A novel class of chemicals that react with abasic sites in DNA and specifically kill B cell cancers

Abstract

Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing

“…The quantification of uracils in DNA was done using AA6 (32,73). Briefly, genomic DNA was digested with HaeIII and treated with O-allylhydroxylamine (10 mM; Sigma) to block the endogenous abasic sites.…”

A Tumor-Promoting Phorbol Ester Causes a Large Increase in APOBEC3A Expression and a Moderate Increase in APOBEC3B Expression in a Normal Human Keratinocyte Cell Line without Increasing Genomic Uracils

“…More recently, we also described another alkoxyamine, AA6, which also reacts with abasic sites and can be attached with a fluorescent tag using copper-free click chemistry ( Fig. 5A) (43). We used AA6 for the quantification of uracils in NOK genome using a genomic DNA standard in which the uracils were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).…”

“…The uracils in the genomic DNA were quantified using an assay based on AA6, an alkoxyamine that can be coupled with a fluorescent dye using copper-free click chemistry (43). This assay is a modification of previously described assay using an alkoxyamine called AA3 (41).…”

Detection of Genomic Uracil Patterns

“…The Ung-ARP assay was developed in Bennett’s group, where specific enzymatic removal of the uracil and detection of the resultant AP sites by biotinylated ARP reagent were combined [ 81 ]. Further developments led to two alkoxyamine reagents, AA3 and AA6, associated with increased reactivity and functional groups, appropriate to conjugate with a wide variety of biochemical labels by click chemistry [ 82 , 83 ]. These reagents were used in different applications [ 84 , 85 , 86 ].…”