Defining the ability of HPV oncogenes to promote mutagenesis - HPV infections must persist for years to decades to cause cervical cancer. During this time, the host genome acquires
mutations that promote tumorigenesis. Do HPV oncogenes (HPV E6 and E7) play an active (causing mutations by
disrupting DNA repair) and/or a passive (via degradation of tumor suppressors like p53 and RB) role in the acquisition of
these mutations? While it is established that HPV oncogenes degrade tumor suppressors, there is evidence that they
may also actively promote genome destabilization. During the viral life cycle, HPV E6 and E7 induce a hyperactivation of
DNA damage signaling and recruit repair factors to sites of viral replication. Our group and others have shown that HPV
oncogenes also change how cells repair double stranded breaks in DNA (DSBs). Our published data and data presented
in this proposal provide further details that show that HPV oncogenes shift DSB repair from error free mechanisms
(homologous recombination or HR) to error prone (microhomology-mediated end joining or MMEJ) mechanisms. These
data suggest that HPV E6 and E7 makes DSB repair more mutagenic, but this has yet to be demonstrated. Other
substantial questions also remain. How do HPV oncogenes promote the shift towards repair by MMEJ? Do HPV
oncogenes promote canonical MMEJ or does the switch to MMEJ result after HPV oncogenes block completion of HR? If
HPV makes DSB repair more mutagenic, is the increase universal or does the presence of non-allelic homology (via
repetitive Alu elements) augment HPV-mediated mutagenesis? Similarly, if HPV oncogenes make DSB repair mutagenic
do they contribute equally to the process? Aim 1 will address mechanistic question regarding how HPV oncogenes
promote the use of MMEJ and how the pathway is initiated, using a combination of live and fixed cell microscopy. Aim 2
will define the extent that HPV oncogenes make DSB more mutagenic and the extent that their mutagenic properties are
further enhanced by the presence of Alu elements. Aim 2 will use a novel reporter construct and droplet digital PCR,
along with deep sequencing approaches and novel bioinformatic pipelines to accomplish these goals. These approaches
have been pioneered by our research team. These innovative approaches will facilitate an improved understanding of
the fundamental processes that drive cervical cancer development. Given that cervical cancers are the 4th most common
cancer in women and kill someone every 90 seconds, this information is important. The knowledge gaps addressed in
this proposal are also relevant to understanding why most HPV infections do not result in cervical cancer and for
developing methods to prevent those that do. Finally, given that cervical cancers are often treated with genotoxic
agents (e.g., cisplatin or radiotherapy), understanding how HPV changes DNA repair could lead to the identification of
ways to augment these interventions. For example, if HPV oncogenes promote MMEJ via a delayed entry into the
pathway, small molecule inhibitors of proteins involved in terminal MMEJ steps would likely result in a tumor-specific
enhancement of radiotoxicity.
