Repair Cycle Research Articles

Structural basis for an early stage of the photosystem II repair cycle in Chlamydomonas reinhardtii

Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.

Targeted inhibition of SUMOylation: treatment of tumors.

SUMOylation is a dynamic and reversible post-translational modification (PTM) of proteins involved in the regulation of biological processes such as protein homeostasis, DNA repair and cell cycle in normal and tumor cells. In particular, overexpression of SUMOylation components in tumor cells increases the activity of intracellular SUMOylation, protects target proteins against ubiquitination degradation and activation, promoting tumor cell proliferation and metastasis, providing immune evasion and increasing tolerance to chemotherapy and antitumor drugs. However, with the continuous research on SUMOylation and with the continued development of SUMOylation inhibitors, it has been found that tumor initiation and progression can be inhibited by blocking SUMOylation and/or in combination with drugs. SUMOylation is not a bad target when trying to treat tumor. This review introduces SUMOylation cycle pathway and summarizes the role of SUMOylation in tumor initiation and progression and SUMOylation inhibitors and their functions in tumors and provides a prospective view of SUMOylation as a new therapeutic target for tumors.

Identification of the CsFtsH genes from Camellia sinensis reveals its potential role in leaf color phenotype

The filamentation temperature-sensitive H (FtsH) metalloprotease participates in the chloroplast photosystem II (PSII) repair cycle, playing a crucial role in regulating leaf coloration. However, the evolutionary history and biological function of the FtsH family in albino tea plants are still unknown. In this study, 35 CsFtsH members, including 7 CsFtsH-like (CsFtsHi1-CsFtsHi7) proteins, mapping onto 11 chromosomes in 6 subgroups, were identified in the ‘Shuchazao2′ tea genome, and their exon/intron structure, domain characteristics, collinearity, protein interaction network, and secondary structure were comprehensively analyzed. Furthermore, real-time fluorescence quantitative PCR (RT-qPCR) analysis revealed that the expression levels of CsFtsH1/2/5/8 were significantly positively correlated with the leaf color of tea plants. The subcellular localization revealed that they were located in the chloroplast. The transgenic Arabidopsis has demonstrated that CsFtsH2 and CsFtsH5 could restore the chlorophyll content and chlorophyll fluorescence intensity in var1 and var2 mutants, respectively. Moreover, protein–protein interactions have confirmed that CsFtsH1 with CsFtsH5, and CsFtsH2 with CsFtsH8 could form a hetero-comples and function in chloroplasts. In summary, this study aims to not only increase the understanding of the underlying molecular mechanisms of CsFtsH but also to provide a solid and detailed theoretical foundation for the breeding of albino tea plant varieties.

Deciphering the correlation between Chr3q26.2 amplification through homologous recombination deficiency and improved survival in ovarian cancer under platinum-based adjuvant therapy.

e17537 Background: Discriminant copy number alterations (CNAs) have been linked to aberrant DNA repair mechanisms. This study aimed to identify CNA-based biomarkers, alongside homologous recombination deficiency (HRD) scores for ovarian cancer patients receiving platinum (Pt)-based chemotherapy, especially in BRCA1/2 wild-type or HRD-low patients. Methods: Fourteen most prevalent and significant focal CNA regions (seven amplification and seven deletion) were examined to investigate their correlations with HRD scores via SHAP values of an XGBoost model and the effectiveness of Pt-based adjuvant chemotherapy in a cohort of high-grade serous ovarian cancer (HGSC) patients (TCGA cohort, N=576), and the results were validated in a separate Chinese ovarian cancer cohort (test cohort, N=457). An in-house developed next-generation sequencing HRD pipeline (GeneseeqPrime HRD) was used to determine HRD scores. Histological subtypes, molecular features, and potential molecular mechanisms associated with the identified biomarkers were further explored. Results: Of 14 high-level CNA regions identified by GISTIC in the TCGA cohort, Chromosome 3q26.2 amplification [ Chr3(q26.2) Amp, a prevalence of 30.7%] was associated with increased HRD scores in both BRCA1/2-altered ( p=0.01) and wild-type HGSCs ( p=0.03). Consistently, in the test cohort, higher HRD scores ( p<0.01) and elevated chromosome instability scores ( p<0.01) were observed in Chr3(q26.2) amplified samples. Among 406 patients administrated with Pt-based adjuvant chemotherapy in the TCGA cohort, those with Chr3(q26.2) Amp exhibited a relatively high sensitivity rate ( p=0.09) and significantly superior disease-free survival after adjusting for HRD score, patient age, tumor stage, and residual disease [hazard ratio (HR): 0.65, 95% confidence interval (CI): 0.48–0.88] when compared to those without, representing Chr3(q26.2) Amp as an independent predictive biomarker. Similar results were obtained in 78 patients using Pt-based adjuvant chemotherapy in the test cohort (HR: 0.68, 95% CI: 0.40–1.15). Moreover, Chr3(q26.2) Amp was more prevalent in HGSCs harboring BRCA1/2 or TP53 aberrations than other histological subtypes driven by PIK3CA, PTEN or KRAS mutations ( p<0.01). In comparison to HGSCs without Chr3(q26.2) Amp, Chr3(q26.2) amplified HGSCs exhibited higher Ragnum hypoxia scores ( p<0.01) and a total of four overexpressed proteins, including p62 ( p<0.01), PIK3CA ( p<0.01), CCNB1 ( p<0.01) and TSC1 ( p<0.01), indicative of suppressed homologous recombination repair pathway and cycle cell arrest. Conclusions: Chr3(q26.2) Amp emerges as a promising biomarker for Pt-based adjuvant chemotherapy in ovarian cancer, potentially reflecting a hypoxia-induced etiology through suppressed homologous recombination repair pathway.

Correlation of hormone receptor positive HER2-negative/MammaPrint high-2 breast cancer with triple negative breast cancer: Results from gene expression data from the ISPY2 trial.

573 Background: A subset of Hormone Receptor positive (HR+), HER2 negative (HER2-) breast cancers (BCs) classified by the MammaPrint (MP) assay as High-2 (MP-H2) show clinical behavior similar to triple-negative (TN) BC. The aim of this analysis was to assess molecular similarities between the HR+/HER2-/MP-H2 (HR+/MP-H2), HR+/HER2-/MP-High-1 (HR+/MP-H1), and TN/MP-H2 (TN/MP-H2) BCs. Methods: Gene expression data used for this analysis [GEO GSE194040] was derived from pre-treatment samples of the I-SPY2 trial, which is an ongoing multicentric randomized Phase II neoadjuvant platform trial for high-risk stage II or III BC. Six hundred eighty-four pretreatment samples were included in this analysis: 274 corresponded to HR+/MP-H1, 105 to HR+/MP-H2, and 305 to TN/MP-H2, accounting for 7 experimental plus a control arm. We used principal component and distance analyses, differentially expressed genes (analysis performed for pairwise comparisons of the 3 subtypes, a linear model, log fold-change and moderated-t p-values relative to a minimum fold-change threshold of fc = 1.5), gene set enrichment analysis using KEGG and GO, and Ingenuity Pathway Analysis, as well as 32 previously defined qualifying biomarkers to study similarities in biology between the subgroups. Results: On principal component and distance analyses, results reveal close similarity between HR+/MP-H2 and TN/MP-H2 indicating shared transcriptomic features. Gene expression analysis revealed that HR+/MP-H2 tumors resemble TN/MP-H2 more than HR+/MP-H1 cancers. Differential gene expression analysis revealed that HR+/MP-H2 and TN/MP-H2 cancers presented only two differentially expressed genes, compared to HR+/MP-H1 and HR+/MP-H2, which presented 577 differentially expressed genes. Upon pathway analysis, HR+/MP-H1 cancers presented the most significant upregulation in the glucocorticoid receptor signaling, DHCR24, and HOTAIR pathways, compared to HR+/MP-H2 cancers. Furthermore, HR+/MP-H2 and TN/MP-H2 shared enrichment of DNA repair and cell cycle pathways. In addition, various biomarkers were analyzed, and among the most representative shared biomarkers for HR+/MP-H2 and TN/MP-H2 were immune features, higher proliferation, and a lower expression of ESR1 co-expressed genes as compared to HR+/MP-H1 cancers. The rates of pathologic complete response were 41% for TN/MP-H2, 31% for HR+/MP-H2, and 11% HR+/MP-H1 cancers among all included patients. Conclusions: A subset of HR+ cancers defined by MP-H2 status closely resemble TN in molecular and clinical features. The similarity of pCR rates for MP-2 observed within either HR+/HER2- and TN is supported by our findings of similarity of active biology between HR+/MP-2 and TN/MP-2. These findings have implications for understanding the biology of HR+/MP-H2 BCs and optimizing treatment strategies.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

A novel method for cycle slip detection and restoration of BDS carrier phase

The chief purpose of this present study is to develop a new approach to cycle slip detection and restoration of the BeiDou Navigation Satellite System (BDS) carrier phase. Based on the RAIM algorithm in the application of aviation safety, we extend the application to ensure the precision of the carrier phase. To stand-alone receivers of a low-cost single frequency, carrier phase data is made different between adjacent epochs. Then least-square residuals were to design a RAIM scheme with differential carrier phase data to find out the epoch with cycle slip, and finally repair cycle slip by Chebyshev polynomial. Results indicate that the approach can detect cycle slip effectively and both big cycle slip and small cycle slip could be repaired.

POLB Regulates Proliferation and Apoptosis of Bovine Primary Myocytes.

DNA polymerase β (DNA polymerase beta (POLB)) belongs to a member of the DNA polymerase X family, mainly involved in various biological metabolic processes, such as eukaryotic DNA replication, DNA damage repair, gene recombination, and cell cycle regulation. In this study, the muscle development-related gene POLB was screened by selection signature and RNA-seq analysis and then validated for the proliferation and apoptosis of bovine primary myocytes. It was also found that overexpression of the POLB gene had a pro-apoptosis effect, but interfering with the expression of the gene had no significant effect on cells. Then, the analysis of related apoptotic genes revealed that POLB overexpression affected CASP9 gene expression.

Research on the toxicological prognostic significance of age-related genes in endometrial cancer unveiling key factors in patient prognosis.

This study investigates the influence of aging-related genes on endometrial cancer, a prominent gynecological malignancy with rising incidence and mortality. By analyzing gene expression differences between cancerous and normal endometrial tissues, 42 aging-related genes were identified as differentially expressed. Utilizing the TCGA-UCEC sample, consensus clustering divided the samples into two molecular subgroups, Aging low and Aging high, based on their expression profiles. These subgroups showed distinct prognoses and survival rates, with the Aging high group associated with DNA repair and cell cycle pathways, and the Aging low group showing suppressed metabolic pathways and increased immune cell infiltration, suggesting a potential for better immunotherapy outcomes. Mutation analysis did not find significant differences in mutation frequencies between the groups, but a high Tumor Mutation Burden (TMB) correlated with better prognosis. A risk score model was also developed, showcasing significant prognostic power. Further analysis of the SIX1 gene revealed its overexpression in cancer cells. Drug sensitivity tests indicated that the low-risk group might respond better to chemotherapy. This research underscores the significance of aging-related genes in endometrial cancer, offering insights into their prognostic value and therapeutic potential, which could lead to personalized treatment approaches and enhanced patient management.

Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy

BackgroundLung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies.MethodsTo overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3’RNA sequencing, real-time PCR, flow cytometry and confocal microscopy.ResultsWe showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level.ConclusionsThis study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

Determining the Underlying Mechanisms of Glioma Progression and Recurrence

INTRODUCTION: Gliomas are the most common malignant brain tumour. Despite standard-of-care therapy, these tumours inevitably recur. Recurrent tumours are often treatment-resistant and associated with a poor prognosis, and there is an urgent need for novel treatments. METHODS: The epigenome-wide methylome, transcriptome, and proteome was analyzed in a total of 271 samples from 176 patients with gliomas, including 166 primary (pre-treatment) and 105 recurrent (post-treatment) tumours. Included is a unique cohort of 81 patients with paired tumour samples collected at both pre- and post- treatment. RESULTS: Epigenomics accounted for the majority of observed variance between samples and was driven by IDH status. Paired analyses identified epigenetic stability in IDH wildtype tumours at recurrence, compared to a loss of methylation at recurrence in IDH mutant tumours. Loss of methylation in IDH mutant gliomas at recurrence was driven by astrocytomas undergoing malignant transformation. Integrated, multi-platform analyses of paired samples undergoing malignant transformation identified novel genetic markers and pathways of transformation. Finally, a novel subgroup of IDH wildtype GBM with improved survival and a distinct proteomics signature was discovered. Multi-platform analyses characterized this novel subgroup as exhibiting hypermethylation, increased DNA mismatch repair and cell cycle regulation, and an altered tumour microenvironment. CONCLUSIONS: In conclusion, this study identifies targetable molecular factors that promote malignant transformation in IDH mutant gliomas and classifies a novel subgroup of IDH wildtype GBM with improved prognosis.

Abstract 4232: Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model

Abstract Pancreatic cancer is marked by a dismal prognosis, with a 5-year survival rate at approximately 10%. A key contributor to its lethality is the development of chemotherapy resistance, followed by early metastasis. However, studies elucidating the mechanisms behind chemotherapy resistance in pancreatic cancer are limited. Previous research has relied on 2D cell line models, which present limitations, such as uniform cell types that fail to capture tumor heterogeneity and an inaccurate representation of clinical realities, wherein resistance often develops gradually rather than through continuous exposure to high drug concentrations. In this study, we developed a more sophisticated organoid model of chemotherapy resistance. Six organoids from three patients were matched to study chemotherapy resistance in a personalized context. Comprehensive whole-exome sequencing and transcriptomic analyses were performed, along with an examination of chemotherapy resistance protein expression and EMT-related markers. Notably, differential expression levels of CK19 and Vimentin were observed between samples. Our analysis also focused on the expression of the ATP-Binding Cassette Subfamily G Member 2 (ABCG2) gene in chemoresistant organoids. Western blot analysis indicated a significant increase in ABCG2 expression in these organoids compared to controls. According to the Moffitt RNA signature, the primary and corresponding chemoresistant organoids retained their molecular subtype classifications both before and after developing chemoresistance. Gene set enrichment analysis uncovered significant upregulation of pathways related to DNA repair and cell cycle regulation, particularly the G2M checkpoint, in chemoresistant organoids. Furthermore, gene ontology functional analysis suggested a notable overrepresentation of activities related to amino acid transmembrane transport and extracellular matrix constituents, underscoring their potential role in the chemoresistance phenotype. Citation Format: Hee Seung Lee. Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4232.

Abstract 1115: Enhancing lung cancer sensitivity to chemotherapy, radiation, and chemo-radiation through inhibition of a mitotic checkpoint kinase BUB1

Abstract Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) represent two major subtypes of lung cancer, marked by challenging treatment resistance. Our study explores the inhibition of BUB1, a critical mitotic checkpoint kinase, in conjunction with radiotherapy and chemotherapy. This approach has the potential to overcome resistance, thus enhancing the efficacy of treatment and improving treatment outcomes for lung cancer patients. We analyzed BUB1 expression in lung cancer using TCGA datasets and patient tissue microarrays (TMAs). In vitro experiments used four NSCLC and two SCLC cell lines with BUB1 inhibitor (BAY1816032), Paclitaxel, Cisplatin and Olaparib. Cells were irradiated (2, 4, or 6 Gy), and assays measured proliferation, long-term effects, DNA damage, and repair. Bioinformatic analysis in NSCLC subtype, lung adenocarcinoma (LUAD) demonstrated marked overexpression of BUB1, correlated with cancer stage, tumor grade and survival. Lung cancer TMAs revealed elevated expression of BUB1 protein in SCLC that correlated with poorer survival. BUB1 inhibition (BUB1i) induced cytotoxicity in LUAD cell lines at micromolar concentrations, while nanomolar concentrations enhanced cell death significantly when combined with radiation. Furthermore, BUB1i sensitized LUAD cells to different classes of chemotherapy (Paclitaxel, Cisplatin), and PARP inhibitor (Olaparib) alone (chemo-sensitization) or with radiation therapy (chemo-radiosensitization). H2030 and H1975 demonstrated higher chemo-radiosensitization efficacy compared to A549, likely due to genetic differences, potentially including a p53 mutation that improves sensitivity by affecting DNA repair and cell cycle regulation. Preliminary investigations revealed DNA double-strand breaks, indicated by prolonged γH2AX foci, and the inhibition of the NHEJ DNA damage repair pathway after BUB1 ablation. Among SCLC cell lines, NCI-H1876 demonstrated higher radiosensitivity upon BUB1 ablation compared to NCI-H2198, potentially due to genetic differences/mutations affecting radiation response. The study highlights the potential of BUB1i in enhancing the sensitivity of NSCLC and SCLC cells to radiotherapy and chemotherapy and provide rationale to explore BUB1i as a novel approach to enhance lung cancer therapy in clinical trials. Citation Format: Shivani Thoidingjam, Sushmitha Sriramulu, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Shirish Gadgeel, Shyam Nyati. Enhancing lung cancer sensitivity to chemotherapy, radiation, and chemo-radiation through inhibition of a mitotic checkpoint kinase BUB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1115.

Abstract 6220: TRIP13 augments pancreatic cancer progression- An integrated systems biology study

Abstract Background- Pancreatic cancer (PanCa) is one of the most aggressive forms of cancer and its incidence rate is continuously increasing every year. It is expected that by 2030, PanCa will become the 2nd leading cause of cancer related deaths in the United States due to the lack of understanding of the complexity of disease, early diagnostic methods, and extremely poor survival. Despite great advancements in biomedical research, there are very limited modalities available for the early detection of PanCa and its treatment. Thus, understanding of disease biology and identification of newer diagnostic and therapeutic modalities are high priority research in the field. Our group has identified Thyroid Receptor Interacting Protein -13 (TRIP13) as an oncogenic protein which contributes to early events of PanCa. Methodology- High dimensional omics and structural elucidation of TRIP13 was conducted using publicly available databases like Consurf, Phosphosite, GTEx and GEPIA2. Molecular biology techniques were applied to cross validate all our bioinformatics data. qPCR, immunoblotting and IHC analysis was performed in progressive PDAC cell lines and human tissues to decipher the expression level of TRIP13 in various pathological staging including functional enrichment analysis using Linkdomics. Results- The structural elucidation analysis revealed that most of the functionally exposed residues, phosphorylation and ubiquitylation sites situated at AAA domain of TRIP13. As compared to normal, TRIP13 is significantly over expressed in pancreatic tumor samples. Pathological staging 2 and 1 (early stages) showed relatively higher expression of TRIP13 as compared advanced stage. Of the total 7 isoforms of TRIP13, only one isoform (TRIP13-001) has shown the over expression in pancreatic tumor samples and shown association with poor survival. In correlation studies, TRIP13 has shown positive association with other pancreatic cancer biomarkers (CEACAM5, S100A4, MUC1, MSLN, CA125). The functional enrichment analyses suggest that TRIP13 is involved in important patho-physiological pathways like DNA repair, viral carcinogenesis, cellular senescence, and cell cycle. Our wet lab experiments also support our computational biology observation. Conclusion- This integrated computational biology study suggests a potential role of TRIP13 in pancreatic cancer progression via modulation of important oncological pathways. Wet lab experimentations also validated the involvement of TRIP13 in pancreatic cancer progression. TRIP13 expression may be associated with patient prognosis. Thus, its inhibitory small molecules can be useful for boosting pancreatic cancer therapies in clinical settings. Key words- TRIP13, Pancreatic cancer, Early events of cancer, Integrative Biology, Transcriptomics, Computational biology Citation Format: Swati Dhasmana, Anupam Dhasmana, Stella Rios, Iris A. Perez, Sheema Khan, Farrukh Afaq, Upender Manne, Murali M. Yallapu, Subhash C. Chauhan. TRIP13 augments pancreatic cancer progression- An integrated systems biology study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6220.

Stratification of glioma based on stemness scores in bulk and single-cell transcriptomes

BackgroundBrain tumours are known to have a high mortality and morbidity rate due to their localised and frequent invasive growth. The concept that glioma resistance could originate from the dissimilarity in the vulnerability of clonogenic glial stem cells to chemotherapeutic drugs and radiation has driven the scientific community to reexamine the comprehension of glioma growth and strategies that target these cells or modify their stemness. MethodsBased on the enrichment scores of 12 stemness signatures, we identified glioma subtypes in both tumour bulks and single cells by clustering analysis. Furthermore, we comprehensively compared molecular and clinical features among the glioma subtypes. ResultsConsistently, in seven different datasets, hierarchical clustering uncovered three subtypes of glioma, termed Stem-H, Stem-M, and Stem-L, with high, medium, and low stemness signatures, respectively. Stem-H and Stem-L exhibited the most unfavorable and favourable overall and disease-free survival, respectively. Stem-H showed the highest enrichment scores of the EMT, invasion, proliferation, differentiation, and metastasis processes signatures, while Stem-L displayed the lowest. Stem-H harboured a greater proportion of late-stage tumours compared to Stem-L. Moreover, Stem-H manifested higher tumour mutation burden, DNA damage repair and cell cycle activity, intratumour heterogeneity, and a more frequent incidence of TP53 and EGFR mutations than Stem-L. In contrast, Stem-L had higher O6-Methylguanine-DNA Methyltransferase (MGMT) methylation levels. ConclusionThe classification of glioma based on stemness may offer new insights into the biology of the tumour, as well as more accurate clinical management of the disease.

Abstract 3368: Combination of a chk1/2 dual inhibitor (prexasertib) and gemcitabine reveals a novel intrinsic synergy mechanism in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) presents a challenging clinical scenario due to its recurrence propensity despite existing treatments. Altered intrinsic signaling pathways significantly contribute to therapeutic resistance, demanding exploration of novel strategies to induce tumor cell demise. Dysregulated cell cycle and DNA damage repair constitute pivotal events driving HNSCC progression, offering potential therapeutic targets.CHK1 and CHK2, pivotal regulators of the DNA replication checkpoint, are promising targets in HNSCC. The convergence of DNA-damaging agents with DNA checkpoint inhibitors represents a burgeoning paradigm in cancer management. Our study delves into elucidating the intrinsic signaling mechanisms triggered by the synergistic action of the CHK1/2 dual inhibitor (Prexasertib) and the nucleoside analog (Gemcitabine) in HNSCC.Our study unveiled a robust synergistic effect between Prexasertib and Gemcitabine, inducing substantial cell death in multiple HNSCC cell lines and mouse model. Remarkably, the combination rapidly induced DNA damage and reduced tumor growth in vivo. To unravel the underlying intrinsic signaling mechanism provoked by the Prexasertib/Gemcitabine combination, we conducted comprehensive molecular analyses.Proteomic profiling uncovered distinct alterations in key signaling pathways upon combination treatment. We identified unique overexpression of an embryonic stem cell-specific transcription factor pivotal in genomic stability regulation, solely induced by single-agent Prexasertib. Specifically, the combination therapy showcased unique modulation of critical genes involved in DNA repair, replication, and cell cycle regulation. These alterations included overexpression or suppression of key genes associated with DNA stability and cell proliferation pathways, accompanied by evidence of CHK1/2 inhibition-mediated DNA damage response accompanied by cleavage of CHK-2 and caspase-3 proteins, validated through Western blots.Additionally, functional assays including the colony formation assays demonstrated markedly reduced survival rates with the combination treatment compared to single-agent therapy. Cell cycle analysis revealed a replication catastrophe and increased pH2AX across multiple HNSCC cell lines following combination treatment.Our findings highlight the significance of CHK1/2 inhibition coupled with nucleoside analog-induced DNA damage in unveiling novel intrinsic signaling mechanisms in HNSCC. These insights offer a promising avenue for targeted therapeutic strategies to enhance treatment efficacy and outcomes in HNSCC.This work has been supported by the MGC, FC, P&MC, and BBSR at the H. Lee Moffitt Cancer Center & Research Institute, a comprehensive cancer center designated by the National Cancer Institute (P30-CA076292). Citation Format: Vinayak C. Palve, Hannah L. Walker-Mimms, Ritu Chaudhary, Victoria Izumi, Neelkamal Chaudhary, Bin Fang, John Koomen, Jose Alejandro Guevara, Paulo C. Rodriguez, Christine H. Chung, Derek R. Duckett. Combination of a chk1/2 dual inhibitor (prexasertib) and gemcitabine reveals a novel intrinsic synergy mechanism in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3368.

Abstract 4350: Characterization of PUF60 splicing activity that is essential for the survival of triple negative breast cancer

Abstract Alternative splicing driven by RNA-binding proteins (RBPs) plays a significant role in TNBC, however, pharmacological modulators of core spliceosomal proteins can disrupt early stages of spliceosome assembly and lead to toxic effects in normal tissues, limiting their utility as safe therapeutics. Here, we used in vivo CRISPR-Cas9 screening to evaluate RBP dependencies in TNBC and identified 17 splicing regulatory candidates that are essential in TNBC but dispensable for normal cell growth and survival. Within this cohort, the alternative splicing factor, PUF60, was the most significantly upregulated candidate in patient TNBC tumors. We found that depletion of PUF60 induced apoptosis in human TNBC cell lines and impeded xenografted tumor growth in vivo while upregulation of PUF60 expression increased TNBC cellular proliferation. Although PUF60 has been shown to also bind DNA and influence transcription, TNBC cells constructed with a previously characterized mutation (L140P) required for 3’ splice site recognition displayed significantly decreased cellular proliferation compared to cells containing wild type PUF60. Integrated eCLIP and KD RNA-sequencing revealed that PUF60 selectively regulates the splicing of mRNAs in TNBC that contribute to essential processes including DNA repair, replication and cell cycle progression. RNA-seq following L140P-mediated inhibition of 3’ ss recognition revealed a cohort of splicing events within genes enriched for similar processes following knockdown of endogenous PUF60 including cell cycle and histone modification. Altogether, our study highlights a critical role for PUF60-mediated splicing in the maintenance of genomic integrity in TNBC as well as the therapeutic potential of identifying and targeting splicing effector RBPs only responsible for TNBC survival. Citation Format: Alexandra Tankka, Trent Gomberg, Cathy Zhou, Vivian Pham, Gene Yeo. Characterization of PUF60 splicing activity that is essential for the survival of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4350.

An Injectable Hydrogel System with Mild Photothermal Effects Combined with Ion Release for Osteosarcoma‐Related Bone Defect Repair

AbstractOsteosarcoma, a common invasive malignant bone disease, presents therapeutic challenges due to the persistent problem of incomplete resection during surgical treatment. This often results in postoperative tumor recurrence and metastasis, and large‐scale bone defects are difficult to self‐repair, seriously affecting patient health. In this study, a dual‐ion doped organic‐inorganic composited SOH1(CP)1 injectable hydrogel system is successfully designed and constructed. This system consists of sericin protein grafted with hydrazide bonds, oxidized chondroitin sulfate, Se and Mg co‐doped HAp nanorods, and polydopamine‐coated CaO2 nanospheres. The system displays strong anti‐tumor activity due to its mild photothermal effects combined with the chemotherapeutic efficacy of SeO32−. Because the degradation behavior of hydrogel matches the bone repair cycle, including the nutritional support of hydrogel skeleton degradation products to promote bone cell proliferation, and the positive regulation of Ca2+, Mg2+, and PO43− released via the degradation of inorganic nanoparticles to promote bone differentiation, the system shows excellent bone defect repair efficacy. Importantly, this system achieves 100% tumor inhibition after 18 days, while ensuring complete bone repair after 12 weeks. Hence, the SOH1(CP)1 injectable hydrogel system, which displays both high anti‐osteosarcoma efficacy and strong bone repair properties, can serve as a new tool for osteosarcoma‐related bone defect repair.

Molecular aspects of cervical cancer: a pathogenesis update.

Cervical cancer (CC) is a significant health problem, especially in low-income countries. Functional studies on the human papillomavirus have generated essential advances in the knowledge of CC. However, many unanswered questions remain. This mini-review discusses the latest results on CC pathogenesis, HPV oncogenesis, and molecular changes identified through next-generation technologies. Interestingly, the percentage of samples with HPV genome integrations correlates with the degree of the cervical lesions, suggesting a role in the development of CC. Also, new functions have been described for the viral oncoproteins E5, E6, and E7, resulting in the acquisition and maintenance of cancer hallmarks, including proliferation, immune response evasion, apoptosis, and genomic instability. Remarkably, E5 oncoprotein affects signaling pathways involved in the expression of interferon-induced genes and EGFR-induced proliferation, while E6 and E7 oncoproteins regulate the DNA damage repair and cell cycle continuity pathways. Furthermore, next-generation technologies provide vast amounts of information, increasing our knowledge of changes in the genome, transcriptome, proteome, metabolome, and epigenome in CC. These studies have identified novel molecular traits associated with disease susceptibility, degree of progression, treatment response, and survival as potential biomarkers and therapeutic targets.