Developing high throughput measurement of thiopurine in DNA by mass spectrometry

PROJECT SUMMARY
Thiopurines such as mercaptopurine are S-substituted antimetabolites used as functional analogs to
natural nucleobase precursors. They are highly effective against hematologic malignancies
(leukemia and lymphoma) and autoimmune diseases (inflammatory bowel diseases [IBD]), with an
estimated patient population andgt;350,000 in the US. However, the cytotoxicity of thiopurines depends
on their conversion into 6-thioguanine (TG) nucleotides (TGN), which are incorporated into DNA,
causing cell death by post-replicative DNA mismatch repair. They have narrow therapeutic indexes
with dose-limiting hematopoietic toxicity whereas low-responders are undertreated with standard
dosing. Therefore, there is enormous clinical benefit from preemptively identifying patients at risk of
thiopurine toxicity and individualizing therapy to mitigate it. We propose here a high throughput
method based on matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) for
quantifying the pharmacological endpoint of thiopurines, TG incorporated in chromosomal DNA
(DNA-TG) of white blood cells, and examine its specificity and accuracy in standard and clinical
samples. To test this strategy, we propose three specific aims: Aim #1. Chemical derivatization,
isolation and detection of 6-thioguanine (6-TG) by MALDI-MS. Aim #2. Examination of linear range,
sensitivity, specificity and reproducibility of the method. Aim #3. Validation of high throughput 6-TG
detection with biological samples. With the aim to individualize thiopurine therapy through reducing
adverse reactions and obtaining optimum therapeutic efficacy for patients on thiopurine treatment,
the high throughput technique facilitates continuous monitoring of thiopurine in DNA before and
during the therapy. This highly translational project will likely have high impact on a large number of
patients who are under chemo-treatment or thiopurine therapy for non-malignant conditions (e.g.,
IBD).PROJECT NARRATIVE
Despite wide use as anti-tumor drugs in patients with hematologic malignancies and as
immunosuppressive drugs in inflammatory bowel diseases, thiopurines have a very narrow
therapeutic index with dose-limiting hematopoietic toxicity that cases extensive morbidity and
possible mortality. Building upon compelling data from our preliminary studies, we propose to
develop a novel mass spectrometry-based high throughput method to accurately and rapidly
measure thiopurines incorporated into DNA to preemptively identify patients at risk of toxicity and
individualize therapy to mitigate it.