Bibliographic record
Abstract
Abstract DNA damaging agents are standard of care therapies for many cancers, but frequently produce poor outcomes in many patients. Using DNA damage checkpoint inhibitors to sensitize cancer cells to standard therapies is a rapidly emerging strategy. Understanding the mechanism behind sensitization will help clinicians develop rational combinations and schedules to improve cancer care. Additionally, understanding how defects in the DNA damage response affect treatment outcomes may help identify which patients will respond to treatment. We have previously demonstrated that inhibition of Checkpoint Kinase 1 (Chk1) with MK8776 sensitizes cells to the DNA damaging agent, gemcitabine, in a schedule-dependent manner. However, the mechanism of sensitization as well as the reason why sensitization increases with time following gemcitabine are not well understood. Gemcitabine inhibits ribonucleotide reductase and rapidly depletes deoxyribonucleotides (dNTPs) leading to S phase arrest. We found that delayed (18-24 h), but not concurrent (0-6 h), Chk1 inhibition following gemcitabine activated the replication machinery. Using chromatin fractionation and western blotting, we found that the helicase co-factor, CDC45, is recruited to DNA during delayed Chk1i after gemcitabine. However, the continued lack of dNTPs prevented functional DNA replication during activation of replicative helicases. This resulted in formation of excessive single-stranded DNA as measured by native BrdU fluorescent microscopy. Consequently, excess single-stranded DNA depleted cellular pools of the DNA protective protein, Replication Protein A. We also confirmed that the treatment results in DNA double-strand breaks, presumably from endonuclease cleavage of unprotected single-stranded DNA. Cyclin Dependent Kinase 2 (CDK2) is a known down-stream target of Chk1 activity. CDK2 is also known to play a role in activating the replicative machinery during normal replication. However, titrating in a CDK2 inhibitor did not prevent activation of replicative helicases, suggesting CDK2 is not required for DNA damage in this model. We identified a separate replication-associated kinase, CDC7, was required for DNA damage mediated by delayed Chk1i following gemcitabine. Treatment with CDC7i prevented Chk1i-mediated activation of the replicative helicases as well as DNA damage 18 h after gemcitabine. We conclude that CDC7-mediated aberrant activation of the replication machinery in the absence of dNTPs is the primary mechanism by which Chk1 inhibitors sensitize cancer cells to gemcitabine. Citation Format: Nicholas JH Warren, Alan R. Eastman. Schedule-dependent activation of DNA helicases by Checkpoint Kinase 1 inhibition following dNTP depletion causes CDK2-independent replication catastrophe [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B20.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".