# Project 1: Synthesis, Characterization and Biomonitoring of DNA Containing Site-Specific Chemotherapy-Induced Complex Alkylation Products

> **NIH NIH P01** · VANDERBILT UNIVERSITY · 2020 · $468,423

## Abstract

Project Summary/Abstract
 The combination of doxorubicin (adriamycin) and cyclophosphamide, so-called AC chemotherapy, is
commonly used for the clinical treatment of breast and other cancers. DNA apurinic/apyrimidinic (AP) sites are
a common result of alkylating agents such as nitrogen mustards. The cytotoxicity of alkylating agents can be
enhanced by methoxyamine, which forms a conjugate with AP sites and inhibits repair. We propose a new
reactivity of doxorubicin and related agents in which they form a covalent conjugate with AP sites in DNA, and
this adduct is highly cytotoxic. The following interconnected Aims are proposed to address this hypothesis.
 Project 1 will demonstrate that the anthracycline antitumor agents doxorubicin, mitoxantrone and
pixantrone form covalent conjugates with AP sites in duplex DNA through an imine linkage (Specific Aim 1).
Imines linkages form reversibily; we therefore propose to synthesize new analogues that form more stable
hydrazine, semicarbazone, and oxime covalent linkages to AP sites. Nitrogen mustards cause complex forms
of DNA damage. In order to evaluate the biological processing of nitrogen mustard DNA adduct, we will
synthesize structurally defined oligonucleotides that contain DNA adducts of nor-nitrogen mustard (the active
form of cyclophosphamide) and thioTEPA. This includes N5-substituted formamidopyrimidine-dG adducts
derived from imidazole ring-opened of the cationic N7-alkylated dG (Specific Aim 2). We will also synthesize
complex secondary products from nitrogen mustards cross-links in which one of the cross-linked dGs has
undergone deglycosylation to an AP site. Based on deglycosylation rates, we predict thioTEPA and nor-
nitrogen mustard will lead to a high burden of AP sites compared to other alkylating agents. The extent to
which these alkylating agents form AP sites in DNA in vitro (calf thymus DNA and cultured human breast
cancer cells) and in vivo (rodent models and white blood cells from breast cancer patients undergoing AC
chemotherapy) as well as AP site conjugates of doxorubicin and related agents will be quantified by ion trap
multistage mass spectrometry (Specific Aim 3).
 Project 1 will work closely with the DNA Synthesis Resource Core to provide site-specifically modified
oligonucleotides containing nor-nitrogen mustard and thioTEPA adducts (Aim 1) as well as covalent drug
conjugates of an AP site (Aim 2) to Project 2 for replication, mutagenesis and repair studies, and Project 3 for
structural analysis. Project 2 will examine the cytotoxicity of the anthracyclines and new analogues in
combination with nor-nitrogen mustard or thioTEPA in cultured human breast cancer cell lines.

## Key facts

- **NIH application ID:** 9982810
- **Project number:** 5P01CA160032-28
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Carmelo J Rizzo
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $468,423
- **Award type:** 5
- **Project period:** — → —

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9982810

## Citation

> US National Institutes of Health, RePORTER application 9982810, Project 1: Synthesis, Characterization and Biomonitoring of DNA Containing Site-Specific Chemotherapy-Induced Complex Alkylation Products (5P01CA160032-28). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9982810. Licensed CC0.

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