(R3041) DNA Polymerase Kappa Acts as a Barrier to Unrestrained Replication in Glioblastoma

Megan Reed (University of Arkansas for Medical Sciences)| Leena Maddukuri (University of Arkansas for Medical Sciences)| Amit Ketkar (University of Arkansas for Medical Sciences)| Maroof Zafar (University of Arkansas for Medical Sciences)| Justin Leung (University of Arkansas for Medical Sciences)| Analiz Rodriguez (University of Arkansas for Medical Sciences)| Robert Eoff (University of Arkansas for Medical Sciences)

Translesion DNA synthesis (TLS) polymerase kappa (hpol k) is overexpressed in glioblastoma multiforme (GBM) and further enriched in recurrent tumors. Expression of hpol k is a prognostic indicator of shorter survival and poor response to the standard-of-care chemotherapeutic, temozolomide (TMZ). However, the effect of high levels of hpol k on DNA replication programs in glioblastoma remains unclear. We have found that either pharmacological inhibition or genetic ablation of hpol k resulted in an accelerated fork rate and increased origin firing. These effects are specific to glioblastoma, as U2OS and hTERT-RPE cells did not exhibit a change in fork rate following inhibition of hpol k activity. Additionally, POLKKO GBM cells exhibited diminished Chk1 activation and increased genomic instability indicative of a defective replication stress response (RSR). Treatment with hydroxyurea (HU) further exacerbated DNA damage relative to POLKWT cells. Furthermore, we observed an increase in ssDNA gap formation, both globally and at sites of replication, in GBM cells lacking hpol k activity.  From these results, we conclude that heightened hpol k activity may be an adaptive response to the chronically elevated levels of replication stress experienced by GBM cells. The fork slowing action of hpol k prevents gap synthesis, possibly preventing depletion of the RPA pool in GBM cells through Chk1-mediated resolution of replication intermediates and by limiting origin activity. Loss of hpol k activity jeopardized the genomic integrity of GBM cells by allowing unrestrained fork progress and a shift towards a gap synthesis mode of replication. These results provide new insights into tumor-specific changes in replication programs and may carry implications for how modulation of the RSR could be leveraged to sensitize GBM cells to gap-inducing therapies.

Support or Funding Information

This work was supported in part by a National Science Foundation grant (NSF-MCB 1903357) and NIH R01(NIH CA183895) award.