Under-replicated DNA Research Articles

Stabilization of expandable DNA repeats by the replication factor Mcm10 promotes cell viability

Trinucleotide repeats, including Friedreich’s ataxia (GAA)n repeats, become pathogenic upon expansions during DNA replication and repair. Here, we show that deficiency of the essential replisome component Mcm10 dramatically elevates (GAA)n repeat instability in a budding yeast model by loss of proper CMG helicase interaction. Supporting this conclusion, live-cell microscopy experiments reveal increased replication fork stalling at the repeat in mcm10-1 cells. Unexpectedly, the viability of strains containing a single (GAA)100 repeat at an essential chromosomal location strongly depends on Mcm10 function and cellular RPA levels. This coincides with Rad9 checkpoint activation, which promotes cell viability, but initiates repeat expansions via DNA synthesis by polymerase δ. When repair is inefficient, such as in the case of RPA depletion, breakage of under-replicated repetitive DNA can occur during G2/M, leading to loss of essential genes and cell death. We hypothesize that the CMG-Mcm10 interaction promotes replication through hard-to-replicate regions, assuring genome stability and cell survival.

53BP1-the ‘Pandora’s box’ of genome integrity

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

TTF2 promotes replisome eviction from stalled forks in mitosis.

When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.

Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity1–4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5–8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

Abstract B020: SF3B1 hotspot mutations confer sensitivity to PARP inhibition through a defective replication stress response

Abstract SF3B1 hotspot mutations are highly prevalent in several tumor types, where they lead to a global disruption of canonical splicing and are associated with a poor clinical outcome. Whilst evidence suggests that spliceosomal inhibitors have a therapeutic benefit for specific SF3B1 mutant cancers, recent trials have failed to report any clinical responses. Here we used a myriad of in vitro and in vivo techniques to identify and explore a synthetic lethal vulnerability in SF3B1 mutant cancers. These included a high throughput drug screen with an in-house curated library of 80 small-molecule inhibitors in an isogenic cell line model with the most prevalent SF3B1 mutation, a resistance CRISPR knockout screen and a range of DNA fibre analyses to gain mechanistic insights into the observed sensitivity. We identified that SF3B1 mutant cells are selectively sensitive to multiple PARP inhibitors (PARPi), which validated in numerous cell lines and patient derived models in vitro and in vivo, whereby they prevent metastasis. SF3B1 selective PARPi sensitivity occurs via a previously undescribed mechanism, as a result of excessive dormant origin firing and altered fork dynamics, due to the loss of the canonical replication stress response (pCHK1, pRPA, 53BP1), when SF3B1 mutant cells are treated with PARPi. This was further substantiated by the lack of MUS81 recruitment to under-replicated DNA intermediates, leading to the accumulation of DNA damage and cell cycle stalling, via the induction of the G2/M checkpoint. These findings demonstrate that SF3B1 mutant cells have wild-type-like replication under normal growth conditions, but fail to safeguard replication when exposed to PARPi. High content proteomic profiling identified that the defective ATR response is a direct consequence of the downregulation of CINP in SF3B1 mutant cells, which was confirmed in multiple isogenic models and in primary tumors. The defective ATR response was rescued with CINP re-expression and phenocopied through CINP silencing. In SF3B1 mutant cells G2/M checkpoint activation under PARP inhibition can be further abrogated by the addition of ATM inhibitors, but not by the inhibition of ATR or Chk1. Our discovery defines a unique mechanism underpinning PARPi sensitivity in SF3B1 mutant cancers. This synthetic-lethal relationship is further validated in a Phase 1 clinical trial, which shows that those patients with SF3B1 mutant relapsed leukemia have the longest progression free survival when treated with single agent PARPi. This novel synthetic lethality extends the utility of PARPi beyond BRCA1/2 mutant cancers and introduces a new therapeutic approach ready for proof-of-concept clinical trials. Citation Format: Philip Bland, Harry Saville, Patty T Wai, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Holly E Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Patrycja Gazinska, Helen N Pemberton, Aditi Gulati, Naomi Guppy, Ioannis Roxanis, Guy Pratt, Ceri Oldreive, Tatjana Stankovic, Samantha Barlow, Helen Kalirai, Sarah E Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti S Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher J Lord, Rachael Natrajan. SF3B1 hotspot mutations confer sensitivity to PARP inhibition through a defective replication stress response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B020.

MutSβ protects common fragile sites by facilitating homology-directed repair at DNA double-strand breaks with secondary structures.

Common fragile sites (CFSs) are regions prone to chromosomal rearrangements, thereby contributing to tumorigenesis. Under replication stress (RS), CFSs often harbor under-replicated DNA regions at the onset of mitosis, triggering homology-directed repair known as mitotic DNA synthesis (MiDAS) to complete DNA replication. In this study, we identified an important role of DNA mismatch repair protein MutSβ (MSH2/MSH3) in facilitating MiDAS and maintaining CFS stability. Specifically, we demonstrated that MutSβ is required for the increased mitotic recombination induced by RS or FANCM loss at CFS-derived AT-rich and structure-prone sequences (CFS-ATs). We also found that MSH3 exhibits synthetic lethality with FANCM. Mechanistically, MutSβ is required for homologous recombination (HR) especially when DNA double-strand break (DSB) ends contain secondary structures. We also showed that upon RS, MutSβ is recruited to Flex1, a specific CFS-AT, in a PCNA-dependent but MUS81-independent manner. Furthermore, MutSβ interacts with RAD52 and promotes RAD52 recruitment to Flex1 following MUS81-dependent fork cleavage. RAD52, in turn, recruits XPF/ERCC1 to remove DNA secondary structures at DSB ends, enabling HR/break-induced replication (BIR) at CFS-ATs. We propose that the specific requirement of MutSβ in processing DNA secondary structures at CFS-ATs underlies its crucial role in promoting MiDAS and maintaining CFS integrity.

Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress in Adult T-Cell Leukemia/Lymphoma

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a disease model to study EP300 deficiency and an EP300-selective PROTAC degrader, as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, transmission of unrepaired inherited DNA lesions in the subsequent G1-phase and accumulation of cytosolic DNA in EP300-deficient cells. However, EP300 deficient cells show a significant defect in innate immune system activation. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress, persistent genomic instability and innate immune system evasion underlie aggressive chemo-resistant tumorigenesis in humans.

Mitotic DNA synthesis in response to replication stress requires the sequential action of DNA polymerases zeta and delta in human cells

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct ‘unscheduled’ DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.

GEN1 promotes common fragile site expression.

Our genomes harbor conserved DNA sequences, known as common fragile sites (CFSs), that are difficult to replicate and correspond to regions of genome instability. Following replication stress, CFS loci give rise to breaks or gaps (termed CFS expression) where under-replicated DNA subsequently undergoes mitotic DNA synthesis (MiDAS). We show that loss of the structure-selective endonuclease GEN1 reduces CFS expression, leading to defects in MiDAS, ultrafine anaphase bridge formation, and DNA damage in the ensuing cell cycle due to aberrant chromosome segregation. GEN1 knockout cells also exhibit an elevated frequency of bichromatid constrictions consistent with the presence of unresolved regions of under-replicated DNA. Previously, the role of GEN1 was thought to be restricted to the nucleolytic resolution of recombination intermediates. However, its ability to cleave under-replicated DNA at CFS loci indicates that GEN1 plays a dual role resolving both DNA replication and recombination intermediates before chromosome segregation.

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

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.

POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps

POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5′ splice site (5′SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5ʹSS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

Conflicts with transcription make early replication late

By sequencing sites of mitotic DNA synthesis in cells lacking homologous recombination, Groelly, Bhowmick, and colleagues show how conflicts between transcription and replication in early S phase can cause under-replicated DNA to persist into mitosis.

Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

WEE1 inhibition augments CDC7 (DDK) inhibitor-induced cell death in Ewing sarcoma by forcing premature mitotic entry and mitotic catastrophe.

Ewing sarcoma is an aggressive childhood cancer for which treatment options remain limited and toxic. There is an urgent need for the identification of novel therapeutic strategies. Our group has recently shown that Ewing cells rely on the S-phase kinase CDC7 (DDK) to maintain replication rates and cell viability and that DDK inhibition causes an increase in the phosphorylation of CDK1 and a significant delay in mitotic entry. Here, we expand on our previous findings and show that DDK inhibitor-induced mitotic entry delay is dependent upon WEE1 kinase. Specifically, WEE1 phosphorylates CDK1 and prevents mitotic entry upon DDK inhibition due to the presence of under-replicated DNA, potentially limiting the cytotoxic effects of DDK inhibition. To overcome this, we combined the inhibition of DDK with the inhibition of WEE1 and found that this results in elevated levels of premature mitotic entry, mitotic catastrophe, and apoptosis. Importantly, we have found that DDK and WEE1 inhibitors display a synergistic relationship with regards to reducing cell viability of Ewing sarcoma cells. Interestingly, the cytotoxic nature of this combination can be suppressed by the inhibition of CDK1 or microtubule polymerization, indicating that mitotic progression is required to elicit the cytotoxic effects. This is the first study to display the potential of utilizing the combined inhibition of DDK and WEE1 for the treatment of cancer. We believe this will offer a potential therapeutic strategy for the treatment of Ewing sarcoma as well as other tumor types that display sensitivity to DDK inhibitors.

Cockayne syndrome group B protein uses its DNA translocase activity to promote mitotic DNA synthesis

Mitotic DNA synthesis, also known as MiDAS, has been suggested to be a form of RAD52-dependent break-induced replication (BIR) that repairs under-replicated DNA regions of the genome in mitosis prior to chromosome segregation. Cockayne syndrome group B (CSB) protein, a chromatin remodeler of the SNF2 family, has been implicated in RAD52-dependent BIR repair of stalled replication forks. However, whether CSB plays a role in MiDAS has not been characterized. Here, we report that CSB functions epistatically with RAD52 to promote MiDAS at common fragile sites in response to replication stress, and prevents genomic instability associated with defects in MiDAS. We show that CSB is dependent upon the conserved phenylalanine at position 796 (F796), which lies in the recently-reported pulling pin that is required for CSB’s translocase activity, to mediate MiDAS, suggesting that CSB uses its DNA translocase activity to promote MiDAS. Structural analysis reveals that CSB shares with a subset of SNF2 family proteins a translocase regulatory region (TRR), which is important for CSB’s function in MiDAS. We further demonstrate that phosphorylation of S1013 in the TRR regulates the function of CSB in MiDAS and restart of stalled forks but not in fork degradation in BRCA2-deficient cells and UV repair. Taken together, these results suggest that the DNA translocase activity of CSB in vivo is likely to be highly regulated by post-translational modification in a context-specific manner.

Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome

In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor. During unperturbed growth, a basal Chk1 activity level is sustained throughout S phase and relies on replication origin firing. Incremental RS triggers stepwise Chk1 over-activation that delays S-phase, suggesting a rheostat-like role for DRC coupled with the replication machinery. Upon RS, Chk1 is inactivated as DNA replication terminates but surprisingly is reactivated in a subset of G2 cells, which relies on Cdk1/2 and Plk1 and prevents mitotic entry. Cells can override active Chk1 signaling and reach mitosis onset, revealing checkpoint adaptation. Cell division following Chk1 reactivation in G2 results in a p53/p21-dependent G1 arrest, eliminating the daughter cells from proliferation.

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

STEM-22. TARGETING REPLICATION STRESS RESPONSE FOR GLIOMA STEM CELL SPECIFIC CYTOTOXICITY

Abstract BACKGROUND Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability. RESULTS Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo. CONCLUSION Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.