DNA Modifications As Biomarkers For Precision Medicine In Patients Treated With Alkylating Drugs

Abstract

DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and oftentimes severe side effects in patients undergoing alkylating-drug based therapies. This observation supports the need for the development of biomarkers able to identify patients who effectively respond to certain therapies and recognize those who instead will develop serious side effects. The use of DNA adducts as predictive biomarkers has been proposed by several studies and herein reviewed. This dissertation focuses on the development and application of analytical methods able to comprehensively screen DNA adducts produced by alkylating drugs. The first study of this dissertation evaluates busulfan reactivity with DNA. Busulfan can promptly react with DNA, therefore, taking advantage of our DNA adductomic approach, DNA adducts formed by reacting busulfan with calf-thymus DNA were characterized. Samples collected from 6 patients undergoing busulfan-based chemotherapy prior to allogeneic hematopoietic cell transplantation were analyzed for the presence of busulfan-derived DNA adducts. Among the 15 adducts detected in vitro, 12 were observed in the patient blood confirming the presence of a large profile of DNA adducts in vivo. Two of the detected adducts were structurally confirmed by comparison with synthetic standards and quantified in patients. Similarly, in the second study, an extensive profile of DNA adducts generated by cyclophosphamide was characterized. Cyclophosphamide is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity. Our DNA adductomic method has been optimized and tailored to maximize the detection of cyclophosphamide-derived adducts. Furthermore, the use of 15N-bacterial DNA served as further confirmation for DNA adduct identification and structural elucidation. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with cyclophosphamide. The last study focused on the synthesis of a cyclophosphamide-derived DNA adduct to be used for quantitation and as an internal standard for future studies. Departing from reported synthetic schemes, several different approaches were tested. This study is currently ongoing, but the reaction schemes tested allowed for a better understanding of dGuo-alkylation regioselectivity. Overall, the work described in this thesis set the stage for the evaluation of a relationship between busulfan and cyclophosphamide DNA adducts and therapy outcome to identify DNA adducts to be used to stratify patients and distinguish who will benefit from therapies from those who may experience severe adverse toxic outcomes.