Impact of Translesion DNA Synthesis on Cancer Therapy and Cellular Metabolism

Abstract

DNA damage is a common challenge in eukaryotes, arising from both exogenous and endogenous sources. If left unrepaired, DNA lesions can stall the replisome and disrupt replication. To address these lesions, cells activate various DNA damage response (DDR) pathways throughout the cell cycle. These pathways include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end-joining (NHEJ). DDR mechanisms are predominantly error-free, for example by utilizing a template-switch strategy during replication, which employs the newly synthesized sister chromatid or repriming downstream of the lesion. This approach allows replication to proceed without introducing mutations, thereby preserving genomic stability. Unfortunately, in cancer cells many DDR genes are mutated, rendering them non-functional, forcing the cells to become dependent on different pathways, such as DNA damage tolerance pathways, to maintain viability. One DNA damage tolerance pathway employed by cancer cells is Translesion DNA Synthesis (TLS), in which a group of specialized group of DNA polymerases known as translesion polymerases replace the replicative polymerase to bypass the lesion. The error rate of TLS polymerases is magnitudes higher than that of the high-fidelity replicases, rendering TLS a predominant source of mutagenesis in cells. Translesion insertion and extension polymerases function together in the form of a translesionsome (also known as mutasome) complex. In the first step of TLS, Proliferating Cell Nuclear Antigen (PCNA) is monoubiquitinated, triggering the recruitment of Y-family TLS polymerases, such as REV1 that further acts as a scaffold for the insertion and extension translesion polymerases. The insertion polymerases include POL?, POL ι, and POL ?, which add a nucleotide opposite to the lesion, whereas the extension function is predominantly carried out by polymerase POL ζ. Pol ζ is a heterotetramer composed of the core subunits REV7 and REV3, and two accessory units PolD2 and PolD3 in human (or Pol31 and Pol 32 in yeast). REV1 provides a critical interaction with Pol ζ’s REV7 subunit through its C-terminal 100 residues. Through my thesis research, I have established that JH-RE-06 not only disrupts TLS but also exhibits remarkable synergy with various cancer therapeutic agents, including chemotherapeutics, kinase inhibitors, and chromatin modifiers. This combination approach amplifies the therapeutic effects, leading to a pronounced reduction in cancer cell viability. These findings suggest that JH-RE-06 could be integrated into combination therapies to enhance treatment efficacy, particularly in cancers that rely heavily on TLS for survival and proliferation. The promising results from these studies underscore the potential usage of JH-RE-06 for the development of innovative cancer treatment strategies. By disrupting the error-prone TLS—a fundamental mechanism contributing to treatment resistance, JH-RE-06 could pave the way for more effective and durable therapeutic outcomes, addressing a critical challenge in oncology. A major discovery of my thesis work is that novel combination treatments for prostate cancer with JH-RE-06 sensitize cells to hormone treatment. Prostate cancer is the most common cancer among men, with one in eight men being diagnosed and one in 44 men dying from it. The vast majority of prostate cancer deaths are due to metastatic, castration-resistant prostate cancer (mCRPC). Standard initial systemic treatment for patients with mCRPC include agents that target androgen receptor (AR) signaling. Despite an initial positive response to these AR pathway inhibitors (ARPIs), acquired resistance remains a significant challenge. We show that treatment of AR-positive prostate cancer cells with the frontline ARPI enzalutamide induces DNA replication stress. Such stress is exacerbated by suppression of translesion DNA synthesis (TLS), leading to aberrant accumulation of single-stranded DNA (ssDNA) gaps and persistent DNA damage biomarkers. We further demonstrate that the TLS inhibitor, JH-RE-06, markedly sensitizes AR-positive prostate cancer cells, but not AR negative benign cells to enzalutamide in vitro. Combination therapy with enzalutamide and JH-RE-06 significantly suppresses cancer growth in a syngeneic allograft murine tumor model over vehicle control or individual treatment groups. These findings suggest that AR inhibition broadly triggers DNA replication stress in hormone- sensitive prostate cancer, thereby exposing a unique vulnerability that can be exploited by a TLS- disrupting adjuvant for targeted therapy. In summary, my research highlights the critical interplay between DDR pathways, DNA damage tolerance mechanisms like TLS, and cancer cell survival under therapeutic stress. By disrupting TLS with the small molecule inhibitor JH-RE-06, I have shown that it is possible to exacerbate replication stress induced by standard treatments such as enzalutamide in prostate cancer and cisplatin or kinase inhibitors in other cancer types, thereby sensitizing cancer cells to these therapies. Furthermore, the unexpected link between TLS and ferroptosis reveals an additional layer of complexity in cellular responses to stress and suggests novel therapeutic opportunities for exploiting this relationship. Collectively, these findings underscore the potential of targeting TLS not only to improve the efficacy of existing cancer therapies but also to uncover new therapeutic strategies aimed at overcoming resistance and driving tumor cell death through complementary pathways. These results lay a foundation for further investigation into TLS as a promising target in cancer treatment.