Non-homologous End-joining Pathway Research Articles

Xrcc5/Ku80 is required for the repair of DNA damage in fully grown meiotically arrested mammalian oocytes

Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging events. Recent studies have shown that DNA double-strand break repair occurs predominantly via the homologous recombination (HR) pathway in small non-growing meiotically arrested oocytes (primordial follicle stage). However, the DNA repair mechanisms employed by fully grown meiotically arrested oocytes (GV-stage) have not been studied in detail. Here we established a conditional knockout mouse model to explore the role of Ku80, a critical component of the nonhomologous end joining (NHEJ) pathway, in the repair of DNA damage in GV oocytes. GV oocytes lacking Ku80 failed to repair etoposide-induced DNA damage, even when only low levels of damage were sustained. This indicates Ku80 is needed to resolve DSBs and that HR cannot compensate for a compromised NHEJ pathway in fully-grown oocytes. When higher levels of DNA damage were induced, a severe delay in M-phase entry was observed in oocytes lacking XRCC5 compared to wild-type oocytes, suggesting that Ku80-dependent repair of DNA damage is important for the timely release of oocytes from prophase I and resumption of meiosis. Ku80 was also found to be critical for chromosome integrity during meiotic maturation following etoposide exposure. These data demonstrate that Ku80, and NHEJ, are vital for quality control in mammalian GV stage oocytes and reveal that DNA repair pathway choice differs in meiotically arrested oocytes according to growth status.

ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.

Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf -/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf -/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.

Non-Homologous End-Joining Pathway Genotypes Significantly Associated with Nasopharyngeal Carcinoma Susceptibility.

Defects in the non-homologous end-joining (NHEJ) DNA repair pathway lead to genomic instability and carcinogenesis. However, the roles of individual NHEJ genes in nasopharyngeal carcinoma (NPC) etiology are not well-understood. The aim of this study was to assess the contribution of NHEJ genotypes, including XRCC4 (rs6869366, rs3734091, rs28360071, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and Ligase4 rs1805388, to NPC risk, with 208 NPC patients and 416 controls. Genotype-phenotype correlations were also investigated by measuring mRNA and protein expression in adjacent normal tissues and assessing the NHEJ repair capacity in blood lymphocytes from 43 NPC patients. The results showed significant differences in the distributions of variant genotypes at XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 between the cases and controls. The variant genotypes of these three polymorphisms were associated with significantly increased NPC risks. NPC patients with the risk genotypes at XRCC6 rs2267437 had significantly reduced expression levels of both mRNA and protein, as well as a lower NHEJ repair capacity, than those with the wild-type genotype. In conclusion, XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 in the NHEJ pathway were associated with NPC susceptibility. XRCC6 rs2267437 can modulate mRNA and protein expression and the NHEJ repair capacity.

Loss of the DNA repair gene RNase H2 and a unique subset of DDR-deficient leiomyosarcomas.

11549 Background: Targeting the DNA damage response (DDR)/Homologous Recombination (HR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS). Loss of RNase H2 decreases DNA repair via the Non-Homologous End-Joining (NHEJ) pathway, leading to increased double stranded breaks, replication stress, and increased cell death. Therefore, we developed an assay to screen for RNase H2 loss in STS patient samples to determine its prevalence and prognostic significance, particularly in LMS patients. Methods: RNASEH2B homozygous deletion (HomDel) calls from TCGA samples were based on the Allele-Specific Copy number Analysis of Tumors (ASCAT2) algorithm. Immunohistochemistry (IHC) of RNase H2 was performed on tissue microarrays (TMAs) of uterine (U-LMS) and soft tissue (ST-LMS) leiomyosarcoma samples from MD Anderson Cancer Center (MDACC) using a selective antibody developed by Repare Therapeutics. RNase H2 loss was defined as < = 10% cells without nuclear IHC staining and scored by experienced pathologists in 2 separate cohorts. Genomic analysis was performed using SNiPDx™, a targeted-NGS assay designed to detect biallelic loss of function in select DDR related genes and whole genome sequencing (WGS). MDACC TMA samples were clinically annotated by retrospective review. Results: Using TCGA data, RNASEH2B HomDels were seen in 6% (5/80) of all LMS cases, with a higher proportion in U-LMS (15%; 4/27) as compared to ST-LMS (2%; 1/53). In a pan-tumor TMA, RNase H2 loss by IHC was found in 3.8% (32/843) of samples overall, and in 10% (9/88) of LMS samples. This proportion of RNase H2 loss was higher in a larger MDACC LMS cohort, where negative staining of RNase H2 was found in 30% (33/110) of U-LMS and 38% (39/102) of ST-LMS cases. 30 MDACC LMS cases (15 U-LMS and 15 ST-LMS) were analyzed for RNASEH2B HomDels by SNiPDx. In U-LMS, RNASEH2B HomDels were detected in 64% (7/11) of RNase H2 IHC loss cases vs. 0% (0/3) in RNase H2 IHC intact cases. No RNASEH2B HomDels were detected in ST-LMS cases (10 IHC loss, 5 intact). The median overall survival (mOS) of MDACC U-LMS patients (n = 109) was 4.3 years. No significant mOS difference was seen in RNase H2 IHC intact cases versus loss (mOS 4.4 v 3.3 years, p = 0.54). In a separate cohort, 45 U-LMS samples were screened by IHC; 22% (10/45) had RNase H2 loss. RNASEH2B HomDels were detected in 70% (7/10) of RNase H2 IHC loss cases using SNiPDx and confirmed using WGS. In contrast, RNASEH2B HomDels were detected in 3% (1/33) of RNase H2 IHC intact cases. In the combined U-LMS cohort with IHC and SNiPDx results (n = 71), the diagnostic accuracy, sensitivity and specificity of RNase H2 IHC for detecting RNASEH2B HomDels was 76%, 93% and 71% respectively. Conclusions: RNase H2 loss via RNASEH 2B HomDels is prevalent in U-LMS and is unique from other DDR pathway alterations. RNase H2 IHC is an effective screening tool and is being developed for future clinical trials targeting DDR in LMS.

Enzalutamide and olaparib synergistically suppress castration-resistant prostate cancer progression by promoting apoptosis through inhibiting nonhomologous end joining pathway.

Recent studies revealed the relationship among homologous recombination repair (HRR), androgen receptor (AR), and poly(adenosine diphosphate-ribose) polymerase (PARP); however, the synergy between anti-androgen enzalutamide (ENZ) and PARP inhibitor olaparib (OLA) remains unclear. Here, we showed that the synergistic effect of ENZ and OLA significantly reduced proliferation and induced apoptosis in AR-positive prostate cancer cell lines. Next-generation sequencing followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed the significant effects of ENZ plus OLA on nonhomologous end joining (NHEJ) and apoptosis pathways. ENZ combined with OLA synergistically inhibited the NHEJ pathway by repressing DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and X-ray repair cross complementing 4 (XRCC4). Moreover, our data showed that ENZ could enhance the response of prostate cancer cells to the combination therapy by reversing the anti-apoptotic effect of OLA through the downregulation of anti-apoptotic gene insulin-like growth factor 1 receptor (IGF1R) and the upregulation of pro-apoptotic gene death-associated protein kinase 1 (DAPK1). Collectively, our results suggested that ENZ combined with OLA can promote prostate cancer cell apoptosis by multiple pathways other than inducing HRR defects, providing evidence for the combined use of ENZ and OLA in prostate cancer regardless of HRR gene mutation status.

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

Polymorphisms in the Nonhomologous End-joining DNA Repair Pathway are Associated with HPV Integration in Cervical Dysplasia.

HPV integration in premalignant lesions and is thought to be an important driver of carcinogenesis. However, it is unclear what factors promote integration. The use of targeted genotyping among women presenting with cervical dysplasia has the potential to be an effective tool in assessing the likelihood of progression to cancer.

DNA Ligase 4 Contributes to Cell Proliferation against DNA-PK Inhibition in MYCN-Amplified Neuroblastoma IMR32 Cells.

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

HR repair pathway plays a crucial role in maintaining neural stem cell fate under irradiation stress.

Environmental stress can cause mutation or genomic instability in stem cells which, in some cases, leads to tumorigenesis. Mechanisms to monitor and eliminate these mutant stem cells remain elusive. Here, using the Drosophila larval brain as a model, we show that X-ray irradiation (IR) at the early larval stage leads to accumulation of nuclear Prospero (Pros), resulting in premature differentiation of neural stem cells (neuroblasts, NBs). Through NB-specific RNAi screenings, we determined that it is the Mre11-Rad50-Nbs1 complex and the homologous recombination (HR) repair pathway, rather than non-homologous end-joining pathway that plays, a dominant role in the maintenance of NBs under IR stress. The DNA damage sensor ATR/mei-41 is shown to act to prevent IR-induced nuclear Pros in a WRNexo-dependent manner. The accumulation of nuclear Pros in NBs under IR stress, leads to NB cell fate termination, rather than resulting in mutant cell proliferation. Our study reveals an emerging mechanism for the HR repair pathway in maintaining neural stem cell fate under irradiation stress.

Radio-resistance of hypoxic tumors: exploring the effects of oxygen and X-ray radiation on non-small lung cancer cell lines

BackgroundSolid tumors are often riddled with hypoxic areas, which develops as a result of high proliferation. Cancer cells willingly adapt and thrive in hypoxia by activating complex changes which contributes to survival and enhanced resistance to treatments, such as photon radiation. Photon radiation primarily relies on oxygen for the production of reactive oxygen species to induce DNA damage. The present in-vitro study aimed at investigating the biochemical responses of hypoxic non-small cell lung cancer (NSCLC) cells, particularly the effects on the DNA damage repair systems contributing to more radioresistant phenotypes and their pro- and anti-oxidant potential, within the first 24 h post-IR.MethodsNSCLC cell lines (H460, A549, Calu-1) were irradiated using varying X-ray doses under normoxia (21% O2) and hypoxia (0.1% O2). The overall cell survival was assessed by clonogenic assays. The extent of irradiation (IR)-induced DNA damage was evaluated by analyzing γ-H2AX foci induction and the altered expression of repair genes involved in non-homologous end joining and homologous recombination pathways. Moreover, cell-altered responses were investigated, including the nuclear and cytosolic hydrogen peroxide (H2O2) production, as well as the associated anti-oxidant potential, in particular some components related to the glutathione system.ResultsAnalysis of clonogenic survival revealed an enhanced radioresistance of the hypoxic NSCLC cells associated with reduced DNA damage and a downregulation of DNA repair genes. Moreover, nuclear H2O2 levels were IR-induced in a dose-dependent manner only under normoxia, and directly correlated with the DNA double-strand breaks. However, the observed nuclear H2O2 reduction in hypoxia appeared to be unaffected by IR, thus highlighting a possible reason for the enhanced radioresistance of the hypoxic NSCLC cells. The cellular antioxidant capacity was upregulated by IR in both oxygen conditions most likely helping to counteract the radiation effect on the cytosolic H2O2.ConclusionsIn conclusion, our data provide insight into the adaptive behavior of radiation-resistant hypoxic NSCLC cells, in particular their DNA repair and oxidative stress responses, which could contribute to lower DNA damage and higher cell survival rates following X-ray exposure. These findings may therefore help to identify potential targets for improving cancer treatment outcomes.

Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

The Influence of Edaphic Factors on DNA Damage and Repair in Wild Wheat Triticum dicoccoides Körn. (Poaceae, Triticeae).

A complex DNA repair network maintains genome integrity and genetic stability. In this study, the influence of edaphic factors on DNA damage and repair in wild wheat Triticum dicoccoides was addressed. Plants inhabiting two abutting microsites with dry terra rossa and humid basalt soils were studied. The relative expression level of seven genes involved in DNA repair pathways-RAD51, BRCA1, LigIV, KU70, MLH1, MSH2, and MRE11-was assessed using quantitative real-time PCR (qPCR). Immunolocalization of RAD51, LigIV, γH2AX, RNA Polymerase II, and DNA-RNA hybrid [S9.6] (R-loops) in somatic interphase nuclei and metaphase chromosomes was carried out in parallel. The results showed a lower expression level of genes involved in DNA repair and a higher number of DNA double-strand breaks (DSBs) in interphase nuclei in plants growing in terra rossa soil compared with plants in basalt soil. Further, the number of DSBs and R-loops in metaphase chromosomes was also greater in plants growing on terra rossa soil. Finally, RAD51 and LigIV foci on chromosomes indicate ongoing DSB repair during the M-phase via the Homologous Recombination and Non-Homologous End Joining pathways. Together, these results show the impact of edaphic factors on DNA damage and repair in the wheat genome adapted to contrasting environments.

Abstract 6204: DNA-PK inhibition augment the cancer specific cytotoxicity of mitotic MTH1 inhibitor OXC-101

Abstract DNA-dependent protein kinase (DNA-PK) plays an important role in the repair of DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. In addition to its role in the NHEJ pathway, DNA-PK is also known to have an emerging role in cell cycle progression and mitosis. Some studies have shown that depletion or inhibition of DNA-PK inhibitors leads to a delay in the transition from metaphase to anaphase. The mitotic MTH1 inhibitor OXC-101(TH1579, karonudib) is a dual inhibitor that both targets MTH1 and disrupts tubulin polymerization in cancer cells, causing DNA damage and mitotic catastrophe in cancer cells. Based on the involvement of DNA-PK in mitosis, we hypothesized that depletion/inhibition of DNA-PK might enhance the DNA damage and mitotic catastrophic effect of OXC-101.To test our hypothesis, we performed an in vitro drug combination experiment using the DNA-PK inhibitor nedisertib with OXC-101 in non-small cell lung carcinoma NSCLC (H460,A549), uveal melanoma (MP -38, MP -41 and MP46), osteosarcoma (U2OS) and) and neuroblastoma (IMR32, Kelly, SKNFI) cancer cell lines and non-cancerous foreskin fibroblasts(VH-10, BjhTERT). We found cancer-specific synergistic drug interactions between nedisertib and OXC-101. Combined treatment of nedisertib and OXC-101 to H460 and MP46 increased expression of s10H3 (mitotic marker) and the apoptosis marker (cPARP) compared with single treatment. However, there was no additional increase in expression of the DNA damage markerγH2AX with combined treatment. We also performed annexin V staining by FACS on MP38 andMP48 cells and observed a significant increase in the apoptotic population with combination treatment compared with single treatment. Imaging live cells, we observed a significant delay or arrest in the mitotic phase followed by death of cancer cells in mitosis in response to combination treatment, whereas cells treated individually with nedisertib or OXC-101 continued to proliferate and progress to the next phase of the cell cycle. These data suggest that the DNA-PK inhibitor may enhance the cytotoxic effect of OXC-101specifically in cancer cells. In addition, we will conduct an in-depth mechanistic study of how depletion/inhibition of DNA-PK enhances the cytotoxicity of OXC-101 and validate the drug combination in an appropriate preclinical mouse model. Citation Format: Akhilesh Nagesh Danda, Helge Gad, Martin Scobie, Laura Martínez-López, Nuria Pastor Carrillo, Manuel Luis Orta, Thomas Helleday, Ulrika warpman berglund, Kumar Sanjiv. DNA-PK inhibition augment the cancer specific cytotoxicity of mitotic MTH1 inhibitor OXC-101 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6204.

Abstract 2816: BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways

Abstract Triple-negative breast cancer (TNBC) is the most lethal type of breast cancer (BC). TNBC has a substantial risk of locoregional recurrence and is difficult to cure due to absence of molecular targets or intrinsic heterogeneity. As radiotherapy (RT) is crucial in the treatment of BC, there is a need to identify novel molecular targets for increasing the efficacy of radiation therapy. BUB1 (Budding uninhibited by benzimidazoles 1) is a serine/threonine kinase implicated in chromosomal segregation during mitosis. However, it is still uncertain how BUB1 contributes to radioresistance in TNBC. By performing expression analysis (Oncomine), we observed that BUB1 is overexpressed in BC and even more in TNBC, indicating that this overexpression could be a prognostic factor for TNBC. MTT assay in several TNBC cell lines demonstrated that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. By performing clonogenic survival assays, we observed that BUB1 ablation led to clinically meaningful radiosensitization in TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468) with radiation enhancement ratios ranging from 1.1 - 1.38. In addition, we showed that inhibition of BUB1 kinase function is essential for the radiosensitization phenotype using siRNAs and CRISPR knockouts by depleting endogenous BUB1 and reintroducing wild-type (WT) or kinase-dead (KD) BUB1. We observed significant increase in tumor doubling time in SUM159 xenografts (mammary fat pads in CB17/SCID mice) when BUB1i was combined with RT (DT=29.6 vs 7.9 days). A reduction in Ki67 was observed in these tumors further confirming the effectiveness of combination treatment. The effect of BUB1 depletion on DNA double strand break repair was assessed using γ-H2AX foci formation assay, which revealed sustained increase in γ-H2AX foci following radiation in BUB1i-treated cells. Further, BLRR assays confirmed that BUB1i reduces non-homologous end joining (NHEJ) efficiency. Biochemical analyses showed that BUB1i increases and stabilizes radiation-induced DNAPKcs phosphorylation (S2056). To evaluate the recruitment of DDR proteins to the chromatin following irradiation (IR), soluble nuclear and the chromatin-enriched fractionation was performed which demonstrated that cells pretreated with BUB1i had increased recruitment of DDR proteins to chromatin suggesting that BUB1 may play a role in the initial recruitment of NHEJ proteins to DSBs. Our findings demonstrate that both genetic and pharmacological inhibition of BUB1 sensitizes TNBC cells to RT and that BUB1 mediated radiosensitization may occur through NHEJ pathway. Our results suggest that BUB1 may represent a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2816.

Abstract 6116: Ablation of mitotic checkpoint kinase BUB1 sensitizes lung adenocarcinoma to different classes of chemotherapy, radiation, and chemo-radiation

Abstract Lung cancer causes the most cancer associated deaths worldwide. About 80 percent patients with inoperable non-small cell lung cancer (NSCLC) develop local recurrence and about 60 percent distant metastasis. NSCLC subtype, lung adenocarcinoma (LUAD) accounts for about 40% of all lung cancers. Recently, targeted therapy with chemotherapy combination has shown to enhance the pathologic complete response rate in a subgroup of NSCLC patients, but most patients do not benefit from therapy due to development of resistance. Thus, identifying molecular drivers of primary and adaptive chemotherapy resistance that can be effectively targeted represents a critical unmet clinical need. Here we aim to evaluate BUB1 (a mitotic checkpoint kinase) as a viable molecular target for increasing the effectiveness of radiotherapy and chemotherapy in LUAD. Bioinformatic analyses were conducted to evaluate BUB1 expression across lung cancer and normal tissues. BUB1 was found to be overexpressed in LUAD compared to normal tissues and BUB1 expression also correlated with cancer stage and tumor grade. LUAD cell lines A549, H1975 and H2030 were used to study the effect of BUB1 inhibition on chemo-radiosensitization. MTT cell proliferation and clonogenic cell survival studies were conducted with pharmacological (BAY1816032) and genomic (CRISPR) BUB1 ablation in combination with Platinum (Cisplatin), Taxol (Paclitaxel) or a PARP inhibitor (Olaparib) without/with radiation. BUB1 inhibition showed cytotoxicity in LUAD cell lines at micromolar concentration and enhanced the radiation mediated cell killing at nanomolar concentration. Moreover, BUB1 inhibition sensitized LUAD cell lines to Cisplatin, Paclitaxel and Olaparib in double or triple chemotherapy combinations. Additionally, BUB1 inhibition significantly increased LUAD cell death when combined with different classes of chemotherapies with radiation (chemo-radiation). Our preliminary mechanistic studies revealed prolonged presence of γH2AX foci, an indicator of DNA double stand breaks. BLRR DNA damage reporter assay indicated that the non-homologous end joining (NHEJ) pathway was inhibited upon BUB1 ablation. Detailed mechanistic studies combining BUB1 ablation with chemo-radiation are underway to delineate signaling cascades involved in BUB1 driven chemo-radiation sensitization. Our data provides evidence that BUB1 inhibition sensitizes lung adenocarcinoma to radiotherapy, different classes of chemotherapy and chemo-radiation through DNA-double strand break repair pathways and provides rationale for clinical trials that combine BUB1 inhibition with chemo-radiation in NSCLC. Citation Format: Shivani Thoidingjam, Sushmitha Sriramulu, Farzan Siddiqui, Benjamin Movsas, Shirish Gadgeel, Shyam Nyati. Ablation of mitotic checkpoint kinase BUB1 sensitizes lung adenocarcinoma to different classes of chemotherapy, radiation, and chemo-radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6116.

Signaling Pathways of the Insulin-like Growth Factor Binding Proteins.

The 6 high-affinity insulin-like growth factor binding proteins (IGFBPs) are multifunctional proteins that modulate cell signaling through multiple pathways. Their canonical function at the cellular level is to impede access of insulin-like growth factor (IGF)-1 and IGF-2 to their principal receptor IGF1R, but IGFBPs can also inhibit, or sometimes enhance, IGF1R signaling either through their own post-translational modifications, such as phosphorylation or limited proteolysis, or by their interactions with other regulatory proteins. Beyond the regulation of IGF1R activity, IGFBPs have been shown to modulate cell survival, migration, metabolism, and other functions through mechanisms that do not appear to involve the IGF-IGF1R system. This is achieved by interacting directly or functionally with integrins, transforming growth factor β family receptors, and other cell-surface proteins as well as intracellular ligands that are intermediates in a wide range of pathways. Within the nucleus, IGFBPs can regulate the diverse range of functions of class II nuclear hormone receptors and have roles in both cell senescence and DNA damage repair by the nonhomologous end-joining pathway, thus potentially modifying the efficacy of certain cancer therapeutics. They also modulate some immune functions and may have a role in autoimmune conditions such as rheumatoid arthritis. IGFBPs have been proposed as attractive therapeutic targets, but their ubiquity in the circulation and at the cellular level raises many challenges. By understanding the diversity of regulatory pathways with which IGFBPs interact, there may still be therapeutic opportunities based on modulation of IGFBP-dependent signaling.

APTX acts in DNA double-strand break repair in a manner distinct from XRCC4.

Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear. Here, we generated APTX knock-out (APTX-/-) cell from human osteosarcoma U2OS through CRISPR/Cas9-mediated genome editing system. APTX-/- cells exhibited increased sensitivity toward ionizing radiation (IR) and Camptothecin in association with retarded DSBR, as shown by increased number of retained γH2AX foci. However, the number of retained 53BP1 foci in APTX-/- cell was not discernibly different from wild-type cells, in stark contrast to XRCC4-depleted cells. The recruitment of GFP-tagged APTX (GFP-APTX) to the DNA damage sites was examined by laser micro-irradiation and live-cell imaging analysis using confocal microscope. The accumulation of GFP-APTX on the laser track was attenuated by siRNA-mediated depletion of XRCC1, but not XRCC4. Moreover, the deprivation of APTX and XRCC4 displayed additive inhibitory effects on DSBR after IR exposure and end joining of GFP reporter. These findings collectively suggest that APTX acts in DSBR in a manner distinct from XRCC4.

LINP1 represses unfolded protein response by directly inhibiting eIF2α phosphorylation to promote cutaneous squamous cell carcinoma

BackgroundEndoplasmic reticulum stress (ER stress) may destroy endoplasmic reticulum homeostasis (ER homeostasis) and leads to programmable cell death. Unfolded protein response (UPR) originally stimulated by ER stress is critical for the survival of tumor cells through trying to re-establish ER homeostasis as an adaption to harsh microenvironment. However, mechanisms involving key regulators in modulating UPR remain underexplored.MethodsThe expression of LINP1 in cutaneous squamous cell carcinoma (cSCC) tissues and cell lines was assessed. Subsequently, LINP1 was knocked out, knocked down or overexpressed in cSCC cells. CCK-8 assays, colony forming assays, transwell migration assays and invasiveness measurement by matrigel-coated transwell were performed to examine the role of LINP1 in cSCC development through gain-of-function and loss-of-function experiments. Transcriptomic sequencing (RNA-Seq) was conducted and indicated the key downstream signaling events regulated by LINP1 including UPR and apoptosis signaling. Furthermore, the direct interaction between LINP1 and eIF2α to modulate UPR and apoptosis was confirmed by RNA pulldown, RNA immunoprecipitation (RIP), ChIP-qPCR and in vitro phosphorylation assays.ResultsIn this study, LncRNA in non-homologous end joining pathway 1 (LINP1) was identified to be one of the top ten highest-expressed LncRNAs in cSCC, the second most common cancer in the world. Functional studies using in vitro and in vivo models revealed that LINP1 functions as an oncogene to promote cell proliferation, colony formation, migration and invasiveness while inhibiting cell apoptosis in cSCC. Transcriptomic sequencing after knockdown of LINP1 indicated LINP1 negatively regulates UPR-related pathways involving key effectors for activating UPR and the apoptosis following the prolonged UPR. Mechanistic study showed LINP1 physically interacts with eIF2α to inhibit its phosphorylation for avoiding unmitigated UPR. Loss of LINP1 followed by enhanced eIF2α phosphorylation led to overactivated UPR and induced DDIT3 expression, contributing to ER stress-induced apoptosis and suppression of cSCC development.ConclusionsOur findings demonstrate a novel regulatory hierarchy of UPR by demonstrating LINP1 as a critical modulator for eIF2α phosphorylation and a suppressor of UPR-mediated apoptosis, which suggests a novel therapeutic target for cSCC treatment.

M6A-mediated nonhomologous end joining (NHEJ) pathway regulates senescence in Brachionus plicatilis (Rotifera)

Epigenetic modifications play an important role in the regulation of senescence. N6-methyladenosine (m6A) is the most abundant modification of mRNA. However, the impact of m6A on senescence remains largely unknown at the animal individual level. Standard model organisms Caenorhabditis elegans and Drosophila melanogaster lack many gene homologs of vertebrate m6A system that are present in other invertebrates. In this study, we employed a small aquatic invertebrate Brachionus plicatilis which has been used in aging studies for nearly 100 years to study how m6A affects aging. Phylogenetic analysis confirmed that rotifers’ m6A pathway has a conserved methyltransferase complex but no demethylases and the m6A reading system was more akin to that of vertebrates than that of D. melanogaster. m6A methyltransferases are highly expressed during development but reduces dramatically during aging. Knockdown of METTL3 results in decreased fecundity and premature senescence of rotifers. Furthermore, RT-qPCR analysis indicates a role for m6A in the nonhomologous end joining (NHEJ) pathway of DNA double-strand breaks (DSBs) repair. Altogether, our work reveals a senescence regulatory model for the rotifer METTL3-m6A-NHEJ pathway.