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after irradiation in the presence or absence of the HSP-90 inhibitor AUY-922 and clonal survival was
assessed. MDA-MB-231 parent cells were irradiated at 2-6 Gy using combinations of AUY-922 and PARP
inhibitor ABT-888 in conjunction with heating (42ºC) for 1 hour before or after irradiation and clonal
survival was assessed. Results: MDA-MB-453 parent and stem-like cells exhibited decreased clonal
survival when subjected to heat following irradiation which was enhanced by inhibiting HSP-90. The effect
of inhibiting HSP-90 with AUY-992 showed no appreciable effect on clonal survival when used with
radiation alone. However, when AUY-992 was combined with radiation followed by heat there was a one
log reduction in clonal survival compared to the non-heated group even at the lowest 2 Gy radiation dose.
Conclusions: Our results indicate that inhibition of HSP-90 enhances the effects of mild heating on
radiosensitization of MDA-MB-453 parent and stem-like cells likely by interfering with the cells ability to
repair sublethal DNA damage seen with 2 Gy radiation, which is clinically relevant. Ongoing studies will
determine the additional role of PARP inhibition on radiosensitivity as well as the treatment response in
the stem-cell rich cell line MDA-MB-231. These results will be presented at the time of the meeting.
(PS1-45) Phosphorylation of Ku results in displacement from DNA ends which plays a role in DNA
double-strand break repair pathway choice. Kyung-Jong Lee; Jingxin Sun; Shu-Chi Wang; Janapriya Saha;
Anthony Davis; and David J. Chen, UT southwestern medical center, Dallas, TX
DNA double-strand breaks (DSBs) are the most lethal lesions in a cell if remain unrepaired. In
mammalian cells, the majority of DSBs are repaired by non-homologous end joining (NHEJ), which is active
at all cell cycle phases. Homologous Recombination (HR) mediated DSB repair can occur in addition to
NHEJ during late S/G2 phase, when a sister chromatid is available to be utilized as a homologous DNA
template. When cells can utilize both NHEJ and HR to repair DSBs, it becomes important for the cell to
properly choose the appropriate DSB repair pathway. One of the current models for pathway choice is
that cell cycle specific chromatin bound proteins, Rif1, PTIP, BRCA1 and 53BP1 interplay to suppress DNA
resection during G1 and enhance HR during S/G2 phases. Another model projects a competition between
NHEJ and HR factors over DNA ends, which is dependent upon DNA end processing/resection. It suggests
that HR factors, MRN/X, CtiP and Exo1 can initiate DNA resection after they displace Ku from DNA ends
by cooperatively producing DNA ends that are not favorable for Ku binding. However, our previous in vitro
data suggest that Ku blocks exo or endonuclease activity of HR factors near DNA ends and dissociation of
bound Ku from DNA ends suggesting there must be a different mechanism to actively displace Ku from
DSB ends. Here we present data that Ku dissociation from DNA ends is mediated by direct phosphorylation
by DNA-PKcs or another PI3K-like kinase in vivo. Purified Ku proteins can be displaced from forked DNA
ends by phosphorylation of active DNA-PKcs, whereas non-phosphorylated Ku cannot. In vivo, Ku are
retained longer at laser-induced DNA damage sites with treatment of DNA-PKcs inhibitor. We, therefore,
suggest that phosphorylation of Ku by one of the PI3K kinases results in active displacement of Ku from
DNA ends, which is the critical step in determining pathway choice, thus allowing DNA end resection to
proceed, favoring HR in S/G2 phases of the cell cycle (Support by NIH and CPRIT).
125 | P a g e
assessed. MDA-MB-231 parent cells were irradiated at 2-6 Gy using combinations of AUY-922 and PARP
inhibitor ABT-888 in conjunction with heating (42ºC) for 1 hour before or after irradiation and clonal
survival was assessed. Results: MDA-MB-453 parent and stem-like cells exhibited decreased clonal
survival when subjected to heat following irradiation which was enhanced by inhibiting HSP-90. The effect
of inhibiting HSP-90 with AUY-992 showed no appreciable effect on clonal survival when used with
radiation alone. However, when AUY-992 was combined with radiation followed by heat there was a one
log reduction in clonal survival compared to the non-heated group even at the lowest 2 Gy radiation dose.
Conclusions: Our results indicate that inhibition of HSP-90 enhances the effects of mild heating on
radiosensitization of MDA-MB-453 parent and stem-like cells likely by interfering with the cells ability to
repair sublethal DNA damage seen with 2 Gy radiation, which is clinically relevant. Ongoing studies will
determine the additional role of PARP inhibition on radiosensitivity as well as the treatment response in
the stem-cell rich cell line MDA-MB-231. These results will be presented at the time of the meeting.
(PS1-45) Phosphorylation of Ku results in displacement from DNA ends which plays a role in DNA
double-strand break repair pathway choice. Kyung-Jong Lee; Jingxin Sun; Shu-Chi Wang; Janapriya Saha;
Anthony Davis; and David J. Chen, UT southwestern medical center, Dallas, TX
DNA double-strand breaks (DSBs) are the most lethal lesions in a cell if remain unrepaired. In
mammalian cells, the majority of DSBs are repaired by non-homologous end joining (NHEJ), which is active
at all cell cycle phases. Homologous Recombination (HR) mediated DSB repair can occur in addition to
NHEJ during late S/G2 phase, when a sister chromatid is available to be utilized as a homologous DNA
template. When cells can utilize both NHEJ and HR to repair DSBs, it becomes important for the cell to
properly choose the appropriate DSB repair pathway. One of the current models for pathway choice is
that cell cycle specific chromatin bound proteins, Rif1, PTIP, BRCA1 and 53BP1 interplay to suppress DNA
resection during G1 and enhance HR during S/G2 phases. Another model projects a competition between
NHEJ and HR factors over DNA ends, which is dependent upon DNA end processing/resection. It suggests
that HR factors, MRN/X, CtiP and Exo1 can initiate DNA resection after they displace Ku from DNA ends
by cooperatively producing DNA ends that are not favorable for Ku binding. However, our previous in vitro
data suggest that Ku blocks exo or endonuclease activity of HR factors near DNA ends and dissociation of
bound Ku from DNA ends suggesting there must be a different mechanism to actively displace Ku from
DSB ends. Here we present data that Ku dissociation from DNA ends is mediated by direct phosphorylation
by DNA-PKcs or another PI3K-like kinase in vivo. Purified Ku proteins can be displaced from forked DNA
ends by phosphorylation of active DNA-PKcs, whereas non-phosphorylated Ku cannot. In vivo, Ku are
retained longer at laser-induced DNA damage sites with treatment of DNA-PKcs inhibitor. We, therefore,
suggest that phosphorylation of Ku by one of the PI3K kinases results in active displacement of Ku from
DNA ends, which is the critical step in determining pathway choice, thus allowing DNA end resection to
proceed, favoring HR in S/G2 phases of the cell cycle (Support by NIH and CPRIT).
125 | P a g e
