Expression Of CDC25A Research Articles

Key Genes Involved in the Beneficial Mechanism of Hyperbaric Oxygen for Glioblastoma and Predictive Indicators of Hyperbaric Oxygen Prolonging Survival in Glioblastoma Patients.

The prognosis of glioblastoma is poor, and therapy-resistance is largely attributed to intratumor hypoxia. Hyperbaric oxygen (HBO) effectively alleviates hypoxia. However, the sole role of HBO in glioblastoma remains controversial. We previously reported that HBO can promote apoptosis, shorten protrusions, and delay growth of glioblastoma, but the molecular mechanism is unclear. We aimed to test candidate genes in HBO-exposed glioblastoma cells and to analyze their correlation with the survival of glioblastoma patients. Glioblastoma cell lines exposed to repetitive HBO or normobaric air (NBA) were collected for RNA isolation and microarray data analysis. GO analysis, KEGG pathway analysis and survival analysis of the differentially expressed genes (DEGs) were performed. HBO not only inhibited hypoxia-inducing genes including CA9, FGF11, PPFIA4, TCAF2 and SLC2A12, but also regulated vascularization by downregulating the expression of COL1A1, COL8A1, COL12A1, RHOJ and FILIP1L, ultimately attenuated hypoxic microenvironment of glioblastoma. HBO attenuated inflammatory microenvironment by reducing the expression of NLRP2, CARD8, MYD88 and CD180. HBO prevented metastasis by downregulating the expression of NTM, CXCL12, CXCL13, CXCR4, CXCR5, CDC42, IGFBP3, IGFBP5, GPC6, MMP19, ADAMTS1, EFEMP1, PTBP3, NF1 and PDCD1. HBO upregulated the expression of BAK1, PPIF, DDIT3, TP53I11 and FAS, whereas downregulated the expression of MDM4 and SIVA1, thus promoting apoptosis. HBO upregulated the expression of CDC25A, MCM2, PCNA, RFC33, DSCC1 and CDC14A, whereas downregulated the expression of ASNS, CDK6, CDKN1B, PTBP3 and MAD2L1, thus inhibiting cell cycle progression. Among these DEGs, 17 indicator-genes of HBO prolonging survival were detected. HBO is beneficial for glioblastoma. Glioblastoma patients with these predictive indicators may prolong survival with HBO therapy. These potential therapeutic targets especially COL1A1, ADAMTS1 and PTBP3 deserve further validation.

Transcriptome analysis reveals that defects in cell cycle regulation contribute to preimplantation embryo arrest

Patients with preimplantation embryo arrest (PREMBA) often experience assisted reproductive failure primarily due to the lack of transferable embryos, and the molecular mechanisms underlying PREMBA remain unclear. In our study, the embryos from five women with recurrent preimplantation embryo arrest and three women with tubal factor infertility were used for single-embryo transcriptome sequencing. Meanwhile, the transcriptomes of normal human preimplantation embryos obtained from GSE36552 were utilized to perform a comparative analysis with the transcriptomes of PREMBA embryos. Our results showed dysregulation of the cell cycle phase transition might be a potential pathogenic factor contributing to PREMBA. Through integrated analysis of the differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA), we identified a number of hub genes using the protein-protein interaction network. The top 5 hub genes were as follows: CCNB2, BUB1B, CDC25A, CCNB3, and PLK3. The expression of hub genes was validated in PREMBA embryos and donated embryos using RT-qPCR. The knockdown of Ccnb2 in mouse zygotes led to an increase in embryo fragmentation, a rise in apoptosis, and a reduction in blastocyst formation. Furthermore, silencing the expression of CDC25A in HEK293T cells resulted in a decrease in cell proliferation and an increase in apoptosis, providing further support for our findings. Our findings could predict the development outcomes of preimplantation embryos and be used as potential therapeutic targets to prevent recurrent failures of IVF/ICSI attempts.

In vivo evaluation of mebendazole and Ran GTPase inhibition in breast cancer model system.

Aim: To investigate the therapeutic potential of mebendazole (MBZ)-loaded nanostructured lipid carriers (NLCs). Methodology: NLC-MBZ was prepared and characterized to evaluate the in vitro and in vivo anticancer effects and the inhibitory effect on RanGTP and its potential as an antimetastatic treatment in vivo. Results: NLC-MBZ exhibited a size and charge of 155±20nm and-27±0.5mV, respectively, with 90.7% encapsulation. Free MBZ and NLC-MBZ had a 50% inhibitory concentrationof 610 and 305nM, respectively, against MDA-MB-231 cell lines. NLC-MBZ decreasedtumor size, suppressed tumor lung metastases, and lowered the expression of CDC25A, SKP2, RbX1and Cullin1while boosting the Rb proteins. Conclusion: NLC-MBZ displayedantiangiogenic potential and resulted in a reduced rate of lung metastasis in vivo.

Machine learning for identifying tumor stemness genes and developing prognostic model in gastric cancer.

Gastric cancer presents a formidable challenge, marked by its debilitating nature and often dire prognosis. Emerging evidence underscores the pivotal role of tumor stem cells in exacerbating treatment resistance and fueling disease recurrence in gastric cancer. Thus, the identification of genes contributing to tumor stemness assumes paramount importance. Employing a comprehensive approach encompassing ssGSEA, WGCNA, and various machine learning algorithms, this study endeavors to delineate tumor stemness key genes (TSKGs). Subsequently, these genes were harnessed to construct a prognostic model, termed the Tumor Stemness Risk Genes Prognostic Model (TSRGPM). Through PCA, Cox regression analysis and ROC curve analysis, the efficacy of Tumor Stemness Risk Scores (TSRS) in stratifying patient risk profiles was underscored, affirming its ability as an independent prognostic indicator. Notably, the TSRS exhibited a significant correlation with lymph node metastasis in gastric cancer. Furthermore, leveraging algorithms such as CIBERSORT to dissect immune infiltration patterns revealed a notable association between TSRS and monocytes and other cell. Subsequent scrutiny of tumor stemness risk genes (TSRGs) culminated in the identification of CDC25A for detailed investigation. Bioinformatics analyses unveil CDC25A's implication in driving the malignant phenotype of tumors, with a discernible impact on cell proliferation and DNA replication in gastric cancer. Noteworthy validation through in vitro experiments corroborated the bioinformatics findings, elucidating the pivotal role of CDC25A expression in modulating tumor stemness in gastric cancer. In summation, the established and validated TSRGPM holds promise in prognostication and delineation of potential therapeutic targets, thus heralding a pivotal stride towards personalized management of this malignancy.

Yifei Sanjie formula alleviates lung cancer progression via regulating PRMT6-YBX1-CDC25A axis.

Lung cancer (LC) is the most prevalent cancer type, with a high mortality rate worldwide. The current treatment options for LC have not been particularly successful in improving patient outcomes. Yifei Sanjie (YFSJ), a well-applicated traditional Chinese medicine formula, is widely used to treat pulmonary diseases, especially LC, yet little is known about its molecular mechanisms. This study was conducted to explore the molecular mechanism by which YFSJ ameliorated LC progression. The A549, NCI-H1975, and Calu-3 cells were treated with the YFSJ formula and observed for colony number, apoptosis, migration, and invasion properties recorded via corresponding assays. The PRMT6-YBX1-CDC25A axis was tested and verified through luciferase reporter, RNA immunoprecipitation, and chromatin immunoprecipitation assays and rescue experiments. Our results demonstrated that YFSJ ameliorated LC cell malignant behaviors by increasing apoptosis and suppressing proliferation, migration, and invasion processes. We also noticed that the xenograft mouse model treated with YFSJ significantly reduced tumor growth compared with the control untreated group in vivo. Mechanistically, it was found that YFSJ suppressed the expression of PRMT6, YBX1, and CDC25A, while the knockdown of these proteins significantly inhibited colony growth, migration, and invasion, and boosted apoptosis in LC cells. In summary, our results suggest that YFSJ alleviates LC progression via the PRMT6-YBX1-CDC25A axis, confirming its efficacy in clinical use. The findings of our study provide a new regulatory network for LC growth and metastasis, which could shed new insights into pulmonary medical research.

Impact of curcumin on ferroptosis-related genes in colorectal cancer: Insights from in-silico and in-vitro studies.

Colorectal cancer (CRC)is responsible for a significant number of cancer-related fatalities worldwide. Researchers are investigating the therapeutic potential of ferroptosis, a type of iron-dependent controlled cell death, in the context of CRC. Curcumin, a natural compound found in turmeric, exhibits anticancer properties. This study explores the effects of curcumin on genes related to ferroptosis (FRGs)in CRC. To gather CRC data, we used the Gene Expression Profiling Interactive Analysis (GEPIA)and Gene Expression Omnibus (GEO)databases, while FRGs were obtained from the FerrDb database and PubMed. We identified 739 CRC differentially expressed genes (DEGs)in CRC and discovered 39 genes that were common genes between FRGs and CRC DEGs. The DEGs related to ferroptosis were enriched with various biological processes and molecular functions, including the regulation of signal transduction and glucose metabolism. Using the Drug Gene Interaction Database (DGIdb),we predicted drugs targeting CRC-DEGsand identified 17 potential drug targets. Additionally, we identified eight essential proteins related to ferroptosis in CRC, including MYC, IL1B, and SLC1A5. Survival analysis revealed that alterations in gene expression of CDC25A, DDR2, FABP4, IL1B, SNCA, and TFAM were associated with prognosis in CRC patients. In SW480 human CRC cells, treatment with curcumin decreased the expression of MYC, IL1B, and EZH2 mRNA, while simultaneously increasing the expression of SLCA5 and CAV1. The findings of this study suggest that curcumin could regulate FRGs in CRC and have the potential to be utilized as a therapeutic agent for treating CRC.

A Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Background and Significance: Patients (pts) with primary or secondary myelofibrosis (MF) experience variable degrees of constitutional symptoms, cytopenias and risk of progression to acute myeloid leukemia leading to a reduced life-expectancy compared to age-matched controls. Although several oral Janus Kinase (JAK) inhibitors such as ruxolitinib (RUX) have been approved by the US FDA based on improvements in symptom burden and spleen volume compared to physician's choice of best available therapy, the duration of response to these agents is generally time-limited and treatment with JAK inhibitors is not considered to have disease-modifying potential. Furthermore, the median overall survival after RUX discontinuation is only 11-14 months highlighting the need for novel mechanism-based therapies for pts with disease progression on RUX. JAK2 V617F mutations promote transition from the G1 to S-phase of the cell cycle via increased expression of CDC25A, which is also upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been implicated in MF pathogenesis and contribute to myeloproliferation. Finally, we and others have shown synergistic effects of the combination of CDK4/6 inhibitors and RUX in murine models of MF (Rampal et al., CCR 2021) including improvement in bone marrow fibrosis suggesting the potential for disease-modifying activity. Several CDK4/6 inhibitors have been approved by the US FDA for the treatment of hormone receptor-positive metastatic breast cancer establishing a robust record of safety data. These preclinical data and known safety profile of CDK4/6 and RUX as single agents, provide rationale for combining CDK4/6 and JAK inhibition in pts with MF. Methods: This multicenter, phase I dose-escalation trial (NCT05714072) evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in primary or secondary MF pts with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. Inadequate response to RUX is defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms, as defined by the presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS. Pts must have been on a stable dose of 10 mg or 15 mg BID of RUX for at least 4 weeks prior to enrollment with adequate platelet (≥75 x 10 9/L) and neutrophil counts (≥1.5 x 10 9/L). Key exclusion criteria include prior treatment with a CDK4/6 inhibitor, accelerated- or blast-phase MPN, platelet count <50 x 10 9/L in the 4 weeks before screening or platelet transfusion(s) within 8 weeks before screening, and absolute neutrophil count <0.5 x 10 9/L in the 4 weeks before screening. Among the currently FDA-approved CDK4/6 inhibitors, abemaciclib was chosen for combination therapy due to less myelosuppressive effects compared with other CDK4/6 inhibitors. This trial uses a conventional “3+3” dose-escalation design ( Figure), pts will be treated with increasing doses of abemaciclib added to a stable dose of RUX (10 mg or 15 mg BID). The pre-study dose of RUX will be maintained to determine the maximum tolerated dose of the combination of RUX and abemaciclib. The primary endpoint is to determine the maximum tolerated dose of RUX + abemaciclib. Secondary endpoints include measures of efficacy such as overall response rate, duration of response and survival endpoints. Exploratory endpoints include changes in allele fraction of driver mutations, gene expression profiling, and clonal architecture to identify biomarkers of response, mechanisms of resistance, and to assess for any disease-modifying effects of the combination therapy. At this time, 2 pts have been enrolled at dose level 0 without any dose-limiting toxicities observed. In line with the known adverse event profile of abemaciclib, grade 1 diarrhea occurred in one patient. One patient developed grade 3 neutropenia which promptly resolved with holding abemaciclib. No unexpected safety signal was noted and enrollment continues. This trial is open to enrollment at Memorial Sloan Kettering Cancer Center and will commence at MD Anderson Cancer Center later this year.

CircCCL22 Regulates CDC25A via Sponging miR-543 and Promotes Proliferation and Metastasis in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecological tumor. Circular RNAs are a novel type of non-coding RNA that have important regulatory functions, particularly in the pathogenic progression of cancer. In this study, we investigated the function of circCCL22, and elucidated its molecular mechanism in EC progresssion. The expression of circCCL22, miR-543 and CDC25A in EC tissues and cells were determined by qRT-PCR and western blot. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing and transwell assays were executed to assess the cell viability, proliferation, migration and invasion. Dual-luciferase report assay was utilized to investigate the interaction of miR-543 with circCCL22 and CDC25A. The role of circCCL22 in EC in vivo was investigated by xenograft assay. CircCCL22 was notably upregulated in EC tissues and cells. Functionally, circCCL22 knockdown suppressed EC cell proliferation, migration and invasion in vitro, and inhibited tumor growth in vivo. Mechanistically, circCCL22 acted as "miR-543 sponges" to regulate its targeted gene CDC25A expression in EC cells. The inhibiting effect induced by circCCL22 knockdown on EC cell proliferation, migration and invasion was greatly reversed by miR-543 inhibition or CDC25A overexpression. Our results revealed that circCCL22 regulated EC progression through targeting miR-543/CDC25A axis, and it could be a novel therapeutic target of EC.

The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Metronomic dosing of ovarian cancer cells with the ATR inhibitor AZD6738 leads to loss of CDC25A expression and resistance to ATRi treatment

ObjectiveATR kinase inhibitors promote cell killing by inducing replication stress and through potentiation of genotoxic agents in gynecologic cancer cells. To explore mechanisms of acquired resistance to ATRi in ovarian cancer, we characterized ATRi-resistant ovarian cancer cells generated by metronomic dosing with the clinical ATR inhibitor AZD6738. MethodsATRi-resistant ovarian cancer cells (OVCAR3 and OV90) were generated by dosing with AZD6738 and assessed for sensitivity to Chk1i (LY2603618), PARPi (Olaparib) and combination with cisplatin or a CDK4/6 inhibitor (Palbociclib). Models were characterized by diverse methods including silencing CDC25A in OV90 cells and assessing impact on ATRi response. Serum proteomic analysis of ATRi-resistant OV90 xenografts was performed to identify circulating biomarker candidates of ATRi-resistance. ResultsAZD6738-resistant cell lines are refractory to LY2603618, but not to Olaparib or combinations with cisplatin. Cell cycle analyses showed ATRi-resistant cells exhibit G1/S arrest following AZD6738 treatment. Accordingly, combination with Palbociclib confers resistance to AZD6738. AZD6738-resistant cells exhibit altered abundances of G1/S phase regulatory proteins, including loss of CDC25A in AZD6738-resistant OV90 cells. Silencing of CDC25A in OV90 cells confers resistance to AZD6738. Serum proteomics from AZD6738-resistant OV90 xenografts identified Vitamin D-Binding Protein (GC), Apolipoprotein E (APOE) and A1 (APOA1) as significantly elevated in AZD6738-resistant backgrounds. ConclusionsWe show that metronomic dosing of ovarian cancer cells with AZD6738 results in resistance to ATR/ Chk1 inhibitors, that loss of CDC25A expression represents a mechanism of resistance to ATRi treatment in ovarian cancer cells and identify several circulating biomarker candidates of CDC25A low, AZD6738-resistant ovarian cancer cells.

Delivery of Therapeutic miRNA via Plasma-Polymerised Nanoparticles Rescues Diabetes-Impaired Endothelial Function.

MicroRNAs (miRNAs) are increasingly recognised as key regulators of the development and progression of many diseases due to their ability to modulate gene expression post-translationally. While this makes them an attractive therapeutic target, clinical application of miRNA therapy remains at an early stage and in part is limited by the lack of effective delivery modalities. Here, we determined the feasibility of delivering miRNA using a new class of plasma-polymerised nanoparticles (PPNs), which we have recently isolated and characterised. We showed that PPN-miRNAs have no significant effect on endothelial cell viability in vitro in either normal media or in the presence of high-glucose conditions. Delivery of a miRNA inhibitor targeting miR-503 suppressed glucose-induced miR-503 upregulation and restored the downstream mRNA expression of CCNE1 and CDC25a in endothelial cells. Subsequently, PPN delivery of miR-503 inhibitors enhanced endothelial angiogenesis, including tubulogenesis and migration, in culture conditions that mimic diabetic ischemia. An intramuscular injection of a PPN-miR-503 inhibitor promoted blood-perfusion recovery in the hindlimb of diabetic mice following surgically induced ischemia, linked with an increase in new blood vessel formation. Together, this study demonstrates the effective use of PPN to deliver therapeutic miRNAs in the context of diabetes.

A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis.

TPS7086 Background: Patients (pts) with myelofibrosis (MF) have a reduced life-expectancy compared to age-matched controls. Several oral Janus Kinase (JAK) inhibitors such as ruxolitinib (RUX) have been approved by the US FDA based on improvements in symptom burden and spleen volume reduction compared to physician’s choice of best available therapy. However, duration of response to these agents is generally time-limited and treatment with JAK inhibitors is not considered to have disease-modifying potential. Additionally, median overall survival after RUX discontinuation is only 11-14 months. Thus, novel mechanism-based therapies for pts with disease progression on RUX are needed. JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A, which is also upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been implicated in MF pathogenesis. Finally, we and others have shown that combination of the CDK4/6 inhibitors and RUX had synergistic effects in murine models of MF (Rampal et al., CCR 2021) providing the preclinical rationale for this trial. Methods: This multicenter, phase I dose-escalation trial (NCT05714072) evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in pts with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. Among the currently FDA-approved CDK4/6 inhibitors, abemaciclib was chosen for combination therapy due to less myelosuppressive effects compared with other CDK4/6 inhibitors. Inadequate response to RUX is defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms, as defined by the presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS. Following a conventional “3+3” dose-escalation design, pts will be treated with increasing doses of abemaciclib and stable doses of RUX (10mg or 15mg BID). The primary endpoint is to determine the maximum tolerated dose of RUX + abemaciclib. Secondary endpoints include measures of efficacy. Exploratory endpoints include changes in allele fraction of driver mutations, gene expression profiling, and clonal architecture to identify biomarkers of response, mechanisms of resistance, and to assess for any disease-modifying effects of the combination therapy. This trial is open to enrollment at Memorial Sloan Kettering Cancer Center and MD Anderson Cancer Center. Clinical trial information: NCT05714072 .

Abstract CT242: A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis

Abstract Introduction: Patients (pts) with primary and secondary myelofibrosis (MF) have a reduced life expectancy. While several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib (RUX) have demonstrated improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy leading to their approval by the US FDA, responses to these agents are generally time-limited. Additionally, these agents are not considered to be disease-modifying and survival in pts after RUX discontinuation is poor with a median survival of 11-14 months. Thus, novel mechanism-based therapies for pts with suboptimal response or progression on RUX are needed. Preclinical Rationale: JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A in preclinical models and CDC25A expression is upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D also play a role in MF pathogenesis and drive myeloproliferation. Finally, combination of the CDK4/6 inhibitor ribociclib and RUX demonstrated reduction in spleen and liver size when compared to RUX alone in murine models, suggesting synergy (Rampal et al., CCR 2021). Methods: This multicenter, phase I dose-escalation trial evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in pts with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of 1 symptom score ≥5 or 2 symptom scores ≥3 using the screening MPN-SAF TSS. This study follows a conventional “3+3” dose-escalation design, in which pts on a stable dose of RUX (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. Abemaciclib was chosen due to less myelosuppression compared with other CDK4/6 inhibitors. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of RUX+ abemaciclib. The primary endpoint is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF pts on a stable dose of RUX. Secondary endpoints include overall response rate, progression-free and overall survival, and duration of response per the modified International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN) 2013 consensus definitions. Correlative studies are planned to identify biomarkers predictive of response to RUX + abemaciclib, mechanisms of resistance (e.g., MAP kinase pathway activation), and any disease-modifying effects of combination therapy. Citation Format: Jan P. Bewersdorf, Srdan Verstovsek, Andriy Derkach, Lucia Masarova, Naveen Pemmaraju, Eytan M. Stein, Michael Mauro, Raajit Rampal, Prithviraj Bose. A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT242.

Cyclin A–CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry

Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken together, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.

Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Introduction and preclinical rationale: Patients with primary and secondary myelofibrosis (MF) have a reduced life expectancy compared to age-matched controls. The prognosis of individual patients is defined by clinical, cytogenetic, and molecular disease characteristics. Several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib are approved by the US FDA for the treatment of patients with intermediate or high-risk MF based on improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy. However, these inhibitors often have a limited duration of response, and survival in patients after ruxolitinib discontinuation is poor (median survival of 13 months). Novel mechanism-based therapies for patients with suboptimal response or progression on ruxolitinib are thus needed. Preclinical studies have demonstrated that JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A. Notably, CDC25A expression has been shown to be upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been demonstrated to play a role in MPN pathogenesis and to drive myeloproliferation. Preclinical studies testing the combination of the CDK4/6 inhibitor ribociclib and ruxolitinib demonstrated reduction in spleen and liver size when compared to ruxolitinib alone in murine models, suggesting synergism (Rampal et al., Clinical Cancer Research 2021). Methods: Based on these promising preclinical data, we hypothesize that combining ruxolitinib with a CDK4/6 inhibitor could have synergistic effects in patients with primary or secondary MF who have an inadequate response to ruxolitinib monotherapy. In this multicenter, phase I dose-escalation trial we evaluate the safety of combination therapy with ruxolitinib and the CDK4/6 inhibitor abemaciclib. Patients with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to ruxolitinib are eligible for enrollment. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS (Emanuel at al. JCO 2012). (Table 1). This study follows a conventional "3+3” dose-escalation design, in which patients on a stable dose of ruxolitinib (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of ruxolitinib and abemaciclib (Figure 1). The primary endpoint of this study is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF patients on a stable, standard dose of ruxolitinib. Secondary endpoints include overall response rate (defined as a composite of complete remission [CR], partial remission [PR], and clinical improvement [CI; defined as anemia, spleen or symptom response]), rates of CR, PR, and CI as well as progression-free survival (PFS), overall survival (OS), and duration of response (DOR) as defined in accordance with the modified 2013 consensus definitions proposed by the International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN). As part of this clinical trial several correlative studies are planned to identify biomarkers predictive of response to ruxolitinib and abemaciclib as well as mechanisms of resistance (e.g., MAP kinase pathway activation). These include evaluation of changes in variant allele fraction of driver mutations, gene expression profile, cell cycle, clonal architecture, cytokine profile, and bone marrow fibrosis, aiming to inform patient selection as well as other alternative combination therapies for future clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

F-Box Protein 11 Suppresses Cell Proliferation and Aerobic Glycolysis in Glioblastomas by Mediating the Ubiquitin Degradation of Cdc25A.

Glioblastoma is a malignant CNS tumor with an extremely poor prognosis. F-box protein 11 (FBXO11) has E3 ubiquitin ligase activity and participates in the pathogenesis of multiple tumors but the role and mechanism of FBXO11 activity in glioblastoma remain unknown. In this study, FBXO11 was first observed to be downregulated in glioblastoma tissues and cell lines. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and colony formation assays and enzyme linked immunosorbent assay (ELISA) demonstrated that overexpression of FBXO11 suppressed proliferation and aerobic glycolysis and induced cell cycle arrest in U251-MG and A172 cells. FBXO1 decreased cell division cycle 25 A (Cdc25A) expression through ubiquitin degradation in a coprecipitation assay. A Western blot assay validated FBXO11 suppression of PKM2 dephosphorylation and c-Myc-mediated aerobic glycolysis via reduction of Cdc25A. In addition, a rescue experiment revealed that FBXO11 suppressed proliferation and aerobic glycolysis, both of which were reversed by overexpression of Cdc25A. FBXO11 overexpression also inhibited tumorigenesis via suppressing Cdc25A expression in vivo. These findings indicate that FBXO11 suppresses cell proliferation and aerobic glycolysis in glioblastomas by mediating the ubiquitin degradation of Cdc25A thereby providing insight into mechanisms of glioblastoma tumorigenesis and identifying a new potential therapeutic strategy.

Protective effect of liposomal nanoparticles-loaded with miR-499 antagonist on cardiomyocyte following acute myocardial infarction through suppression of CDC25A

Abnormal expression of miR-499 is related to progression of acute myocardial infarction (MI). This study aimed to explore the effect of liposomal nanoparticles carrying miR-499 antagonist on proliferation of cardiomyocytes and myocardial injury, to provide evidence for in-depth analysis of pathogenesis. With 10 sham-operated rats as control group, 10 rats were induced MI. The cardiac function and myocardial tissue morphology were detected. Myocardial cells were transfected with liposomal nanoparticles (blank group), miR-499 agonist (agonist group), liposomal nanoparticles carrying miR-499 agonist (carrier+antagonist group), or CDC25A activator and inhibitor. CDC25A, Akt, and mTOR expressions were determined in the myocardial cells upon treatments, as targeting relationships between miR-499 and CDC25A were detected by dualluciferase reporter gene assay. Myocardial cell proliferation and apoptosis were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Myocardial infarction (MI) rats exhibited myocardial damage and had irregular cardiac function indexes, and oxidative stress indexes with inflammatory cell infiltration and disordered myocardial architecture. miR-499 antagonist-loaded liposomal nanoparticles significantly elevated the ratio of viable cells, while cell viability was not altered in the other groups (P < 0.05). The miRNA-loaded nanomaterials induced decreased cell apoptosis, and overexpression of miR-499 increased apoptosis (P < 0.05). The expressions of CDC25A, Akt and mTOR proteins were increased by presence of miR-499 antagonist-loaded liposomal nanoparticles. However, silencing of CDC25A induced decreased viability, while the ratio of viable cells was increased in the CDC25A activator group (P < 0.05). There was a direct targeting relationship between miR-499 and CDC25A. It was found that Liposomal nanoparticles carrying miR-499 antagonist down-regulated the expression of CDC25A by down-regulating the expression of miR-499 to activate the Akt/mTOR signaling pathway, and enhanced the cardiomyocyte proliferation following MI.

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

The treatment with all-trans retinoic acid causes apoptosis without chromosomal instability in adipose-derived stem cells and might act starting the browning process

Introduction: The knowledge about the biology of obesity and apoptosis induction of adipose-derived stem cells (ADSCs) may support in the development of new therapies. Objective: The present research aimed to investigate the toxicogenic effect of all-trans retinoic acid (ATRA) as well as its influence in the adipogenic differentiation and expression of genes related to DNA damage, cell cycle and thermogenesis. Methodology: Cell cultures of human adipose-derived stem cells were treated for 12 hours with ATRA (20.75 μM) and were performed the adipogenic differentiation, comet assay, micronucleous, cell death, cell cycle and qPCR. Results: The ATRA treatment decreased the capacity of adipogenic differentiation of ADSCs. The comet assay demonstrated increase in nucleoid frequency with genomic damage and scoring. However, these damages were not fixed at the chromosome level, since the tail moment was not significant. The treatment with ATRA increased cytotoxicity and increased cell death by apoptosis. The relative expression of CHEK-1, CHEK-2, CDC25A, CDC25C, ATM and ATR decreased and only UCP1 increased significantly. Conclusion: The results of the present study demonstrate that the use of ATRA induces genomic damage in ADSCs, although it was eliminated in the apoptosis process. Therefore, administration of ATRA in ADSCs did not cause chromosomal instability, which suggests toxicogenic safety. Also it was considered that ATRA might active the browning effect. Thus, this compound is considered a promising candidate for the development of novel therapies for the treatment of obesity and / or localized fat by the use of mesotherapy.