Access Type

Open Access Dissertation

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Steve M. Patrick

Abstract

ERCC1/XPF is a DNA endonuclease with variable expression in primary tumor specimens, and has been investigated as a predictive biomarker for efficacy of platinum-based chemotherapy in non-small cell lung cancers where up to 30-60% of tumors harbor low to undetectable ERCC1 expression. The failure of an international, randomized Phase III clinical trial utilizing ERCC1 expression to predict response to platinum-based chemotherapy suggests additional mechanisms underlying the basic biology of ERCC1 in the response to platinum-DNA damage remain unknown. In this work, we aimed to characterize a panel of ERCC1 knockout cell lines generated via CRISPR-Cas9 where we identified a synthetic viable phenotype in response to intestrand crosslinks (ICLs) with ERCC1 deficiency. Characterization of these ERCC1 knockout cell lines revealed loss of ERCC1 hypersensitized cells to cisplatin when wildtype (WT) p53 is retained, while there was only modest sensitivity in cell lines that were p53mutant/null. Additionally, when p53 was disrupted by CRISPR-Cas9 (p53*) in ERCC1 knockout/p53WT cells, there was reduced apoptosis and increased viability after platinum treatment. These results were recapitulated in two patient data sets utilizing p53 mutation analysis and ERCC1 expression to assess Overall Survival. We also show that kinetics of ICL-repair differed between ERCC1 knockout/p53WT and ERCC1 knockout/p53* cells. Finally, we provide evidence that cisplatin tolerance in the context of ERCC1 deficiency relies on DNA-PKcs and BRCA1 function as well as timely entry into S phase suggesting that replication dependent mechanisms are likely involved in promoting platinum tolerance.

Building upon these observations, we utilized our established lung cancer cell line models of ERCC1 deficiency to find that platinum tolerance with ERCC1 deficiency relies upon ATR signaling. ATR inhibition by M6620 selectively and synergistically enhanced platinum sensitivity of platinum tolerant ERCC1-deficient cells. Interestingly, this increased sensitivity was independent of Chk1 and Wee1 kinase inhibition, suggesting that ATR may support platinum-tolerance in the absence of ERCC1 by suppressing global replication fork collapse independent of activating the G2/M cell cycle checkpoint. Additionally, dual treatment led to increased formation of DNA double strand breaks and was associated with increased levels of pulverized chromosomes. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATRi treatment may also enhance response to immunotherapy in ERCC1-deficient tumors harboring a p53 mutation.

Our findings implicate p53 as a potential confounding variable in clinical assessments of ERCC1 as a platinum biomarker via promoting an environment in which error-prone mechanisms of ICL-repair may be able to partially compensate for loss of ERCC1. Additionally, results of this study have led to the identification of a feasible therapeutic strategy combining M6620 with cisplatin to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.

Download

Share

COinS