6 research outputs found
Mechanism of Action Studies of Lomaiviticin A and the Monomeric Lomaiviticin Aglycon. Selective and Potent Activity Toward DNA Double-Strand Break Repair-Deficient Cell Lines
(−)-Lomaiviticin A (<b>1</b>) and the monomeric lomaiviticin
aglycon [aka: (−)-MK7-206, (<b>3</b>)] are cytotoxic
agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate
the cellular responses to these agents and identify synthetic lethal
interactions with specific DNA repair factors. Toward this end, we
first characterized the kinetics of DNA damage by <b>1</b> and <b>3</b> in human chronic myelogenous leukemia (K562) cells. DSBs
are rapidly induced by <b>3</b>, reaching a maximum at 15 min
post addition and are resolved within 4 h. By comparison, DSB production
by <b>1</b> requires 2–4 h to achieve maximal values
and >8 h to achieve resolution. As evidenced by an alkaline comet
unwinding assay, <b>3</b> induces extensive DNA damage, suggesting
that the observed DSBs arise from closely spaced single-strand breaks
(SSBs). Both <b>1</b> and <b>3</b> induce ataxia telangiectasia
mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent
production of phospho-SER139-histone H2AX (γH2AX) and generation
of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of
exposure, which is indicative of activation of nonhomologous end joining
(NHEJ) and homologous recombination (HR) repair. Both compounds also
lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production
of γH2AX at later time points (6 h post addition), which is
indicative of replication stress. <b>3</b> is also shown to
induce apoptosis. In accord with these data, <b>1</b> and <b>3</b> were found to be synthetic lethal with certain mutations
in DNA DSB repair. <b>1</b> potently inhibits the growth of
breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese
hamster lung fibroblast cell line derivative (VC8), and phosphatase
and tensin homologue deleted on chromosome ten- (PTEN-) deficient
human glioblastoma (U251) cell lines, with LC<sub>50</sub> values
of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities
of >11.6 versus the isogenic cell lines transfected with and expressing
functional BRCA2 and PTEN genes. <b>3</b> inhibits the growth
of the same cell lines with LC<sub>50</sub> values of 6.0 ± 0.5
and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2
and PTEN mutants, respectively. These data argue for the evaluation
of these agents as treatments for tumors that are deficient in BRCA2
and PTEN, among other DSB repair factors
Abstract 4319: Lupus antibody-based cancer therapy
A subset of autoantibodies produced by patients with systemic lupus erythematosus (SLE) penetrates into cell nuclei and binds DNA, and we believe that such antibodies may have applications in cancer therapy. We have discovered that the cell-penetrating, nuclear-localizing anti-DNA lupus antibody 3E10 inhibits key steps in DNA single- and double-strand break repair and has potential for development as a targeted therapy for tumors harboring deficiencies in DNA repair. 3E10 preferentially binds DNA substrates with free single-strand tails and interferes with both base excision repair and homology-directed repair (HDR) in vitro, and HDR efficiency is reduced in cells treated with 3E10 as measured in the chromosome-based DR-GFP fluorescent reporter assay. The binding of 3E10 to DNA can be directly visualized under electron microscopy (EM), and EM studies confirmed that 3E10 interferes with RAD51 filament formation, which is a critical step in HDR. The 3E10 single chain variable fragment penetrates into human tumor xenografts in nude mice, and 3E10 sensitizes cancer cells and tumors to DNA-damaging therapy. In addition, 3E10, by itself, is toxic to BRCA2-deficient cancer cells but not to repair-proficient cells, and when combined with a DNA-damaging agent, 3E10 has a very large cytotoxic effect on BRCA2-deficient cancer cells. The synthetically lethal effect of 3E10 on BRCA2-deficient cancer cells is consistent with our finding that 3E10 inhibits DNA repair, and it suggests that 3E10 has potential as a targeted therapy for tumors harboring deficiencies in DNA repair, such as certain breast, ovarian, and prostate cancers. Of note, patients with SLE have lower than expected incidence rates of breast, ovarian, and prostate cancers, and it is tempting to speculate that the circulating cell-penetrating anti-DNA autoantibodies provide patients with SLE some protection against the development of DNA repair-deficient tumors. In summary, our work with the 3E10 antibody has provided proof of principle for the development of a lupus antibody as a cancer therapy and opened up new avenues for exploration into the biology of lupus antibodies