PROJECT SUMMARY / ABSTRACT
The DNA in all eukaryotic cells is wrapped around the four core histone proteins H2A, H2B, H3 and H4 to form
nucleoprotein filaments called chromatin. Histones help package the DNA to fit it in the nucleus and regulate
access to the genetic information contained within the DNA. Hence, all aspects of DNA metabolism, including
DNA damage and repair, as well as diseases such as cancer are likely to be affected by chromatin structure.
Improper or inefficient DNA repair is closely linked to cancer formation. Not surprisingly, aberrant chromatin
structure or assembly results in genomic instability, which is characterized by the increased rate of acquisition
of alterations in the genome and is associated with most human cancers. Chromatin structure and function are
regulated by posttranslational histone modifications and sequence variants of the canonical histones present at
specific loci or under certain conditions. Recently, certain mutations in the histone variant H3.3 were shown to
drive specific cancers such as glioblastomas, chondroblastomas and large cell tumor of the bone, primarily in
children and young adults. How H3.3 mutations lead to tumors is unclear, although aberrant transcription due
to altered histone modifications are believed to contribute to carcinogenesis. Our preliminary data strongly
suggests that H3.3 plays a crucial role in promoting homologous recombination (HR) mediated DNA repair,
defects in which are likely to make a strong contribution to carcinogenesis. We find that histone H3.3 is rapidly
recruited to sites of laser induced DNA damage in live human cells, whereas the cancer associated H3.3
mutants are defective in this response. Further, H3.3 knockdown results in accumulation of spontaneous DNA
damage, enhanced sensitivity to DNA damaging agents and poor recruitment of several HR factors to DNA
damage sites. Based on these data, we hypothesize that cells carrying cancer-associated H3.3 mutations
are defective in HR and rely on Non-Homologous End Joining (NHEJ) for DNA Double Strand Break
(DSB) repair. Hence, inhibition of NHEJ in the presence of DSBs should selectively eliminate H3.3
mutant cancer cells while sparing normal cells. We will test our hypothesis using cell biological assays and
a mouse xenograft tumor model along with normal and H3.3 mutant tumor derived cell lines in 3 aims:
Aim 1.) Determine the precise role of histone variant H3.3 in DNA DSB repair.
Aim 2.) Measure the sensitivity of H3.3 mutant cells to NHEJ inhibitors with/without exogenous DSBs.
Aim 3.) Test the efficacy of NHEJ inhibition on H3.3 mutant tumor growth in a mouse xenograft model.
Relevance to public health: The studies proposed here will define the role of H3.3 in DNA DSB repair as well
as the contribution of DNA repair defects associated with H3.3 mutations to childhood cancers. This will enable
a better understanding of the overall contribution of H3.3 in cancer prevention. Moreover, the proposed studies
can potentially lead to the development of targeted therapeutic strategies in the near future for H3.3 mutant
tumors that would spare non-tumor cells.
