# Novel approaches to enhance tumor cell cytotoxicity of alkylating agents

> **NIH NIH R01** · UNIVERSITY OF SOUTH ALABAMA · 2020 · $350,572

## Abstract

Project Summary / Abstract
DNA repair pathways maintain the integrity of the genome and help prevent the onset of cancer. Conversely, DNA
repair deficiencies engender tumor heterogeneity and promote the selection of highly aggressive subtypes (i.e.,
`mutator' phenotype). Importantly, DNA repair proteins have emerged as potential synthetic lethal targets for
improving the selective response to currently available anti-cancer regimens. Key to advancing such a strategy is
a detailed mechanistic understanding of the select DNA repair pathway in question, an appreciation of repair
pathway redundancy & crosstalk and a molecular characterization of processes that regulate the function and
stability of the proteins essential for repair. This completely revised R01 proposal builds on our recent discovery
that Polß is ubiquitylated on lysines 206/244 in a manner that is dependent on the cell cycle and the type of DNA
damage. Further, we reported that (i) Polß ubiquitylation may govern pathway crosstalk, (ii) select oxidized DNA
lesions require/promote Polß degradation and (iii) Polß-mediated BER facilitates DNA repair pathway crosstalk
that may be regulated by steady-state levels of Polß protein. Therefore, our first task was to identify the E3
ligases and de-ubiquitylation enzymes (DUBs) that may regulate Polß. High-resolution mass spectrometry
proteomic analysis of Polß-interacting proteins and in response to PARP-activation allowed us to identify several
E3 ligases and DUBs likely to target Polß to regulate stability and function. Our new data supports a role for the
E3 ubiquitin ligase TRIP12 contributing to the regulation of Polß stability and governing Polß chromatin retention.
Further, Polß ubiquitylation appears to govern BER repair complex dynamics. Most importantly, our preliminary
data suggests that ubiquitylation facilitates trafficking of Polß to participate in late-phase replication associated
repair (RAR) foci in response to complex DNA lesions. These RAR foci are XRCC1 dependent but devoid of DSB
markers such as γH2AX or 53BP1. The Aims detailed in the proposal will use purified proteins, cancer cell lines,
tumor stem cells, high-resolution proteomics, live-cell fluorescent imaging and mouse xenografts to address our
hypothesis that ubiquitylation/de-ubiquitylation regulates Polß stability, Polß-dependent BER repair complex
dynamics and facilitates a coordinated trafficking mechanism to promote Polß involvement yet suppress 53BP1
involvement at complex lesions following alkylation, cisplatin and radiation damage.

## Key facts

- **NIH application ID:** 9878768
- **Project number:** 5R01CA148629-10
- **Recipient organization:** UNIVERSITY OF SOUTH ALABAMA
- **Principal Investigator:** Robert W Sobol
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $350,572
- **Award type:** 5
- **Project period:** 2010-07-02 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9878768

## Citation

> US National Institutes of Health, RePORTER application 9878768, Novel approaches to enhance tumor cell cytotoxicity of alkylating agents (5R01CA148629-10). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9878768. Licensed CC0.

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