Replication Stress in Lung Cancer
Abstract
Innovation: Results will, for the first time, identify at base pair resolution where replication fork progression is compromised genome-wide in a genotype-specific manner in lung cancer lines. Results will reveal classes of fork impediments susceptible to FDA-approved or candidate drugs for treating lung cancer or that become hotspots for toxicity in normal cells. This fundamental work will provide new frameworks for understanding normal functions of DNA replication and repair machinery and where and why different responses to replication stress occur. This work will provide insight into how replication fork-related defects contribute to lung cancer plus provide novel replication integrity defect-related biomarkers predictive of drug susceptibility in individual lung cancers. The long-term goal is to guide custom treatments that take advantage of creating synthetic defects in fork integrity at impediments that become enriched in a tumor-dependent manner. Rationale: During replication stress, forks stall or collapse (the latter resulting single or double stranded DNA breaks) at non-random sites across the genome and must be restarted or repaired using mechanisms that are distinct for different kinds of impediments. This project’s working hypothesis is that genetic defects in lung cancers lead to DNA replication stress and cause stalled or broken replication forks to accumulate at cancer- specific hotspots in addition to sites found in normal cells. Such hotspots could arise from either a defect in repair/restart of forks or a defect in another biological process that results in increased frequency of formation of certain types of impediments (e.g., R loops) within the genome. Such hotspots are predicted to reduce replication fidelity, contribute to tumorigenesis via errors during fork restart, as well as influence sensitivity to distinct classes of therapeutic drugs that compromise fork restart and/or impediment removal/prevention. Inhibition of pathways required for replication through identified impediments is predicted to disrupt proliferation and promote cancer cell death or senescence. The rationale for mapping stalled and collapsed forks in lung cancer lines from the NCI-60 panel is that data can be leveraged by comparing to extensive publicly available NCI-60 datasets in CellMiner and DTP databases (NCI). Genome-wide mRNA or miRNA expression, gene copy numbers, protein levels, and whole exome sequencing data (which identified normal and cancer-specific genetic variants such as insertion, deletion, missense mutations, splice site defects, etc.) is available. Similarly, thousands of compounds have been screened in these lines and their 50% growth inhibition (GI50) are available for cross- comparison. Comparing mutations unique to each cancer to responses to drugs targeting DNA repair or chromatin biology machinery and to tumor-specific stalled fork hot spots will facilitate identifying pathways critical for responding to different impediments. Paired tumor/normal lines will also reveal tumor-specific defects. Objectives: Objective 1. To map genome-wide sites where replication forks stall or collapse in lung cancer cells by TrAELseq in the nine NCI-60 lung cancer cell lines (A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-322M, NCI-H460, and NCI-H522) +/- FDA-approved or candidate drugs used to treat lung cancer. Objective 2. To map genome-wide sites where forks stall or collapse in matched lung tumor/normal cells by TrAELseq using two pairs of matched cell lines (NCI-H1395/NCI-BL1395, NCI-H1437/NCI-BL1437) +/- FDA-approved or candidate drugs used to treat lung cancer. Methods for Both Objectives: To identify stalled and collapsed forks, genomic locations where 3’ ends of DNA strands accumulate (e.g., sites of partial or full fork reversal, breaks) will be determined to base pair resolution by a new NGS strategy called TrAELseq. For example, accumulation of 3’ end peaks in c
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310356
Entities
People
- Ann L Kirchmaier
Organizations
- Purdue University
- United States Army