KML001 targets telomeres in prostatecancer cells
19thEORTC-NCI-AACR San Francisco, CA
Abstract
KML001 (sodium meta arsenite, Kominox) is an orally bioavailable, trivalent arsenic compound that has entered phase I/II
clinical trials in prostate cancer. Arsenicals have been described to cause chromosome abnormalities as a result of the
generation of reactive oxygen species (ROS) in cells. Recent studies however, have shown that trivalent arsenicals can
specifically alter telomere length, telomerase activity and telomere binding proteins, suggesting that the telomere
/telomerase complex might be a direct target of sodium meta arsenite. This study was initiated to investigate whether
KML001 targets telomeres and telomerase in prostate cancer cells. The prostate cancer cell lines PC3 and DU145, which
have relatively short telomeres (2.5 and 4.2 kb, respectively) were chosen for in vitro experiments. KML001 concentrations
needed to inhibit cell growth to 50% (IC50) and 100% (IC100) were determined by MTT assay. The IC50 for PC3 was found
to be 0.23 µM and for DU145 2 µM, the IC100s were 9 µM and 5 µM, respectively; the IC50s and IC100s were used for all
mechanistic analyses. The TeloTAGGG Quantification Kit was employed to measure mRNA expression of the telomerase
catalytic subunit hTERT; hTERT protein expression, induction of telomere-associated DNA damage by histone H2AX
phosphorylation, and 8-oxodeoxyguanosine formation were studied by immunofluorescence microscopy. Direct binding of
KML001 to 5'-(TTAGGG)x-3' telomericsequences was assessed by high resolution MALDI (Matrix-assisted Laser
Desorption Ionization) mass spectrometry, and by metaphase fluorescence in situhybridization (FISH). KML001 treatment
of prostate cancer cells did down-regulate hTERT mRNA expression after 24 hrs of continuous exposure to drug. hTERT
protein expression in the nucleus was also reduced and a translocation to the cytoplasm was observed, suggesting the
displacement of hTERT from the telomeres. Consistent with these observations, we found that KML001 can bind to
telomeric repeat sequences at a ratio of 1 molecule drug per 3 telomeric repeats and that these lesions result in a rapid
induction of γ-H2AX, a marker of DNA-damage response and telomere-initiated senescence. γ-H2AX phosphorylation (γ-H2AX)
occurred earlier (after 2 hrs) and at lower KML001 (IC50) concentrations compared to the ROS-induced formation of
8- oxodeoxyguanosine, which was not seen prior to 24 hrs and only at the IC100. Moreover, direct telomere binding by
KML001 resulted in a gradual telomere loss with two telomeres per chromosome missing at 48 hrs and a complete telomere
loss seen at 72 hrs in IC100 treated PC3 cells. Our findings indicate that KML001 can specifically target telomeres and
telomerase. The latter might provide biomarkers to assess drug response and should be considered in clinical
trials of KML001 in prostate and other cancers
