Regulation Of Cyclin-dependent Kinases Research Articles

Sanggenol L Induces Apoptotic Cell Death by Activating the Caspase/AIF/p53 Signaling Pathway in Breast Carcinoma

Background Breast carcinoma is a deadly malignancy with a greater risk of morbidity and death, which has contributed significantly to the sharp rise in cancer cases. It has a recognized impartial effect on women. The need for a variety of treatment choices is prompted by the current therapy modules’ lack of effectiveness and adverse effects. Purpose In the present investigation, we examined the therapeutic value of Sanggenol L (San L) in human breast carcinoma cells. San L impacts on carcinoma of the breast cells are not fully understood in terms of its molecular and cellular pathways. Methods In order to assess the effectiveness of San L, this investigation employs a number of qualitative approaches, such as cytotoxicity analysis, sulforhodamine B (SRB), 4′,6-diamidino-2-phenylindole (DAPI), reactive oxygen species (ROS), and cell cycle assessment, which were conducted using breast cancer BT-474 cells. Additionally, cell death caused by San L through caspase-dependent and independent apoptosis has been investigated with western blot analysis. Results and Conclusion San L therapy significantly diminished the ability of BT-474 breast carcinoma cells to proliferate and impeded colony formation while having no impact on HBL-100 normal breast epithelial cells in a dose- and time-dependent way. San L treatment resulted in cell cycle arrest and cell death in breast carcinoma cells, which were characterized by acridine orange/ethidium bromide (AO/EtBr), DAPI, and flow cytometry at time-dependent way. The present study also explained that the half-maximal inhibitory concentration (IC50) concentration of (21 and 17.3 µM/mL) San L treatment induced caspase-dependent apoptosis [enhanced the expression of Bax and decreased the expression of B-cell lymphoma 2 (Bcl-2), procaspase-3, -8, and -9], stimulation of caspase-independent apoptosis [upregulation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) on cytosol], and suppression of cell cycle [downstairs regulation of cyclin-dependent kinase 1/2 (CDK1/2), cyclin D1, cyclin E, or upregulation of p53] in breast tumor cells. According to these findings, San L causes both caspase-dependent and independent apoptosis by triggering p53 in breast carcinoma cells.

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50=3.94nM) with high efficacy (Dmax=96%) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

The cell cycle controls spindle architecture in Arabidopsis by activating the augmin pathway

To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on mitotic spindles. Here, we measured prime characteristics of the Arabidopsis mitotic spindle and built a three-dimensional dynamic model using Cytosim. We identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle morphology in Arabidopsis. We found that the augmin component ENDOSPERM DEFECTIVE1 (EDE1) is a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant rescue of ede1 resembled the spindle phenotypes of cycb3;1 and cdkb1 mutants and the protein associated less efficiently with spindle microtubules. Accordingly, reducing the level of augmin in simulations recapitulated the phenotypes observed in the mutants. Our findings emphasize the importance of cell-cycle-dependent phospho-control of the mitotic spindle in plant cells and support the validity of our model as a framework for the exploration of mechanisms controlling the organization of the eukaryotic spindle.

Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Myeloid cannabinoid CB1 receptor deletion confers atheroprotection in male mice by reducing macrophage proliferation in a sex-dependent manner.

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signalling in atherosclerosis. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1-deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on oestrogen receptor (ER)α-oestradiol signalling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing, and mitochondrial respiration among the key processes affected by CB1 signalling, which was supported by metabolic flux assays. Chronic administration of the peripherally restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism, and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. Impaired macrophage CB1 signalling is atheroprotective by limiting their arterial recruitment, proliferation, and inflammatory reprogramming in male mice. The importance of macrophage CB1 signalling appears to be sex-dependent.

Acquired control over proliferation and differentiation of ventricular cardiomyocytes to break down the mechanisms of cardiac repair

Abstract Funding Acknowledgements None. Loss of myocardial tissue remains a leading cause of morbidity and mortality by causing heart rhythm disturbances and heart failure. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. In order to find new curative options for these disabling cardiac diseases a shift of focus towards steering the heart to regenerate itself is needed. The aim of this study was to generate a robust ventricular cardiomyocyte model in which proliferation and differentiation could be tightly controlled to investigate cardiac regeneration. To accomplish this we have generated a line of immortalized neonatal rat ventricular cardiomyocytes (nrVMs) through expression of the cell cycle regulators cyclin-dependent kinase 4 and cyclin D1 (CDK4-D1). The CDK4-D1 expressing nrVMs were positive for the proliferation marker Ki67 in contrast to control nrVMs. Cardiomyogenic differentiation could still be induced after multiple passages (> passage 10) as evidenced by immunocytochemistry and optical voltage mapping. These contractile and excitable cells were indeed positive for the cardiac-specific sarcomeric proteins alpha actinin and troponin T in a sarcomeric pattern similar to native nrVMs. Passaging of the control nrVM cultures, however, ultimately led to a senescent cell population lacking sarcomeric markers after passage 3. Electrophysiological analysis of immortalized monolayers, after inducing cardiomyogenic differentiation, showed not only uniform propagation of electrical signal throughout the cultures, but also re-entrant activity although at low frequency. In conclusion, these data show that were able to immortalize ventricular cardiomyocytes by preserving their contractile and excitable phenotype upon significant expansion. Further optimisation of this model will lead to a research model of ventricular cardiomyocytes which will give us the opportunity to unravel the process of cardiomyogenesis and uncover mechanisms which could allow us to enhance the ability of the heart to regenerate itself.

Anti-pancreatic cancer activity of cassane diterpenoids isolated from the seeds of Caesalpinia sappan mediated by autophagy activation via ROS/AMPK/mTORC1 pathway

Three undescribed cassane diterpenoids, caesalpanins D-F (1-3), and seven known ones were isolated from the seeds of Caesalpinia sappan. Structures and absolute configurations of 1-3 were elucidated based on the extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, and ECD calculations. Structurally, compound 1 was the first example of 18-norcassane diterpenoid and 2 was a rare 20-norcassane diterpenoid having an unusual five-membered oxygen bridge between C-10/C-18. The anti-proliferative activity of 1, 3, and 4-10 against PANC-1cells (pancreatic ductal adenocarcinoma cell line) was evaluated, and phanginin H (4) was found to exhibit anti-cancer activity with IC50 value of 18.13±0.63μM. Compound 4 inhibited PANC-1cell growth by arresting the cell cycle at G2/M phase via regulation of cyclin-dependent kinases, and the self-renewal and metastasis of PANC-1cells by suppressing cancer cell stemness. Furthermore, compound 4 induced ROS generation and subsequently activated autophagy, which was demonstrated by the formation of autophagic vacuoles and dynamic change of autophagic flux. The induced ROS accumulation resulted in AMPK activation and subsequently regulation of mTORC1 activity and ULK phosphorylation, indicating that 4 triggered autophagy through ROS/AMPK/mTORC1 pathway. These findings suggested that 4 might potentially be an autophagy inducer for the therapy of pancreatic cancer.

CDK activity at the centrosome regulates the cell cycle

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis.

Stellera chamaejasme L. extract inhibits adipocyte differentiation through activation of the extracellular signal-regulated kinase pathway.

Stellera chamaejasme L. (SCL) is a perennial herb with demonstrated bioactivities against inflammation and metabolic dysfunction. Adipocyte differentiation is a critical regulator of metabolic homeostasis and a promising target for the treatment of metabolic diseases, so we examined the effects of SCL on adipogenesis. A methanol extract of SCL dose-dependently suppressed intracellular lipid accumulation in adipocyte precursors cultured under differentiation induction conditions and reduced expression of the adipogenic transcription factors PPARγ and C/EBPα as well as the downstream lipogenic genes fatty acid binding protein 4, adiponectin, fatty acid synthase, and stearoyl-CoA desaturase. The extract also promoted precursor cell proliferation and altered expression of the cell cycle regulators cyclin-dependent kinase 4, cyclin E, and cyclin D1. In addition, SCL extract stimulated extracellular signal-regulated kinase (ERK) phosphorylation, while pharmacological inhibition of ERK effectively blocked the inhibitory effects of SCL extract on preadipocyte differentiation. These results suggest that SCL extract contains bioactive compounds that can suppress adipogenesis through modulation of the ERK pathway.

Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells.

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERβ agonist, and genistein, an isoflavonoid from soybeans known to have ERβ-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

Live-cell imaging defines a threshold in CDK activity at the G2/M transition

Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.

The Dysregulation of Cyclin Dependent Kinase Regulators Role in SV40 Related Renal Cell Carcinoma

The purpose of this study was to explore the possible involvement of SV40 polyomavirus in the development of renal cell carcinoma (RCC) in patients from the province of Al-Najaf. The study analyzed 75 paraffin-embedded block tissues of RCC, collected from archives of AL-Sader Medical City, and some private histopathology laboratories in Najaf governorate. The patients included 45 males and 30 females, aged between 22 and 70 years. The study used advanced scientific techniques, including Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC), to detect the presence of SV40 and evaluate the expression state of Cyclin-Dependent Kinase Regulators (KAP or cyclin-dependent kinase inhibitor 3 (CDKN3) & Cyclin E1 markers). Hematoxylin and Eosin staining was used for diagnosing RCC. The study found that RCC is associated with the dysregulation of Cyclin-Dependent Kinase regulators (CDK), caused by the SV40 polyomavirus. The results of the IHC analysis showed an increased positive percentage for KAP or CDKN3 marker and a decreased positive percentage of Cyclin E1 marker. Additionally, the clear cell type was found to be the most common, accounting for 56% of the cases, while grade I was the most prevalent, representing 41.3% of the cases. Tumor stage type I was found to be higher, with 25 cases. PCR detected the presence of SV40 in 20 cases, accounting for 26.7% of the total cases studied. The study concluded that the Simian Virus 40 (SV40), particularly its Large T Antigen (Tag), affects CDK regulators and disrupts the delicate equilibrium of cell cycle regulation systems. Therefore, the study suggests a possible link between the development of renal cell carcinoma and the SV40 polyomavirus. The study recommends routine testing for the detection of RCC using PCR and IHC methods.

Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.

Myeloid cannabinoid receptor CB1 deficiency confers atheroprotection in a sex-specific manner

Abstract Aim Cardiovascular diseases (CVDs) are the leading cause of death globally, claiming an estimated 17.9 million lives each year. A better understanding of the underlying chronic inflammatory disease mechanisms in atherosclerosis might help finding novel treatments and reduce CVD deaths. In particular, the cannabinoid receptor CB1 has been implicated in atherosclerosis, while its cell-specific effects in this disease are not well understood. Methods Myeloid Cnr1 (CB1 encoding gene) knockout mice were generated on apolipoprotein E deficiency (Apoe-/-) background by crossing CB1flox mice with transgenic mice carrying Cre under control of the lysozyme M (LysM) promoter for selective expression in myeloid cells. Age - and sex-matched groups were analyzed at baseline, 4 and 16 weeks Western diet (0,15% cholesterol). For in vitro experiments, murine bone marrow-derived macrophages (BMDM) were used. Results Male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores compared to controls, while only minor effects in females were observed. Male Cnr1 deficient mice had reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in vitro in BMDMs and blunted by female hormone estradiol treatment. Array-based kinase activity profiling of BMDMs stimulated with a synthetic CB1 agonist revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling via bulk RNA-sequencing of CB1 agonist-stimulated BMDMs further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, again only in male mice. Conclusion Impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The biological effects of macrophage CB1 signaling seem to be more pronounced in male mice. This highlights the need to consider the biological sex as an important variable in preclinical studies.

Expression analysis of cytoskeleton regulator RNA and Cyclin Dependent Kinase Inhibitor 2B genes in breast cancer.

Breast cancer has been found to be associated with deregulation of several non-coding genes and mRNA coding genes. To assess expressions of CYTOR and CDKN2B in breast cancer and adjacent samples and find their relevance with clinical data. We enumerated expression level of CDKN2B and CYTOR in 43 newly diagnosed breast cancer samples and their adjacent specimens using real-time PCR method Expression data was judged using Wilcoxon matched-pairs signed rank test. CYTOR level was higher in tumors compared with adjacent tissues. Nevertheless, there was no difference in expression of CDKN2B between these two sets of tissues. ROC curve analysis showed that CYTOR levels can differentiate between tumoral and adjacent tissues with AUC, specificity and sensitivity values of 0.65, 37% and 92% (P= 0.017). There was a positive correlation between expression levels of CYTOR and CDKN2B genes in breast cancer tissues (r= 0.5 and P= 0.0008) as well as adjacent tissues (r= 0.79 and P< 0.0001). Relative expression level of CDKN2B in normal tissues was associated with clinical stage (P= 0.014). Moreover, relative expression level of CDKN2B in tumor tissues was associated with the body weight. There was no other association between expressions of CYTOR and CDKN2B and clinical or pathological variables. Cumulatively, this study offers evidence for involvement of these genes in the pathoetiology of breast cancer.

Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1

p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCFSKP2 (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCFSKP2. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.

Inhibition of cell cycle-dependent hyphal and biofilm formation by a novel cytochalasin 19,20‑epoxycytochalasin Q in Candida albicans

Biofilm-mediated drug resistance is a key virulence factor of pathogenic microbes that cause a serious global health threat especially in immunocompromised individuals. Here, we investigated the antihyphal and antibiofilm activity of 19,20‑epoxycytochalasin Q (ECQ), a cytochalasin actin inhibitor isolated from medicinal mushroom Xylaria sp. BCC1067 against Candida albicans. Remarkably, 256 µg/ml of ECQ inhibited over 95% of C. albicans hyphal formation after 24 h-treatment. Combined ECQ and lipid-based biosurfactant effectively enhanced the antihyphal activity, lowering required ECQ concentrations. Hyphal fragmentation and reduction of biofilm biomass, shown by SEM and AFM visualization of ECQ-treated biofilms, were well corelated to the reduced metabolic activities of young and 24 h-preformed C. albicans biofilms. Induced intracellular accumulation of reactive oxygen species (ROS) also occurred in accompany with the leakage of shrunken cell membrane and defective cell wall at increasing ECQ concentrations. Transcriptomic analyses via RNA-sequencing revealed a massive change (> 1300 genes) in various biological pathways, following ECQ-treatment. Coordinated expression of genes, associated with cellular response to drugs, filamentous growth, cell adhesion, biofilm formation, cytoskeleton organization, cell division cycle, lipid and cell wall metabolisms was confirmed via qRT-PCR. Protein–protein association tool identified coupled expression between key regulators of cell division cyclin-dependent kinases (Cdc19/28) and a gamma-tubulin (Tub4). They coordinated ECQ-dependent hyphal specific gene targets of Ume6 and Tec1 during different phases of cell division. Thus, we first highlight the antihyphal and antibiofilm property of the novel antifungal agent ECQ against one of the most important life-threatening fungal pathogens by providing its key mechanistic detail in biofilm-related fungal infection.

Mechanistic Investigation of a Combination of a Cell Cycle Inhibitor and an HDAC Inhibitor in Pancreatic Cancer Cells

<b>Abstract ID 18966</b> <b>Poster Board 433</b> Currently, pancreatic cancer has a 5-year survival rate of about 11%, making it one of the deadliest malignancies. Better treatment strategies are required for pancreatic cancer due to late diagnosis, early metastasis, and drug resistance. Tumor growth and metastasis are facilitated by changes in the epigenetic profile caused by DNA and histone alterations. Another key feature of the cancer is the ability to elude the control of growth suppressors that monitor cell cycle. We aimed to investigate the combination of drugs that therapeutically target histone deacetylase (HDAC) and the key cell cycle regulator cyclin-dependent kinase (CDK) in pancreatic cancer. We used panobinostat, a pan-HDAC inhibitor and abemaciclib, an FDA-authorized CDK4/6 inhibitor for breast cancer. Our preliminary <i>in&nbsp;vitro</i> results in pancreatic cancer cells shows synergistic cytotoxicity of the combination when administered concurrently. Additionally, the proliferation and clonogenic survival of pancreatic cancer cells significantly decreased as a result of the combined therapy. Our mechanistic research on how this potent combination affects histone modifications, gene expression, and cell cycle regulation will shed light on interactions between epigenetic modifications and cell cycle in pancreatic cancer cell death. Our <i>in&nbsp;vitro</i> findings indicate that combining medications that pharmacologically target two crucial components of pancreatic cancer proliferation has a significant potential for treating this deadly condition and, as such, merits additional research. The College of Pharmacy and Health Sciences at St. John9s University in New York provided funding for this study together with the support from the National Institute of General Medical Sciences (Award Number R16GM145557).

Abstract 1557: Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer

Abstract Breast cancer (BC) is the most prevalent cancer in women worldwide. Despite initial sensitivity to endocrine therapies in hormone receptor positive BC, high mortality is driven by treatment-resistant disease. While several highly effective targeted treatments have been developed, resistance is common in the metastatic setting, highlighting the need for new therapies. Cyclin-dependent kinases (CDKs) have emerged as important targets for cancer treatment due to their integral role in driving the cell cycle. CDK4/6 inhibitors have advanced strongly in ER+ BC and are now becoming standard-of-care. However, resistance is a major problem. To address this, we have targeted CDK7, a master regulator of the cell cycle CDKs and transcription processes, both commonly dysregulated in cancer. CDK7 inhibition selectively inhibits cancer cell growth due to their reliance on aberrant gene expression and/or unregulated cell cycle progression. ICEC0942 (Samuraciclib), discovered in our lab at Imperial College London, has recently progressed to Phase 2 clinical trials. Considering the current clinical development of this and other CDK7 inhibitors (CDK7i), it is important to determine which patients will best respond to Samuraciclib. Towards identifying modulators of response to Samuraciclib, we performed a genome-wide CRISPR/Cas9-knockout screen in MCF7 cells. The mTOR/PI3K pathway was identified as important for response to CDK7i. Knockout of mTOR activators reduced sensitivity to Samuraciclib, while knockout of mTOR repressors increased sensitivity. Next, we studied the role of mutations in the catalytic subunit of PI3K (PIK3CA), which occur in nearly 30% of BC cases, in response to Samuraciclib. We utilized an isogenic panel of MCF10A cell lines, which were either wild-type for PIK3CA or had a heterozygous knock-in of either of the two most common mutations (E545K or H1047R). Cell growth was monitored following long-term (42-day) treatment with Samuraciclib. The drug was washed out (day 15) to determine the role of PIK3CA mutations in drug-induced growth arrest. We find that although Samuraciclib effectively inhibits growth of all cell lines, regardless of PIK3CA mutation status, the longevity of growth arrest post-washout is promoted by activating PIK3CA mutations. Finally, we assessed the role of mTOR and its downstream effectors in Samuraciclib response. Cell proliferation of MCF7 cells treated with Samuraciclib or in combination with mTOR/PI3K/AKT inhibitors was measured via flow cytometry. Samuraciclib treatment arrested cell proliferation; however, proliferation was reverted in combination with an mTOR/PI3K/AKT inhibitor. In conclusion, our findings highlight the importance of an active mTOR/PI3K pathway in promoting response to Samuraciclib. This may help guide patient treatments and development of effective combination therapies. Citation Format: Kaste Jurgaityte, Georgina Sava, Chun Fui Lai, Hailing Fan, Van Nguyen, Laki Buluwela, Simak Ali. Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1557.

Dynamic changes of 3'UTR length during oocyte-to-zygote transition of in vitro pig embryos.

Alternative polyadenylation (APA) generates different 3'-untranslated regions (3'UTRs) to regulate gene expression and localization, and affects a variety of biological processes. Here, we characterized the 3'UTR dynamics during the oocyte-to-zygote transition by analysing our previously reported porcine single-cell RNA-seq (scRNA-seq) datasets (in vitro matured metaphase II (MII) oocytes, in vitro fertilized zygotes (IVF1) and parthenogenetically activated 1-cell embryos (PA1)). After IVF1 versus MII comparison, dynamic analyses of APA from RNA-seq (DaPars) method identified 139 mRNAs with significantly different 3'UTRs (padj .≤ .05), mainly enriched in cell cycle, regulation of cyclin-dependent protein kinase activity, histone modification, mRNA surveillance, and regulation of actin cytoskeleton. For PA1 versus MII comparison, 105 mRNAs with significantly different 3'UTRs (padj .≤ .05) were identified to be mainly enriched in intracellular transport, mitotic spindle organization, cell cycle, pyruvate metabolism and glycolysis/gluconeogenesis. Furthermore, there were 7 mRNAs with more significant 3'UTR differences (|△PDUI| ≥ 0.45 and |log2 [PDUI ratio]| ≥ 0.59) respectively in IVF1 versus MII (Lrp2bp, Mtfr2, Nhlrc2, Psip1, Smu1, Ssr1 and Wtap) and PA1 versus MII (Asf1b, Dimt1, Nap1l1, Ncoa4, Nudt21, Pnn and Rpl15) comparisons. Integrative genomics viewer analysis further identified that 3'UTRs of Psip1, Smu1, Ssr1 and Wtap had more than 140 nt average length changes, whereas those of Dimt1, Nap1l1 and Rpl15 were shortened with more than 460 nt. Regulatory elements (PAS, CPE, microRNA binding sites and m6 A sites) in 3'UTRs of different lengths were predicted. Our findings provide useful information to further investigate the molecular mechanism of 3'UTR in regulating the oocyte-to-zygote transition of pig embryos.