posted on 2022-01-20, 18:06authored byMorwena
J. Solivio, Alessia Stornetta, Julie Gilissen, Peter W. Villalta, Sofie Deschoemaeker, Arne Heyerick, Ludwig Dubois, Silvia Balbo
Many chemotherapeutic drugs exert
their cytotoxicity through the
formation of DNA modifications (adducts), which interfere with DNA
replication, an overactive process in rapidly dividing cancer cells.
Side effects from the therapy are common, however, because these drugs
also affect rapidly dividing noncancerous cells. Hypoxia-activated
prodrugs (HAPs) have been developed to reduce these side effects as
they preferentially activate in hypoxic environments, a hallmark of
solid tumors. CP-506 is a newly developed DNA-alkylating HAP designed
to exert strong activity under hypoxia. The resulting CP-506-DNA adducts
can be used to elucidate the cellular and molecular effects of CP-506
and its selectivity toward hypoxic conditions. In this study, we characterize
the profile of adducts resulting from the reaction of CP-506 and its
metabolites CP-506H and CP-506M with DNA. A total of 39 putative DNA
adducts were detected in vitro using our high-resolution/accurate-mass
(HRAM) liquid chromatography tandem mass spectrometry (LC–MS3) adductomics approach. Validation of these results was achieved
using a novel strategy involving 15N-labeled DNA. A targeted
MS/MS approach was then developed for the detection of the 39 DNA
adducts in five cancer cell lines treated with CP-506 under normoxic
and hypoxic conditions to evaluate the selectivity toward hypoxia.
Out of the 39 DNA adducts initially identified, 15 were detected,
with more adducts observed from the two reactive metabolites and in
cancer cells treated under hypoxia. The presence of these adducts
was then monitored in xenograft mouse models bearing MDA-MB-231, BT-474,
or DMS114 tumors treated with CP-506, and a relative quantitation
strategy was used to compare the adduct levels across samples. Eight
adducts were detected in all xenograft models, and MDA-MB-231 showed
the highest adduct levels. These results suggest that CP-506-DNA adducts
can be used to better understand the mechanism of action and monitor
the efficacy of CP-506 in vivo, as well as highlight a new role of
DNA adductomics in supporting the clinical development of DNA-alkylating
drugs.