HR-mediated DNA Repair Research Articles

First-in-human, phase 1/2 study of GSK4524101, an oral DNApolymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors.

TPS3174 Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0–2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Data presented on behalf of the original authors with their permission. Presented at the American Association for Cancer Research (AACR) Annual Meeting 2024; April 5-10, 2024; San Diego, CA. Final publication number: 9686. Reused with permission. Clinical trial information: NCT06077877 .

Abstract CT169: First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors

Abstract Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0-2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Citation Format: Vivek Samnotra, Veronica Moroz, Luda Shtessel, Mita Kuchimanchi, Patrick Hanafin, Malar Pannirselvam, Aishwarya Bhaskar, Andrew Liu, Haluk Yuzugullu, Minal Barve, David Sommerhalder, Timothy Yap, Michele Sanicola-Nadel. First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT169.

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair.

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.

LCK facilitates DNA damage repair by stabilizing RAD51 and BRCA1 in the nucleus of chemoresistant ovarian cancer

Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success of this therapy in the treatment of HR repair deficient cancers has not translated to HR-proficient cancers. Our studies identify the novel role of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in the regulation of HR repair in endometrioid epithelial ovarian cancer (eEOC) model. We show that DNA damage leads to direct interaction of LCK with the HR repair proteins RAD51 and BRCA1 in a kinase dependent manner RAD51 and BRCA1 stabilization. LCK expression is induced and activated in the nucleus in response to DNA damage insult. Disruption of LCK expression attenuates RAD51, BRCA1, and BRCA2 protein expression by hampering there stability and results in inhibition of HR-mediated DNA repair including suppression of RAD51 foci formation, and augmentation of γH2AX foci formation. In contrast LCK overexpression leads to increased RAD51 and BRCA1 expression with a concomitant increase in HR DNA damage repair. Importantly, attenuation of LCK sensitizes HR-proficient eEOC cells to PARP inhibitor in cells and pre-clinical mouse studies. Collectively, our findings identify a novel therapeutic strategy to expand the utility of PARP targeted therapy in HR proficient ovarian cancer.Graphical

Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability.

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.

The BRCA2 and CDKN1A-interacting protein (BCCIP) stabilizes stalled replication forks and prevents degradation of nascent DNA.

DNA replication stress is characterized by impaired replication fork progression, causing some of the replication forks to collapse and form DNA breaks. It is a primary cause of genomic instability leading to oncogenic transformation. The repair-independent functions of the proteins RAD51 and BRCA2, which are involved in homologous recombination (HR)-mediated DNA repair, are crucial for protecting nascent DNA strands from nuclease-mediated degradation. The BRCA2 and CDKN1A-interacting protein (BCCIP) associates with BRCA2 and RAD51 during HR-mediated DNA repair. Here, we investigated the role of BCCIP during the replication stress response. We find that in the presence of replication stress, BCCIP deficiency increases replication fork stalling and results in DNA double-strand break formation. We show that BCCIP is recruited to stalled replication forks and prevents MRE11 nuclease-mediated degradation of nascent DNA strands.

Impairment of RAD17 Functions by miR-506-3p as a Novel Synthetic Lethal Approach Targeting DNA Repair Pathways in Ovarian Cancer.

Epithelial ovarian cancer (EOC) remains the most lethal gynecological cancer and development of chemo-resistance is a major factor in disease relapse. Homologous recombination (HR) is a critical pathway for DNA double strand break repair and its deficiency is associated to a better response to DNA damage-inducing agents. Strategies to inhibit HR-mediated DNA repair is a clinical need to improve patients’ outcome. MicroRNA (miRNAs) affect most of cellular processes including response to cancer treatment. We previously showed that miR-506-3p targets RAD51, an essential HR component. In this study we demonstrated that: i) another HR component, RAD17, is also a direct target of miR-506-3p and that it is involved in mediating miR-506-3p phenotypic effects; ii) the impairment of miR-506-3p binding to RAD17 3’ UTR reverted the miR-506-3p induced platinum sensitization; iii) miR-506-3p/RAD17 axis reduces the ability of EOC cell to sense DNA damage, abrogates the G2/M cell cycle checkpoint thus delaying the G2/M cell cycle arrest likely allowing the entry into mitosis of heavily DNA-damaged cells with a consequent mitotic catastrophe; iv) RAD17 expression, regulated by miR-506-3p, is synthetically lethal with inhibitors of cell cycle checkpoint kinases Chk1 and Wee1 in platinum resistant cell line. Overall miR-506-3p expression may recapitulate a BRCAness phenotype sensitizing EOC cells to chemotherapy and helping in selecting patients susceptible to DNA damaging drugs in combination with new small molecules targeting DNA-damage repair pathway.

Abstract 5688: RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers

Abstract Genome instability has long been considered the primary driver of most cancer types. A double strand break (DSB) in DNA can have deleterious consequences for a cell, which if not repaired faithfully, can lead to mutations and chromosomal rearrangements, or even cell death. DSBs can be processed by several DNA repair pathways, of which homologous recombination (HR) is the preferred method due to its error-free nature. HR uses an intact homologous DNA sequence as a template for recovering the information lost at the break site. A significant proportion of all cancers, especially triple-negative breast, ovarian pancreatic and prostate cancers, have loss of function alterations affecting genes involved in HR-mediated DNA repair. Alternate repair pathways operate when HR is defective in tumors, but the pathways operative in this context remain a matter of contention. Previous work in vivo in yeast and in vitro systems has established a new role of RNA in DNA repair. Owing to its abundance in the cell and its sequence similarity to parental DNA, we sought to define whether RNA can act as a template for the repair of DSBs in human cells. We developed a novel high throughput assay to test if DNA breaks can be repaired using RNA as an alternative template in mammalian cells. Human cells were transfected with a guide RNA cloned in a Cas9 expression vector to generate a site-specific DSB at the AAVS1 locus, a safe harbour, in the human genome. Furthermore, a donor template in the form of DNA or RNA (homologous to the DSB locus) containing a unique mutational signature was provided at the time of transfection. The unique mutational signature enables us to determine if the donor has been utilized as a template for DNA repair. Using this assay, we demonstrate that cells can use a spliced RNA transcript as a functional template to repair a DSB. We have identified that Rev3L, a key component of the translesion synthesis polymerase Pol Zeta (ζ), has a novel reverse-transcriptase activity in human cells and can help repair the DSB using RNA as a template. Further characterization of this repair pathway and its associated mutational scar will provide new insights into the mutational signatures seen in HR-defective cancers, enabling a better understanding of the DNA repair pathways upregulated in these tumours. The proposed studies could help prioritize novel therapeutic approaches by exploiting synthetic lethality in HR-deficient cancers as well as HR-proficient cancers when used in combinatorial cancer therapy. Citation Format: Manisha Jalan, Juber Patel, Kyrie S Olsen, Sana Ahmed-Seghir, Daniel S Higginson, Jorge S Reis-Filho, Nadeem Riaz, Simon N Powell. RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient 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 5688.

Abstract 3237: Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance

Abstract Background: BRCA1/2 mutant tumors cells are deficient for homologous recombination (HR)-mediated DNA repair and are particularly sensitive to PARP inhibitors (PARPi). PARPi have proved efficacy in breast, ovarian, prostate, and pancreatic cancers, particularly in HR-deficient tumors, while their activity is limited in HR-proficient tumors. However, PARPi resistance is inevitable and therapeutic resistance resulting from restoration of HR repair is a pressing clinical problem. Identifying combination treatments to sensitize tumors cells to PARPi and/or overcome PARPi resistance is critical to expand the benefit of these therapies. The Polo-like kinase 1 (PLK1), a serine threonine kinase, is a master regulator of mitosis, overexpressed in many cancers. PLK1 is also involved in the DNA damage response through the promotion of HR-mediated DNA repair and the recovery from the G2/M checkpoint. PLK1 roles in HR repair suggest that PLK1 inhibition may reverse PARPi resistance. Methods: To test the effect of PLK1 and PARP inhibitors combination, we used onvansertib, a highly selective, ATP-competitor PLK1 inhibitor currently in clinical development and the FDA-approved PARPi olaparib. The antitumor effect of the single and combined drug treatments was tested in 2 BRCA1 mutated high-grade serous ovarian cancer (HGSOC) patient derived (PDX) models resistant to olaparib. Orthotopically PDX transplanted mice were treated for 4 weeks and followed for survival. Results: The combination of onvansertib and olaparib was well tolerated and showed strong anti-tumor activity in both PDX models. The combination significantly increased mice survival in comparison to vehicle, olaparib and onvansertib, and showed that onvansertib can re-sensitize PARPi-resistant tumors to olaparib. Median survival increased by 2.7-fold and 8.1-fold respectively in the 2 PDX models in the combination group versus vehicle and the Kaplan Meyer survival curves of mice treated with the combination showed a statistically survival advantage versus control and single agent treated mice. Pharmacodynamic analyses showed an increase in mitotic, apoptotic and DNA damage markers in tumors treated with the combination versus vehicle. Conclusions: The combination of the PLK1 inhibitor onvansertib and the PARPi olaparib showed potent anti-tumor activity in olaparib-resistant BRAC1 mutant HGSOC PDX models. Additional studies are ongoing to further assess the potential of the combination in BRCA wild-type and mutant ovarian, prostate, pancreatic and breast cancer preclinical models. Citation Format: Michela Chiappa, Federica Guffanti, Alessandra Decio, Alessandro Aliverti, Francesca Ricci, Eugenio Scanziani, Federica Camin, Ilaria Craparotta, Maria Chiara Barbera, Marco Bolis, Maya Ridinger, Giovanna Damia. Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance [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 3237.

205 Targeting Homologous Repair to Overcome Genotoxic Therapy Resistance in Pancreatic Cancer

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is upregulated in PDAC cells. METHODS/STUDY POPULATION: Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Drug targeting of RAD18 was achieved through the use of a USP-7 inhibitor, P5091. Cells with or without stable knockdown of RAD18 were implanted orthotopically in the pancreas of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. RESULTS/ANTICIPATED RESULTS: Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF)=1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by the increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via an HR-GFP reporter assay (p=0.039), and increased tumor growth delay following radiation in vivo (p<0.001). P5091 treatment of both MP2 and PANC-1 resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. P5091 increased radiosensitization, yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was also reduced (p=0.014). DISCUSSION/SIGNIFICANCE: Herein, we show the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. USP7 inhibition successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

FDI-6 and olaparib synergistically inhibit the growth of pancreatic cancer by repressing BUB1, BRCA1 and CDC25A signaling pathways

Inducing homologous recombination (HR) deficiency is a promising strategy to broaden the indication of PARP1/2 inhibitors in pancreatic cancer treatment. In addition to inhibition kinases, repression of the transcriptional function of FOXM1 has been reported to inhibit HR-mediated DNA repair. We found that FOXM1 inhibitor FDI-6 and PARP1/2 inhibitor Olaparib synergistically inhibited the malignant growth of pancreatic cancer cells in vitro and in vivo. The results of bioinformatic analysis and mechanistic study showed that FOXM1 directly interacted with PARP1. Olaparib induced the feedback overexpression of PARP1/2, FOXM1, CDC25A, CCND1, CDK1, CCNA2, CCNB1, CDC25B, BRCA1/2 and Rad51 to promote the acceleration of cell mitosis and recovery of DNA repair, which caused the generation of adaptive resistance. FDI-6 reversed Olaparib-induced adaptive resistance and inhibited cell cycle progression and DNA damage repair by repressing the expression of FOXM1, PARP1/2, BUB1, CDC25A, BRCA1 and other genes-involved in cell cycle control and DNA damage repair. We believe that targeting FOXM1 and PARP1/2 is a promising combination therapy for pancreatic cancer without HR deficiency.

RNF19A-mediated ubiquitination of BARD1 prevents BRCA1/BARD1-dependent homologous recombination

BRCA1-BARD1 heterodimers act in multiple steps during homologous recombination (HR) to ensure the prompt repair of DNA double strand breaks. Dysfunction of the BRCA1 pathway enhances the therapeutic efficiency of poly-(ADP-ribose) polymerase inhibitors (PARPi) in cancers, but the molecular mechanisms underlying this sensitization to PARPi are not fully understood. Here, we show that cancer cell sensitivity to PARPi is promoted by the ring between ring fingers (RBR) protein RNF19A. We demonstrate that RNF19A suppresses HR by ubiquitinating BARD1, which leads to dissociation of BRCA1-BARD1 complex and exposure of a nuclear export sequence in BARD1 that is otherwise masked by BRCA1, resulting in the export of BARD1 to the cytoplasm. We provide evidence that high RNF19A expression in breast cancer compromises HR and increases sensitivity to PARPi. We propose that RNF19A modulates the cancer cell response to PARPi by negatively regulating the BRCA1-BARD1 complex and inhibiting HR-mediated DNA repair.

GLI1-targeting drugs induce replication stress and homologous recombination deficiency and synergize with PARP-targeted therapies in triple negative breast cancer cells

Triple negative breast cancer (TNBC), an aggressive and highly metastatic subtype of breast cancer. Glioma-associated oncogene 1 (GLI1) is a transcription factor and effector of the Hedgehog (Hh) signaling pathway, and is predictive of poor survival for TNBC patients. A nanostring DNA Damage Response (DDR) mRNA panel was used to identify GLI1-induced regulation of DDR genes. Western blots, immunohistochemistry and immunofluorescence were used to evaluate protein expression. Colony assays and mammosphere formation assays were utilized to assess survival of cancer cells. Flow cytometry analyses were employed to evaluate changes in the cell cycle profile, and DNA fiber assays were used to analyze alterations in replication dynamics in TNBC cells. The UALCAN portal and Ensemble programs were used for computational analysis of TCGA data. CompuSyn software was used to calculate combination index (CI) values to assess synergism in drug combination experiments. Inhibition of GLI1 in TNBC cells transcriptionally downregulate expression of FANCD2 and its foci formation, and causes a homologous recombination repair (HR) deficiency. As HR-deficient cancer cells are sensitive to PARP-targeted therapies, we evaluated a combination of the GLI1 inhibitor, GANT61, and a PARP inhibitor (olaparib) in TNBC cells. Combination of GANT61 and olaparib elevated DNA damage levels and these drug combinations caused synergistic lethality to TNBC cells. Aberrantly activated GLI1 regulates HR-mediated DNA repair by transcriptionally regulating FANCD2 to overcome chemotherapy-induced replication stress and DNA damage, and it contributes to resistance of TNBC cells to therapeutics.

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

Oct4 confers stemness and radioresistance to head and neck squamous cell carcinoma by regulating the homologous recombination factors PSMC3IP and RAD54L

Head and neck squamous cell carcinoma (HNSCC) is often being diagnosed at an advanced stage, conferring a poor prognosis. The probability of local tumor control after radiotherapy depends on the eradication of cancer stem cells (CSCs) with activated DNA repair. This study provides evidence that the CSC-related transcription factor Oct4 contributes to HNSCC radioresistance by regulating DNA damage response and the CSC phenotype. Knockdown of Oct4 A isoform reduced self-renewal capacity in HNSCC and led to partial tumor cell radiosensitization caused by transcriptional downregulation of the cell cycle checkpoint kinases CHK1 and WEE1 and homologous recombination (HR) repair genes PSMC3IP and RAD54L. Besides, PARP inhibition with Olaparib selectively radiosensitized Oct4 A knockout, but not wild-type HNSCC cells. This finding links Oct4 A to the HR-mediated DNA repair mechanisms. In turn, knockdown of PSMC3IP and RAD54L reduced the HNSCC self-renewal capacity and clonogenic cell survival after irradiation, suggesting the interplay between DNA repair and the CSC phenotype. Similar to the effect of Oct4 knockdown, overexpression of Oct4 also resulted in significant HNSCC radiosensitization and increased DNA damage, suggesting that Oct4-dependent regulation of DNA repair depends on its fine-tuned expression. In line with this observation, HNSCC patients with high and low nuclear Oct4 expression at the invasive tumor front exhibited better loco-regional tumor control after postoperative radio(chemo)therapy compared to the intermediate expression subgroup. Thus, we found that the Oct4-driven transcriptional program plays a critical role in regulating HNSCC radioresistance, and a combination of radiotherapy with PARP inhibitors may induce synthetic lethality in Oct4-deregulated tumors.

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper-resection.

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

Radio-sensitizing effects of microRNA-27a elevation in lung cancer cells by inhibiting ZEB1 expression and activating DNA damage repair pathway.

MicroRNAs (miRNAs or miRs) exert either as tumor-inhibiting or oncogenic roles in tumorigenesis of lung cancer. In the present study, we identified a novel microRNA (miR)-27a as being involved in the radiosensitivity of lung cancer cells. Therefore, we sought to characterize its potential underlying mechanism in lung cancer cell sensitivity to radiotherapy. To this end, A549 and H460 cells irradiated with 8 Gy irradiation (IR) were used as a cell model. RT-qPCR exhibited that the expression of miR-27a increased, whereas ZEB1 was poorly expressed in A549 and H460 cells exposed to IR. As reflected by dual-luciferase reporter gene assay, miR-27a could target and inversely modulate ZEB1 expression. Gain- and loss-of-function experiments exhibited that miR-27 inhibition promoted proliferation of IR-treated A549 and H460 cells and reduced the sensitivity of A549 and H460 cells to radiotherapy, which was rescued by silencing of ZEB1. Further, miR-27a inhibition disrupted the homologous recombination (HR)-mediated DNA repair, evidenced by reduced ATM, pCHK2 and Rad51 levels. Collectively, miR-27a activates HR-mediated DNA repair by inhibiting ZEB1 expression to enhance the radiosensitivity of lung cancer cells, highlighting a therapeutic target for lung cancer radiosensitivity.

ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that threaten genome integrity. To address DSBs, eukaryotic cells of model organisms have evolved a complex network of cellular pathways that are able to detect DNA damage, activate a checkpoint response to delay cell cycle progression, recruit the proper repair machinery, and resume the cell cycle once the DNA damage is repaired. Cell cycle checkpoints are primarily regulated by the apical kinases ATR and ATM, which are conserved throughout the eukaryotic kingdom. Trypanosoma brucei is a divergent pathogenic protozoan parasite that causes human African trypanosomiasis (HAT), a neglected disease that can be fatal when left untreated. The proper signaling and accuracy of DNA repair is fundamental to T. brucei not only to ensure parasite survival after genotoxic stress but also because DSBs are involved in the process of generating antigenic variations used by this parasite to evade the host immune system. DSBs trigger a strong DNA damage response and efficient repair process in T. brucei, but it is unclear how these processes are coordinated. Here, by knocking down ATR in T. brucei using two different approaches (conditional RNAi and an ATR inhibitor), we show that ATR is required to mediate intra-S and partial G1/S checkpoint responses. ATR is also involved in replication fork stalling, is critical for H2A histone phosphorylation in a small group of cells and is necessary for the recruitment and upregulation of the HR-mediated DNA repair protein RAD51 after ionizing radiation (IR) induces DSBs. In summary, this work shows that apical ATR kinase plays a central role in signal transduction and is critical for orchestrating the DNA damage response in T. brucei.

A KRAS-RAD18-miR-296-3p Regulated Network Mediates Radioresistance in Pancreatic Cancer Through Homologous Recombination

Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is increased upon induction of KRAS mutation and reduced following KRAS suppression. Therefore, we hypothesized targeting RAD18 in KRAS mutant PDAC with its predicted inhibitor, miR-296-3p, will increase radiosensitization. Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins was performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Forced expression of miR-296-3p mimics was performed using lipofectamine transfection. Cells with or without stable knockdown of RAD18 were implanted subcutaneously in the hind limb of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. Doxycycline treatment of KRAS inducible human pancreatic ductal epithelial (HPDE) cells increased both RAD18 protein and RNA expression. In contrast, knockdown of KRAS by siRNA reduced RAD18 protein and RNA expression in PANC-1 and MP2 cell lines (3.5 and 4.2-fold, respectively). Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF) = 1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via a HR-GFP reporter assay (p = 0.039), and increased tumor growth delay following radiation in vivo (p<0.001). Overexpression of RAD18 resulted in increased radiation clonogenic survival (DEF = 0.88). We identified that miR-296-3p has a conserved target sequence complimentary to the 3’UTR region of the RAD18 mRNA. MiR-296-3p expression via qRT-PCR was absent in a panel of 6 KRAS mutant PDAC cell lines and highly expressed in 2 normal pancreatic cell lines. MiR-296-3p mimic transfection of the high RAD18 expressing PANC-1 and MP2 cells resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. Following the transfection of miR-296-3p mimics into PANC-1 or MP2 cells, radiosensitization increased (DEF = 1.15 and 1.14, respectively), yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was reduced (p = 0.014). Herein, we show that KRAS mutation in PDAC positively regulates expression of the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. MiR-296-3p successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

Targeting Mutant PPM1D Sensitizes Diffuse Intrinsic Pontine Glioma Cells to the PARP Inhibitor Olaparib.

Diffuse intrinsic pontine glioma (DIPG) is an invariably fatal brain tumor occurring predominantly in children. Up to 90% of pediatric DIPGs harbor a somatic heterozygous mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3.3 (H3F3A, 65%) or H3.1 (HIST1H3B, 25%). Several studies have also identified recurrent truncating mutations in the gene encoding protein phosphatase 1D, PPM1D, in 9%-23% of DIPGs. Here, we sought to investigate the therapeutic potential of targeting PPM1D, alone or in combination with inhibitors targeting specific components of DNA damage response pathways in patient-derived DIPG cell lines. We found that GSK2830371, an allosteric PPM1D inhibitor, suppressed the proliferation of PPM1D-mutant, but not PPM1D wild-type DIPG cells. We further observed that PPM1D inhibition sensitized PPM1D-mutant DIPG cells to PARP inhibitor (PARPi) treatment. Mechanistically, combined PPM1D and PARP inhibition show synergistic effects on suppressing a p53-dependent RAD51 expression and the formation of RAD51 nuclear foci, possibly leading to impaired homologous recombination (HR)-mediated DNA repair in PPM1D-mutant DIPG cells. Collectively, our findings reveal the potential role of the PPM1D-p53 signaling axis in the regulation of HR-mediated DNA repair and provide preclinical evidence demonstrating that combined inhibition of PPM1D and PARP1/2 may be a promising therapeutic combination for targeting PPM1D-mutant DIPG tumors. IMPLICATIONS: The findings support the use of PARPi in combination with PPM1D inhibition against PPM1D-mutant DIPGs.