Replication Gap Suppression by Translesion Synthesis Promotes Drug Resistance in hard-to-treat cancers

Abstract public domain (NIH RePORTER)

Project Summary/Abstract Glioblastoma and pancreatic cancers are among the most aggressive and drug-resistant cancers with poor prognosis and low survival rates. As current therapeutic interventions, which consist of surgical resection followed by radiation and chemotherapy, rely on DNA damage effects, there is an urgent need for new therapeutic strategies to improve outcomes for these patients. A key mechanism contributing to drug resistance in cancer is replication gap suppression (RGS), which helps maintain genomic stability by controlling single-stranded DNA (ssDNA) gaps that affect cell fitness during replication stress. Translesion synthesis (TLS), a process involving specialized DNA polymerases that bypass DNA lesions, plays a central role in RGS, allowing cancer cells to continue proliferating despite DNA damage. However, the mechanism remains unknown. The premise of this study is supported by published and preliminary data that shows: 1) a TLS polymerase kappa (Pol κ) is overexpressed in glioblastoma. 2) early doctoral findings show that Pol κ regulates RGS and fork speed in glioblastoma, processes essential for cancer cell survival. 3) my doctoral research has identified two novel roles of Pol κ: a) Pol κ prevents the accumulation of ssDNA gaps in glioma cells by preventing repriming by Primase DNA polymerase (PrimPol). b) Pol κ slows replication fork speed by promoting the fork reversal repair pathway. This research aims to test the central hypothesis that TLS polymerases enable tumor survival by facilitating the continuation of DNA replication without generating ssDNA gaps that limit cell fitness. Inhibiting TLS promotes ssDNA gap formation and enhances the sensitivity of cancers to standard therapeutics that generate extensive DNA damage that blocks the normal replicative polymerases. During the F99 phase, Aim 1a. will investigate whether Pol κ reduces ssDNA gaps produced from PrimPol by filling these gaps. In this approach, I first use polymerase extension assay to determine the role of Pol κ in extending synthetic substrates with ssDNA gaps. Aim1b. will determine the Pol κ’s mechanism in fork reversal by using enzymatic activity assays to measure changes in the specificity constant (kcat/KM) for Pol κ-mediated fork reversal. Aim 1c. will determine Pol κ interacting proteins during DNA replication using quantitative proteomics approaches TAP-MS and iPOND-MS. During the K00 phase, I will shift my focus to characterizing the architecture of ssDNA gaps and assessing TLS polymerase activity in pancreatic cancer cells. I will involve nanopore sequencing to determine the positions, numbers, and size of the ssDNA gaps in the context of TLS polymerase inhibition to enhance cancer cell sensitivity to DNA-damaging agents. To complete these aims, I have assembled a mentoring team of leaders in cancer research and drug resistance with expertise in enzymology, proteomics, and genomics approaches. These new approaches, combined with my already strong background in genetics, cell biology, molecular biology, and biochemistry, will allow me to address the most challenging issues facing standard therapeutics in challenging cancers and prepare me to pursue a career as an independent academic investigator.

Public health relevance public domain (NIH RePORTER)

Project Narrative The studies proposed in this application will investigate the molecular mechanism of translesion polymerase kappa (Pol κ) in replication gap suppression (RGS) in glioblastoma, which is one of the key drug resistance mechanisms in cancer (F99 phase), as well as characterizing single- stranded DNA (ssDNA) gaps in pancreatic cancer (K00 phase) that affects tumor cell fitness and cell survival. Completion of the proposed research will provide the rationale for reducing replication gaps and will uncover novel therapeutic targets to improve patient drug response, and overall outcomes and reduce healthcare costs associated with these challenging cancers in the United States.