Cell Cycle

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

Objectives Low grade serous ovarian cancer (LGSOC) is characterized by frequent TP53 wild type status and limited responsiveness to conventional chemotherapy. This study investigated the cytotoxic effects of oxaliplatin combined with 5-fluorouracil on patient derived LGSOC cells. Results Clinically relevant concentrations of oxaliplatin and 5-fluorouracil had minimal effects on LGSOC cell viability but markedly reduced cellular proliferation and clonogenic recovery. Combined treatment induced pronounced accumulation of cells in the G0/G1 phase of the cell cycle and increased expression of p53 and p21, accompanied by reduced pRB phosphorylation, consistent with activation of the p53-p21-pRb pathway. Oxaliplatin plus 5-fluorouracil also increased senescence-associated beta-galactosidase activity, cellular enlargement and flattening, and reactive oxygen species production, supporting a senescence-like phenotype. However, after drug withdrawal, treated cells progressively regained proliferative capacity, indicating that the senescence phenotype was transient rather than fully irreversible. Together, these findings show that oxaliplatin plus 5-fluorouracil primarily arrest rather than kill LGSOC cells and induces a transient senescence-like phenotype and G0/G1-phase arrest by activating the p53-p21-pRb pathway.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

Abstract The canonical WNT signaling pathway guides cell growth throughout the human lifespan. In adults, WNT plays an essential role in tissue homeostasis by maintaining tissue stem cells. In the intestine, the WNT transcription factor, TCF4, is necessary for stem cell maintenance. Aberrant activation of WNT signaling is generally accepted as an initiating event in colon cancer, leading to constitutive TCF4 activity. To investigate the role of this key transcription factor in global gene regulation, we abrogated TCF4 expression in colon cancer cells. Loss of TCF4 function resulted in the over-expression of the WNT transcription factor, LEF1. LEF1 was transcriptionally competent and over-compensated for TCF4 (TCF7L2) silencing in a WNT reporter assay. TCF4 enlisted the transcriptional repressor CtBP1, and bound the LEF1 promoter, indicative of direct repression. TCF4 and LEF1 drove different transcriptional programs, with TCF4 favoring MYC and cell cycle progression, while LEF1 favored immune-related gene signatures. TCF4 was the primary regulator of MYC expression, yet also the mediator of LEF1 repression, suggestive of competition centered on TCF4 transcriptional output despite the {beta}-catenin saturated colon cancer nucleus. We conclude that TCF4 has dual activator-repressor activity in colon cancer.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

Advanced-stage high grade serous ovarian cancer (HGSC) initially responds to platinum-based chemotherapy; however, most HGSC tumors recur and develop chemotherapy-resistance, making recurrent ovarian cancer (OC) a fatal disease. Ovarian cancer stem cells (OCSCs), a subpopulation of cells capable of surviving treatment and repopulating tumors, contribute to disease relapse, and understanding the mechanisms(s) responsible for OCSC survival is critical for developing strategies to overcome acquired resistance and prevent tumor recurrence. However, whether platinum-induced OCSC enrichment results from the selection of pre-existing OCSCs or chemotherapy-induced cellular plasticity, and the molecular mechanisms underlying these processes, remain poorly understood. Here, we demonstrated that cisplatin treatment not only enriched for OCSCs but promoted the conversion of ALDH- cells to ALDH+ cells, revealing chemotherapy-induced plasticity as a potential mechanism for OCSC expansion. This conversion was associated with nuclear receptor signaling pathways, vitamin D receptor signaling (VDR/RXR) and retinoic acid receptor signaling (RAR/RXR). Importantly, activation of VDR/RXR signaling with vitamin D reduced stemness phenotypes and the cisplatin induced conversion to ALDH+ cells. In addition, vitamin D combined with cisplatin decreased tumor volume in vivo compared to either treatment alone. In contrast, cisplatin treatment increased RAR/RXR signaling, supporting an associative role for ligand-dependent nuclear receptor signaling in OCSC plasticity. Together, these findings reveal that platinum chemotherapy can actively promote the acquisition of stem-like properties and identifies VDR/RXR signaling as a potential regulatory mechanism of OCSC plasticity. Targeting this pathway with vitamin D may provide a therapeutic strategy to target OCSCs and improve responses to platinum-based chemotherapy.

A central question in developmental biology is how spatiotemporal embryo morphological events and cell differentiation are precisely coordinated to eventually develop into a mature organism. Previous research has shown cell positions and asymmetric division stages are related to cell lineage specification since morula stage. However, the detailed spatiotemporal dynamics and histories of embryonic cells and their relations with cell lineage specification remain incompletely understood from the onset of embryo development onward. My study on in-vitro embryos showed that continuous live cell tracking mapped community-like patterns in cell origin and differentiation. Cell positions, cell temporal factors (such as cell division order and cell cycle) and their histories intricately interacted over pre- and peri-implantation stages. These interactions of spatiotemporal cellular activities directed embryo morphological events and asymmetric cell lineage origin and differentiation throughout 2-cell to around 100-cell stage. In conclusion, this study provides comprehensive insights into the cellular spatiotemporal dynamics of cell inheritance and differentiation during pre- and peri-implantation morphological events and cell lineage specification. By longitudinally integrating spatial and temporal parameters relating to early embryogenesis, the findings not only bridge divergent explanations stemmed from different studies in the field but also offers a refined foundation for evaluating embryo potential and improving outcomes in assisted reproductive technologies, stem cell research, and regenerative medicine.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Telomeres, the ends of linear chromosomes, form a cap composed of specific telomeric DNA associated with specialized proteins that ensures efficient protection against chromosome degradation and, consequently, overall genome instability. Three main complexes have been implicated in chromosome end protection, namely telomerase, shelterin, and the CST complex (CTC1/Cdc13-STN1-TEN1). In a recent study, we uncovered mutants of the S. cerevisiae CST in which damage was sensed both by the two major spindle checkpoints and the major DNA damage checkpoint. In the best-studied of these mutants, stn1-sz2, the stability and organization of the mitotic tubulin spindle was profoundly affected, as were the organization of the centrosomes and the kinetochores. Here, we find that the stn1-sz2 mutant is hypersensitive to deletion mutants of two members of the kinetochores, Ctf19 and Mcm21, which correlated with mis-localization of their associated subunit in the COMA complex, Okp1, during anaphase. Defects in interactions between the kinetochores and the tubulin spindle are entirely sensed by the SAC (spindle assembly checkpoint). Here, we find that some of the stn1-sz2 damage is recognized by the Mad2 checkpoint protein independently from the SAC. The data presented here contribute to a better understanding of the damage in stn1-sz2, the prototype of novel mutants of telomeric CST characterized by mitotic-spindle-damage in addition to the classical telomeric-DNA-damage.

Perturbations that cause DNA replication stress can induce lineage-specific changes in gene expression. Specifically, DNA replication stress activates primed loci in euchromatin that are epigenetically silenced, but the mechanism linking stalled replication forks to the activation of distal genes remains unknown. Using reactivation of fetal hemoglobin (HbF) by hydroxyurea in erythroid progenitors as a model, we find that redistribution of the repressive histone methyltransferase G9a from cis-regulatory elements to stalled replication forks increases transcription of the gamma-globin genes HBG1 and HBG2, explaining how hydroxyurea increases HbF expression and benefits patients with sickle cell anemia. In addition to loss of HBG1/2 silencing, redistribution of G9a also increases expression of a primed, GATA1-driven transcriptional program. G9a and H3K9me2 accumulate at stalled replication forks in a manner dependent on the DNA-binding protein WIZ. Overexpression of G9a partially reduces the impact of G9a redistribution on HBG1/2 transcript levels. Together, these findings support a model in which stalled replication forks recruit corepressors away from primed genes, thereby facilitating their activation and explaining how DNA replication stress promotes lineage-specific changes in gene expression.

Purpose: Uveal melanoma (UM) is a rare but aggressive intraocular malignancy originating in the uvea (choroid, iris, and ciliary body). Despite primary treatment, 50% of UM patients develop metastases to the liver, lungs, bones, and skin. Topotecan, a topoisomerase I inhibitor, and SP141, an MDM2 inhibitor, have been studied in cancers, including retinoblastoma. This study explores a combined approach with these drugs in the 92-1 UM cell line. Methods: Primary choroidal melanoma-derived 92-1 cells were treated with topotecan and SP141 individually to determine IC50 values. Cell viability and proliferation were assessed using the MTT assay and the xCELLigence real-time monitoring system. The synergistic effects were analyzed quantitatively using the Loewe Additivity and Highest Single-Agent (HSA) models. Flow cytometry was employed to analyze cell cycle distribution, while cell death and apoptosis were monitored via high-throughput, real-time quantitative analysis using the IncuCyte live-cell kinetic imaging system. Results: Both Topotecan and SP141 induced significant cell death in 92-1 cells. xCELLigence showed dose- and time-dependent inhibition of proliferation. Combining sub-IC50 concentrations of topotecan and SP141 exhibited significant synergistic effects, markedly reducing growth. Cell cycle analysis revealed that topotecan causes a dose-dependent biphasic arrest in G2 and S phases, while SP141 causes G2/M arrest, leading to cell death. Cytotoxicity assays corroborated increased cell death following combination therapy, and the Caspase 3/7 assay indicated that apoptosis is the mechanism underlying cell death. Conclusions: The multi-targeted approach combining topotecan and SP141 drugs demonstrated synergistic therapeutic effects in 92-1 cells, suggesting a potential treatment strategy for managing UM.

Abstract Polycystic ovarian syndrome and Type 2 diabetes mellitus are complex multivariate diseases that share well-established clinical pathophysiology, yet the molecular basis of their overlap is unknown. Prior bioinformatics studies have identified common differentially expressed genes between these two conditions. However, they have not accounted for the directionality of the gene expression changes observed, potentially obscuring any biological distinctions they may have. This study applied a direction-aware approach to classify molecular signatures between PCOS and T2DM as concordant or discordant. Transcriptomic analysis of GEO datasets GSE138518 (ovarian granulosa tissue, PCOS) and GSE25724 (pancreatic islet tissue, T2DM) identified 225 and 1,302 DEGs, respectively. Venn diagram analysis showed that only three genes (SLC6A8, RGS4, and SORL1) were shared between PCOS and T2DM from the total of 1,527 genes, and all 3 genes were regulated in opposing directions. Disease gene retrieval from the Comparative Toxicogenomics Database, Online Mendelian Inheritance in Man database, and GeneCards showed 214 shared disease-associated genes, with 311 genes unique to PCOS and 496 genes unique to T2DM. Protein-protein interaction construction using STRING (version 12.0) identified 70 interacting nodes. CytoHubba analysis across 6 scoring methods identified 18 high-confidence hub genes, including INS, BCL2, MTOR, LEP, MFN2, and PIK3CD. Functional enrichment analysis identified biological processes including immune activation, apoptosis, and insulin signaling, which were confirmed as the main pathways in KEGG pathway analysis. These findings show that while PCOS and T2DM share limited DEGs, they share common pathogenic pathways.

Cells and tissues have the ability to respond to different stresses and maintain their homeostasis. The b-Zip transcription factor Xrp1 was found to play a pivotal role in this context in Drosophila. Xrp1 is required for the response to irradiation, in genetic conditions of ribosomal protein (Rp) deficiency leading to cell competition, and more generally in mediating the Integrated Stress Response (ISR). However, how these diverse stresses converge to and activate Xrp1 is still unclear. Our results demonstrate the existence of three separate mechanisms of Xrp1 induction. In response to Rp stress, a short isoform of Xrp1 is produced and requires the function of the ribosomal protein RpS12. In response to irradiation, a specific mRNA isoform is transcriptionally induced, which encodes the Xrp1-short protein isoform, in a p53-dependent manner. In response to the ISR, the long isoform of Xrp1 is translated by the derepression of a uORF present on the Xrp1 5'UTR, a mechanism that requires eIF2 phosphorylation, RpS12, and a transcriptional induction by ATF4. Therefore, our work provides a comprehensive understanding of the role of Xrp1 in different situations of cellular stress, illustrating the complex level of integration of the mechanisms controlling this central gene.

The circadian clock allows living systems to anticipate environmental daily variations by scheduling cellular mechanisms according to the time of the day. The reciprocal interplay between metabolism and circadian clock is essential for energy homeostasis, and the disruption of metabolic clock inputs contributes to dysfunction of clocks in peripheral tissues. However, the molecular mechanisms through which the feeding/fasting cycle entrains peripheral clocks remain incompletely understood. Here, we investigate experimentally and theoretically the role of AMP-dependent kinase (AMPK), a key fasting sensor, in metabolic regulation of the circadian clock. Using a luciferase reporter driven by the Bmal1 promoter to monitor molecular clock activity in human U2OS cells, and thanks to a signal processing method based on the Hilbert transform to retrieve the instantaneous phase and period of circadian signals, we show that pharmacological activation of AMPK by AICAR significantly elevates Bmal1 promoter activity and markedly lengthens the clock period in a dose-dependent manner. Conversely, the clock period is shortened by the pharmacological inhibition by SR18292 of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1-alpha). Importantly, the action of AICAR is abolished by FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, demonstrating that AMPK-dependent modulation of Bmal1 promoter activity requires NAD+ availability. These experimental results are recapitulated by numerical simulations of our previously published mathematical model, where PGC-1-alpha plays a key role in mediating AMPK activity to the clock. Moreover, the comparison of experimentally measured and theoretically predicted phase response curves, following AMPK activation at different circadian phases, provides an additional validation of the model. By integrating biological experiments with mathematical modeling, our results identify a key mechanism through which metabolic factors can entrain and regulate the circadian clock via NAD+-dependent AMPK-mediated regulation of Bmal1 promoter activity.

Ovarian cancer stands out as one of the most lethal gynecological cancers among women in the United States. While patients with ovarian cancer often show positive responses to treatment, it is common for the cancer to reappear and develop resistance to treatment, so it is important to explore and create novel combinations to increase patient survival rates. ATRi (Ataxia telangiectasia and Rad3-related inhibitors) and ONC201 monotherapies have shown efficacy in ovarian cancer in preclinical models. We hypothesized that combining ATRi with ONC201 would enhance this efficacy. ONC201 induces apoptosis through the TRAIL-pathway following activation of the integrated stress response and inactivation of Akt which is upregulated in ovarian cancer. Six human ovarian cancer cell lines were treated alone and with the novel drug combination of ceralasertib (ATRi) and ONC201. We found an IC50 range of 0.88-20.16 microM for ONC201 and IC50 range of 0.55-54.75 microM for ceralasertib. The combination of ONC201 plus ceralasertib resulted in PARP cleavage consistent with apoptosis along with reduction in Bcl-XL, Xiap and p-Akt expression after 72 hours of treatment. Cytokine profiling showed a decrease in markers of inflammation and tumor progression. Our data provide evidence of synergy from the combination of ATRi ceralasertib and ONC201 against Ovarian cancer cells that merits further investigation including clinical translation.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.