POLQ Research Articles

DNA Polymerase Theta Protects Leukemia Cells from Metabolic-Induced DNA Damage

DNA Polymerase Theta Protects Leukemia Cells from Metabolic-Induced DNA Damage

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Identification of RP-6685, an Orally Bioavailable Compound that Inhibits the DNA Polymerase Activity of Polθ.

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.

DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers.Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

Poly(ADP) ribose polymerase promotes DNA polymerase theta-mediated end joining by activation of end resection

The DNA polymerase theta (Polθ)-mediated end joining (TMEJ) pathway for repair of chromosomal double strand breaks (DSBs) is essential in cells deficient in other DSB repair pathways, including hereditary breast cancers defective in homologous recombination. Strand-break activated poly(ADP) ribose polymerase 1 (PARP1) has been implicated in TMEJ, but the modest specificity of existing TMEJ assays means the extent of effect and the mechanism behind it remain unclear. We describe here a series of TMEJ assays with improved specificity and show ablation of PARP activity reduces TMEJ activity 2-4-fold. The reduction in TMEJ is attributable to a reduction in the 5’ to 3’ resection of DSB ends that is essential for engagement of this pathway and is compensated by increased repair by the nonhomologous-end joining pathway. This limited role for PARP activity in TMEJ helps better rationalize the combined employment of inhibitors of PARP and Polθ in cancer therapy.

Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks.

Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.

POLQ suppresses genome instability and alterations inDNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ–/– cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ–/– than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ–/– cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ–/– cells. In conjunction with the expanded telomere lengths of POLQ–/– cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

Synthetic Lethality Targeting Polθ.

Research studies regarding synthetic lethality (SL) in human cells are primarily motivated by the potential of this phenomenon to be an effective, but at the same time, safe to the patient’s anti-cancer chemotherapy. Among the factors that are targets for the induction of the synthetic lethality effect, those involved in DNA repair seem to be the most relevant. Specifically, when mutation in one of the canonical DNA double-strand break (DSB) repair pathways occurs, which is a frequent event in cancer cells, the alternative pathways may be a promising target for the elimination of abnormal cells. Currently, inhibiting RAD52 and/or PARP1 in the tumor cells that are deficient in the canonical repair pathways has been the potential target for inducing the effect of synthetic lethality. Unfortunately, the development of resistance to commonly used PARP1 inhibitors (PARPi) represents the greatest obstacle to working out a successful treatment protocol. DNA polymerase theta (Polθ), encoded by the POLQ gene, plays a key role in an alternative DSB repair pathway—theta-mediated end joining (TMEJ). Thus, it is a promising target in the treatment of tumors harboring deficiencies in homologous recombination repair (HRR), where its inhibition can induce SL. In this review, the authors discuss the current state of knowledge on Polθ as a potential target for synthetic lethality-based anticancer therapies.

Abstract 796: Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers

Abstract DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. While inhibitors of targets in the Non-Homologous End Joining (NHEJ) DNA repair pathway, such as DNA-dependent protein kinase (DNA-PK), are available for clinical use, it remains unclear which cancers are vulnerable to these agents. In a genome-wide CRISPR knockout screen with the DNA-PK inhibitor M3814, we identify loss of POLQ, encoding polymerase theta, and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition, whereas loss of TP53 conferred resistance to DNA-PK inhibition. Inhibition of DNA-PK led to increased DNA double strand break end-resection, increased expression of polymerase theta, and activation of MMEJ repair. Combined DNA-PK inhibition by M3814 and polymerase theta inhibition by novobiocin resulted in synthetic lethality mediated by the accumulation of resected DNA and apoptosis. Significantly, this drug combination efficiently killed TP53-deficient human patient-derived xenografts and the corresponding tumor organoids. Taken together, our results provide a rationale for the combination of an inhibitor of DNA-PK mediated NHEJ and an inhibitor of polymerase theta mediated MMEJ in an anti-cancer trial. If DNA-PK is inhibited, cancers develop a hyper-dependence on MMEJ and an upregulation of DNA double strand break end resection and polymerase theta expression. Similarly, P53-deficiency which confers resistance to DNA-PK inhibition, also leads to a hyper-dependence on MMEJ and an upregulation of polymerase theta expression. Thus, a combination of DNA-PK and polymerase theta inhibitors may provide a precision treatment strategy for TP53-mutant solid tumors, known to account for 50% of newly diagnosed cancers. Citation Format: Jeffrey Patterson-Fortin, Arindam Bose, Wei-Chih Tsai, Carter Grochala, Huy Nguyen, Jia Zhou, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Kelsey McQueen, Geoffrey I. Shapiro, David Kozono, Alan D. D'Andrea. Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 796.

Abstract 1519: ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression

Abstract Aldehyde dehydrogenase (ALDH) is a superfamily of 19 known enzymes that participated in the metabolism of endogenous and exogenous aldehydes. ALDH1A1 is a major subtype contributing to high ALDH activity out of all ALDH isoforms and plays a critical role in the maintenance of cancer stem cells. Our previous studies have shown that ALDH1A1 is able to reduce the sensitivity of BRCA2-/- ovarian cancer cells to PARP inhibitors by enhancing microhomology-mediated end joining (MMEJ). MMEJ uses 5-25 base pair micro homologous sequences to align the broken strands before joining, and this repair pathway requires DNA polymerase θ (Pol θ, encoded by POLQ). Here we show that ALDH1A1 can increase the expression of Pol θ, at both mRNA and protein levels, in ovarian cancer cell lines. ALDH1A1 is reported to catalyze the production of all-trans retinoic acid (ATRA), which can bind to the retinoic acid receptor (RAR), and promote transcription of the downstream target genes that possess retinoic acid response element (RARE) in their promoter regions. Interestingly we identified several RAREs in the promoter region of the POLQ gene. Thus, we hypothesized that ALDH1A1 promotes Pol θ expression via activation of the RAR signaling pathway. We found that ATRA and RAR agonist (CH55) increased but pan RAR antagonist (AGN193109) treatment decreased the expression of POLQ, indicating that the RAR pathway is involved in the transcription of POLQ. We also demonstrated that RARα can bind to the promoter region of Pol Q, activation of RAR signaling by treatment with ATRA or CH55 increased the enrichment of K27 acetylated histone H3 in this promoter region. Moreover, we showed that mutated ALDH1A1 lacking the enzyme activity was unable to enhance POLQ transcription. These data suggest that ALDH1A1 enhances the Pol Q expression by increasing the production of ATRA which further activates the RAR signaling pathway. Citation Format: Kousalya Lavudi, Ananya Banerjee, Shurui Cai, Na Li, Xuetao Bai, Wang Qi-En. ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1519.

OC-0428 Development of DNA polymerase theta inhibitors as tumour specific radiosensitisers

OC-0428 Development of DNA polymerase theta inhibitors as tumour specific radiosensitisers

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends.

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double-stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

Abstract P056: Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance

Abstract To target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight, allosteric inhibitors of the polymerase function of DNA polymerase Theta (Polθ), including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining (TMEJ), without targeting Non-Homologous End Joining. Moreover, we show that exposure to ART558 can elicit DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which are a cause of PARP inhibitor resistance, result in in vitro and in vivo sensitivity to Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells. The inhibition of DNA nucleases that promote end-resection reversed these effects, suggesting that resection via Exo1 or Blm-Dna2 being a cause, at least in part, of the ART558 sensitivity phenotype. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Citation Format: Diana A. Zatreanu, Helen M. R. Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall M. B. Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luned M Badder, Daniela Novo, Eleanor G. Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Katherine Ewings, Andre Nussenzweig, Marcel Tijsterman, Andrew Tutt, Simon J. Boulton, Geoff S. Higgins, Steve Pettitt, Graeme C. M. Smith, Christopher J. Lord. Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P056.

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.

Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair.

The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5′–dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5′-resected substrates by locating and pairing microhomologies present in 3′-overhanging single-stranded tails, cleaving the extraneous 3′-DNA by dNTP-dependent end-processing, before extending the nascent 3′ end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions.

Opposite Roles for ZEB1 and TMEJ in the Regulation of Breast Cancer Genome Stability.

Breast cancer cells frequently acquire mutations in faithful DNA repair genes, as exemplified by BRCA-deficiency. Moreover, overexpression of an inaccurate DNA repair pathway may also be at the origin of the genetic instability arising during the course of cancer progression. The specific gain in expression of POLQ, encoding the error-prone DNA polymerase Theta (POLθ) involved in theta-mediated end joining (TMEJ), is associated with a characteristic mutational signature. To gain insight into the mechanistic regulation of POLQ expression, this review briefly presents recent findings on the regulation of POLQ in the claudin-low breast tumor subtype, specifically expressing transcription factors involved in epithelial-to-mesenchymal transition (EMT) such as ZEB1 and displaying a paucity in genomic abnormality.

DNA Polymerase θ: A Cancer Drug Target with Reverse Transcriptase Activity.

The emergence of precision medicine from the development of Poly (ADP-ribose) polymerase (PARP) inhibitors that preferentially kill cells defective in homologous recombination has sparked wide interest in identifying and characterizing additional DNA repair enzymes that are synthetic lethal with HR factors. DNA polymerase theta (Polθ) is a validated anti-cancer drug target that is synthetic lethal with HR factors and other DNA repair proteins and confers cellular resistance to various genotoxic cancer therapies. Since its initial characterization as a helicase-polymerase fusion protein in 2003, many exciting and unexpected activities of Polθ in microhomology-mediated end-joining (MMEJ) and translesion synthesis (TLS) have been discovered. Here, we provide a short review of Polθ‘s DNA repair activities and its potential as a drug target and highlight a recent report that reveals Polθ as a naturally occurring reverse transcriptase (RT) in mammalian cells.