Negative Regulator Of Cell Cycle Research Articles

2-Methoxyestradiol, an Endogenous 17β-Estradiol Metabolite, Induces Antimitogenic and Apoptotic Actions in Oligodendroglial Precursor Cells and Triggers Endoreduplication via the p53 Pathway.

The abnormal growth of oligodendrocyte precursor cells (OPCs) significantly contributes to the progression of glioblastoma tumors. Hence, molecules that block OPC growth may be of therapeutic importance in treating gliomas. 2-Methoxyestradiol (2ME), an endogenous tubulin-interacting metabolite of estradiol, is effective against multiple proliferative disorders. Based on its anti-carcinogenic and anti-angiogenic actions, it is undergoing phase II clinical trials. We hypothesize that 2ME may prevent glioma growth by targeting OPC growth. Here, we tested this hypothesis by assessing the impact of 2ME on the growth of an OPC line, "Oli-neu", and dissected the underlying mechanism(s). Treatment with 2ME inhibited OPC growth in a concentration-dependent manner, accompanied by significant upregulation in the expression of p21 and p27, which are negative cell-cycle regulators. Moreover, treatment with 2ME altered OPC morphology from multi-arm processes to rounded cells. At concentrations of 1uM and greater, 2ME induced apoptosis, with increased expressions of caspase 3, PARP, and caspase-7 fragments, externalized phosphatidylserine staining/APOPercentage, and increased mitochondrial activity. Flow cytometry and microscopic analysis demonstrated that 2ME triggers endoreduplication in a concentration-dependent fashion. Importantly, 2ME induced cyclin E, JNK1/2, and p53 expression, as well as OPC fusion, which are key mechanisms driving endoreduplication and whole-genome duplication. Importantly, the inhibition of p53 with pifithrin-α rescued 2ME-induced endoreduplication. The pro-apoptotic and endoreduplication actions of 2ME were accompanied by the upregulation of survivin, cyclin A, Cyclin B, Cyclin D2, and ppRB. Similar growth inhibitory, apoptotic, and endoreduplication effects of 2ME were observed in CG4 cells. Taken together, our findings provide evidence that 2ME not only inhibits OPC growth and triggers apoptosis, but also activates OPCs into survival (fight or flight) mode, leading to endoreduplication. This inherent survival characteristic of OPCs may, in part, be responsible for drug resistance in gliomas, as observed for many tubulin-interacting drugs. Importantly, the fate of OPCs after 2ME treatment may depend on the cell-cycle status of individual cells. Combining tubulin-interfering molecules with drugs such as pifithrin-α that inhibit endoreduplication may help inhibit OPC/glioma growth and limit drug resistance.

The cell cycle regulator p16 promotes tumor infiltrated CD8+ T cell exhaustion and apoptosis

The therapeutic efficacy of adoptive T cell therapy is largely restricted by reduced viability and dysfunction of CD8+ T cells. Continuous antigen stimulation disrupts the expansion, effector function, and metabolic fitness of CD8+ T cells, leading to their differentiation into an exhausted state within the tumor microenvironment (TME). While the function of the cell cycle negative regulator p16 in senescent cells is well understood, its role in T cell exhaustion remains unclear. In this study, we demonstrated that TCR stimulation of CD8+ T cells rapidly upregulates p16 expression, with its levels positively correlating with TCR affinity. Chronic TCR stimulation further increased p16 expression, leading to CD8+ T cell apoptosis and exhaustion differentiation, without inducing DNA damage or cell senescence. Mechanistic investigations revealed that p16 downregulates mTOR, glycolysis, and oxidative phosphorylation (OXPHOS) associated gene expression, resulting in impaired mitochondrial fitness, reduced T cell viability, and diminished effector function. Furthermore, the deletion of p16 significantly enhances the persistence of CD8+ T cells within tumors and suppresses the terminal exhaustion of tumor-infiltrating T cells. Overall, our findings elucidate how increased p16 expression reshapes T cell intracellular metabolism, drives T cell apoptosis and exhaustion differentiation, and ultimately impairs T cell anti-tumor function.

Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy.

During the last years, there has been an increased effort in the discovery of selective and potent kinase inhibitors for targeted cancer therapy. Kinase inhibitors exhibit less toxicity compared to conventional chemotherapy, and several have entered the market. Mirk/Dyrk1B kinase is a promising pharmacological target in cancer since it is overexpressed in many tumors, and its overexpression is correlated with patients' poor prognosis. Mirk/Dyrk1B acts as a negative cell cycle regulator, maintaining the survival of quiescent cancer cells and conferring their resistance to chemotherapies. Many studies have demonstrated the valuable therapeutic effect of Mirk/Dyrk1B inhibitors in cancer cell lines, mouse xenografts, and patient-derived 3D-organoids, providing a perspective for entering clinical trials. Since the majority of Mirk/Dyrk1B inhibitors target the highly conserved ATP-binding site, they exhibit off-target effects with other kinases, especially with the highly similar Dyrk1A. In this review, apart from summarizing the data establishing Dyrk1B as a therapeutic target in cancer, we highlight the most potent Mirk/Dyrk1B inhibitors recently reported. We also discuss the limitations and perspectives for the structure-based design of Mirk/Dyrk1B potent and highly selective inhibitors based on the accumulated structural data of Dyrk1A and the recent crystal structure of Dyrk1B with AZ191 inhibitor.

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state before initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators—including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (β-TrCP)—resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.

GSK-3β inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats.

Brain injury in preterm infants is a major cause of disability and mortality in children. GSK-3β is a common pathogenic factor for cognitive dysfunction and involves in neuronal proliferation and differentiation. However, GSK-3β affected neuronal differentiation and its molecular pathogenesis after hypoxic-ischemic brain damage in neonatal rats remains unclear. This study investigated the effects of GSK-3β inhibitor (TWS119) on cell cycle regulatory proteins, a neuronal differentiation factor (CEND1), maturation neurons, T-box brain transcription factor 1 (TBR1)-positive neurons to clarify the mechanisms of hypoxic-ischemic brain damage in neonatal rats. We used hypoxic-ischemic Sprague-Dawley neonatal rats with brain damage as models. These rats were used for investigating the effect of GSK-3β on cell cycle regulatory proteins, neuronal differentiation factor (CEND1), maturation neurons, TBR1-positive neurons by western blot and immunofluorescence. Cyclin D1 (a positive cell cycle regulator) expression decreased, and p21 (a negative cell cycle regulator) expression increased in the TWS119 group compared to the hypoxia-ischemia (HI) group 7 days after HI. Additionally, compared to the HI group, TWS119 treatment up-regulated CEND1 expression and promoted neuronal differentiation and cortex development based on NeuN and TBR1 expression. Our study suggests that the GSK-3β inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats by inhibiting cell cycle pathway.

Extracellular vesicle small RNA cargo discriminates non-cancer donors from pediatric B-lymphoblastic leukemia patients.

Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates.

Single-Cell Longitudinal Characterization of FL Heterogeneity and Residual Disease in the Bone Marrow from Low-Tumor Burden FL Enrolled in the Flirt Clinical Trial

Follicular Lymphoma (FL) remains a clinically challenging disease as most patients undergo a succession of clinical response/relapse cycles suggesting that current therapies do not eradicate residual tumor cells. Genetic studies suggest that FL relapses seed from a reservoir of rare Cancer Precursor Cells (CPCs), which likely remain resistant to treatment due to their homing to propitious niches such as the bone marrow (BM) and/or to the acquisition of a specific functional B-cell state. Yet, the rarity of such CPCs and the use of bulk sequencing technologies have so far hampered their direct characterization. Profiling CPC remains thus a major challenge in the FL field. Here, we identify and characterize FL cells before/after rituximab monotherapy within the BM of low tumor burden FL, looking for CPC-like cells supporting minimal residual disease. We harnessed a unique collection of longitudinal BM aspirates from 15 newly diagnosed low-tumor burden FL patients enrolled in the phase III clinical trial FLIRT (NCT02303119) evaluating Rituximab monotherapy and sampled at diagnosis (M0) and at one-year post-treatment start (M12). We generated a multi-modal single-cell dataset combining gene expression, B-cell receptor (BCR) and T-cell receptor (TCR) repertoire (Fig1A). We used BCR sequence analysis as a pivotal method to assign the clonal malignant identity within variably infiltrated BM at diagnosis and track the clonally related residual disease FL cells at M12. We compared FL B cell transcriptional heterogeneity after rituximab therapy. We also integrated BM T cell transcriptomes from both timepoints to study compositional changes of the BM microenvironment linked to a B-cell depletion treatment. We profiled 9,222 BM infiltrating FL cells. Single cell clustering showed a unique transcriptional signature for malignant cells of each FL patient, indicating a major inter-patient heterogeneity. By contrast, the non-malignant B cell clusters were mixed across patients and organized into transcriptional signatures encompassing normal B-cell differentiation steps (Fig1B, left). We then used unsupervised metaclustering of malignant B cells to uncover shared transcriptional states across patients, yielding 6 metaclusters (Mcl) expressing distinct gene expression programs. Mcl gene expression signatures observed in FL BM shared some similarities with the transcriptional programs observed in FL lymph nodes such as non-cycling Germinal Center-like (Mcl.1) or Mem-like (Mcl.3 and Mcl.4) states. This suggests that FL transcriptional heterogeneity is conserved between distinct niches. BM FL cells also upregulated several pathways, including glycolytic metabolism ( GAPDH), NF-κB signaling, negative regulators of cell cycle, or entry into quiescence programs. By analyzing BM samples obtained at M12, we were able to track and profile very rare clonally-related FL cells in 80% of FL patients with a range of 2 to 65 residual FL cells per patient. Those residual FL cells present a heterogeneous cellular phenotype partly redundant with the transcriptional heterogeneity found at diagnosis indicating that residual cells cover a plurality of cell states whose links with clinical outcome is underway. Differential expression analysis in one case also revealed a transcriptional shift in residual FL cells (including overexpression of BCL2) compared to BM tumor cells at diagnosis (Fig1B, right), providing novel insights into putative CPC identity with possible implications for personalized therapies. Finally, exploration of the FL T-cell microenvironment revealed abundant TFH-like cells at diagnosis that were barely detectable in healthy donors. Interestingly, high frequency of the TFH-like subset was maintained at remission suggesting a potential role as a niche for therapy-resistant B cells and later relapses. In summary, our single cell RNAseq analysis in the frame of a clinical trial provide the first longitudinal map of FL heterogeneity in the BM and characterize putative CPCs resistant to Rituximab-therapy. Ongoing efforts are now focusing on the microenvironment drivers of this intra-patient BM diversity and on specific molecular features of BM residual cells that could represent therapeutic vulnerabilities in this particular low-tumor burden FL population.

OR2802 A Novel CABLES1 Missense Variant Associated With Cushing's Disease Disrupts Protein Structure And Stability

Abstract Disclosure: A.L. Franco-Álvarez: None. M. Torres Morán: None. R.G. Rebollar-Vega: None. N. Pankratz: None. J. Lane: None. F.R. Faucz: None. P. Chittiboina: None. D.M. Kay: None. J.L. Mills: None. C.A. Stratakis: None. L.C. Hernández-Ramírez: None. Introduction. The adrenal-pituitary feedback loop is disrupted in Cushing's disease (CD). One of the glucocorticoid-responsive genes participating in this regulatory pathway is the negative cell cycle regulator CABLES1. We have previously reported five germline missense CABLES1 variants associated with CD and macroadenomas: two in young adults (p.E178K and p.L240F), two in children (p.G312D and p.D463G), and one in a middle-aged adult (p.P31L). The first four variants resulted in loss of the cell growth inhibition activity, but the fifth one has not been functionally validated. Aims. To report in detail a case of CD associated with the CABLES1 p.P31L variant and to characterize the molecular consequences of this variant using in silico and in vitro approaches. Methods. Variant detection was done via exome sequencing. Immunohistochemistry for CDKN1B and CABLES1 was performed on pituitary tumor slides. Loss of heterozygosity for CABLES1 p.P31L and common somatic variants were investigated in the tumor tissue. Protein stability studies were done for the CABLES1 variants p.P31L, p.L240F, p.G312D and p.D463G transiently expressed in HEK293 cells, using the cycloheximide chase method. The interaction of the CABLES1 variants with CDK2 was analyzed by transient expression in HEK293 cells and co-immunoprecipitation. Results. The p.P31L variant was found in a 53-year-old woman with CD. She achieved remission after resection of a macroadenoma (reported as a null-cell tumor). Recurrence one year later prompted a reintervention, and this a 60 mm Crooke cell adenoma was resected, again with postoperative remission. The p.P31L variant is of uncertain significance according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. The affected residue (P31) is part of a PPP motif that lies within a proline-rich region of CABLES1 (residues 30-52) within the N-terminal disorder, with unknown functional role. By immunohistochemistry, the corticotropinoma retained CABLES1 expression, but had lost 70% of CDKN1B expression. Loss of heterozgosity for CABLES1 p.P31L, as well as and hotspot BRAF, USP8, and USP48 were negative in the tumor. The p.P31L variant had an increased degradation constant (0.13±0.04) and a reduced half-life (5.80±1.93 hours), compared with wild type CABLES1 (degradation constant of 0.06± 0.02 and half-life of 13.52 ±5.11 hours; P=0.0036 for degradation constant and P=0.0091 for half-life). The rest of variants showed no significant differences. All variants retained their ability to interact with CDK2. Conclusions. The CABLES1 p.P31L variant encodes an unstable protein with a reduced half-life, which accordingly may result into a reduced tumor suppressor effect. Further studies are required to fully understand how CABLES1 loss of function impacts corticotroph cell growth. Presentation: Sunday, June 18, 2023

A novel small molecule inhibitor of p38⍺ MAP kinase augments cardiomyocyte cell cycle entry in response to direct cell cycle stimulation.

Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of four-cell cycle factors, CDK1, CDK4, cyclin B1 and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridine-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38 MAP kinase activity assay showed that NDPPC inhibits p38⍺ with 5-10 times lower IC50 compared to the other P38 isoforms in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. NDPPC is a novel inhibitor for p38 MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.

Identification of quiescent FOXC2+ spermatogonial stem cells in adult mammals

In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2+ SSCs and the ablation of FOXC2+ SSCs led to the depletion of the undifferentiated spermatogonia pool. During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.

Identification of quiescent FOXC2+ spermatogonial stem cells in adult mammals.

In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2+ SSCs and the ablation of FOXC2+ SSCs led to the depletion of the undifferentiated spermatogonia pool. During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.

Study on the mechanism of action of colchicine in the treatment of coronary artery disease based on network pharmacology and molecular docking technology.

Objective: This is the first study to explore the mechanism of colchicine in treating coronary artery disease using network pharmacology and molecular docking technology, aiming to predict the key targets and main approaches of colchicine in treating coronary artery disease. It is expected to provide new ideas for research on disease mechanism and drug development. Methods: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Swiss Target Prediction and PharmMapper databases were used to obtain drug targets. GeneCards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), DrugBank and DisGeNET databases were utilized to gain disease targets. The intersection of the two was taken to access the intersection targets of colchicine for the treatment of coronary artery disease. The Sting database was employed to analyze the protein-protein interaction network. Gene Ontology (GO) functional enrichment analysis was performed using Webgestalt database. Reactom database was applied for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Molecular docking was simulated using AutoDock 4.2.6 and PyMOL2.4 software. Results: A total of 70 intersecting targets of colchicine for the treatment of coronary artery disease were obtained, and there were interactions among 50 targets. GO functional enrichment analysis yielded 13 biological processes, 18 cellular components and 16 molecular functions. 549 signaling pathways were obtained by KEGG enrichment analysis. The molecular docking results of key targets were generally good. Conclusion: Colchicine may treat coronary artery disease through targets such as Cytochrome c (CYCS), Myeloperoxidase (MPO) and Histone deacetylase 1 (HDAC1). The mechanism of action may be related to the cellular response to chemical stimulus and p75NTR-mediated negative regulation of cell cycle by SC1, which is valuable for further research exploration. However, this research still needs to be verified by experiments. Future research will explore new drugs for treating coronary artery disease from these targets.

Association of gene expression in ovarian granulosa cells with embryonic development potential among patients with polycystic ovary syndrome

To assess the association of gene expression with development potential of early embryos derived from patients with polycystic ovary syndrome (PCOS). Three pairs of infertile patients with respectively matched age, body mass index, ovarian reserve and treatment with gonadotrophin-releasing hormone (GnRH) antagonists were selected. Patients with fewer embryos were assigned as the case group (n = 3), whilst the remainders were assigned as the control group (n = 3). Ovarian granulosa cells from patients were collected for the extraction of total RNA and subjected to RNA sequencing. The results were subjected to differential gene expression and functional enrichment analyses. Compared with the control group, 76 genes were up-regulated and 110 genes were down-regulated in the case group. The level of estradiol (E2) was significantly higher in the control group on the trigger day with the injection of human chorionic gonadotrophin (HCG). Compared with the control group, the KRT7 gene was most significantly up-regulated, whilst the CCNYL2 gene was most significantly down-regulated in the case group. Gene ontology (GO) entries enrichment has found those associated with chromosome segregation, cell cycle regulation, and fatty acid metabolism to be significantly enriched. The genes participating in the regulation of cell assembly, differentiation, negative regulation of cell cycle, negative regulation of development, extracellular regulated protein kinases (ERK), ERK1 and ERK2 signaling pathways to be significantly down-regulated. KEGG enrichment analysis of cell signaling pathways revealed that steroid hormone biosynthesis-related genes were enriched. Among patients treated with GnRH antagonists, the significant difference in the number of oocytes fertilized in vitro and the number of available embryos are associated with the difference in the expression of genes of ovarian granulosa cells.

Abstract 11801: Glycoprotein Nonmetastatic Melanoma Proteinbbregulates Lysosomal Integrity and Lifespan of Senescent Endothelial Cells

Introduction: The accumulation of senescent cells during aging relates to the development of various age-related pathologies including atherosclerosis and heart failure, which can be improved by specific elimination of senescent cells, so called “senolysis”. We have identified glycoprotein nonmetastatic melanoma protein B (GPNMB) as an antigen specifically expressed by senescent endothelial cells, so called “seno-antigen”. Also, we previously developed the vaccine against Gpnmb, eliminated senescent cells in mice, leading to an improvement of age-related pathologies. However, detailed function of GPNMB in senescent cells is still uncertain. Purpose: To elucidate the role of GPNMB in senescent cells and cardiovascular-related pathologies. Methods: Overexpression or depletion of GPNMB in endothelial cells (ECs) were generated. Cellular senescence was induced in ECs by doxorubicin. Gpnmb-knockout or overexpressed (Gp-KO or Gp-OE) mice were generated and imposed to hind-limb ischemia treatment or high-fat-diet feeding to evaluate vascular functions. Double transgenic mice (ApoE-KO and Gp-KO or Gp-OE) were also generated and imposed to high-fat diet to develop atherosclerosis. Results: The depletion of GPNMB in ECs was proved to shorten their replicative lifespan, and increase the expression of negative cell cycle regulators including p53, p21 and p16. Conversely, overexpressed GPNMB protected ECs from senescence stress. In vivo studies showed the impairment of vascular function, atherogenesis, and endothelium-dependent vasodilatation were enhanced in Gp-KO mice, but attenuated in Gp-OE mice. Furthermore, GPNMB was found to localize on lysosomal membrane. Cells with depleted GPNMB demonstrated the accumulation of dysfunctional lysosomes, indicated the contribution of GPNMB in maintaining lysosome integrity. Under senescence-associated lysosomal stress, elevated GPNMB expression was detected, contributing to senescent cells survival. Conclusions: GPNMB acts as a protective factor to senescent cells. Seno-antigen targeting GPNMB is possibly considered as an efficient strategy against cardiovascular diseases and other age-associated disorders with higher selectivity and fewer off-target effects.

In Silico Studies on GCP-Lys-OMe as a Potential 14-3-3σ Homodimer Stabilizer.

14-3-3 sigma is a vital negative cell cycle regulator. Its expression is consistently downregulated in many types of cancer through gene promoter hypermethylation or proteasomal degradation. 14-3-3 sigma needs to form a homodimer to be functional, while dimers are less prone to degradation than monomers. This suggests that a homodimer stabilizer may increase the tumor suppressive activities of 14-3-3 sigma. However, no known homodimer stabilizer of 14-3-3 sigma has been reported to date. Therefore, this study attempts to test the potential capability of GCP-Lys-OMe (previously reported to bind at the dimer interface of 14-3-3 zeta isoform), to bind and stabilize the 14-3-3 sigma homodimer. In silico docking of GCP-Lys-OMe on 14-3-3 sigma showed more favorable interaction energy (−9.63 kcal/mole) to the dimer interface than 14-3-3 zeta (−7.73 kcal/mole). Subsequent 100 ns molecular dynamics simulation of the GCP-Lys-OMe/14-3-3 sigma complex revealed a highly stable interaction with an average root-mean-square deviation of 0.39 nm (protein backbone) and 0.77 nm (ligand atoms). More contacts between residues at the homodimer interface and a smaller coverage of conformational space of protein atoms were detected for the bound form than for the apo form. These results suggest that GCP-Lys-OMe is a potential homodimer stabilizer of 14-3-3 sigma.

Antiproliferative effects of dried Moringa oleifera leaf extract on human Wharton’s Jelly mesenchymal stem cells

Mesenchymal stem cells (MSCs) have seen an elevated use in clinical works like regenerative medicine. Its potential therapeutic properties increases when used in tandem with complementary agents like bio-based materials. Therefore, the present study is the first to investigate the cytotoxicity of a highly valued medicinal plant, Moringa oleifera, on human Wharton’s Jelly mesenchymal stem cells (hWJMSCs) and its effects on the cells’ gene expression when used as a pre-treatment agent in vitro. M. oleifera leaves (MOL) were dried and subjected to UHPLC-QTOF/MS analysis, revealing several major compounds like apigenin, kaempferol, and quercetin in the MOL, with various biological activities like antioxidant and anti-cancer properties. We then treated the hWJMSCs with MOL and noticed a dose-dependant inhibition on the cells’ proliferation. RNA-sequencing was performed to explain the possible mechanism of action and revealed genes like PPP1R1C, SULT2B1, CDKN1A, mir-154 and CCNB1, whose expression patterns were closely associated with the negative cell cycle regulation and cell cycle arrest process. This is also evident from gene set enrichment analysis where the GO and KEGG terms for down-regulated pathways were closely related to the cell cycle regulation. The Ingenuity pathway analysis (IPA) software further predicted the significant activation of (p < 0.05, z-score > 2) of the G2/M DNA damage checkpoint regulation pathway. The present study suggests that MOL exhibits an antiproliferative effect on hWJMSCs via cell cycle arrest and apoptotic pathways. We believe that this study provides an important baseline reference for future works involving MOL’s potential to accompany MSCs for clinical works. Future works can take advantage of the cell’s strong anti-cancer gene expression found in this study, and evaluate our MOL treatment on various cancer cell lines.

Abstract P1029: Improving Cardiomyocyte Cell Cycle Entry In Response To Direct Cell Cycle Stimulation Using A Novel Small Molecule Inhibitor Of P38

Background: Myocardial infarction is the leading cause of mortality globally, due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we showed that overexpression of a combination of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), induced cell division in ~15% of post-mitotic mouse, rat, and human CMs. The aim of the current study is to identify novel small molecules that further augment CM cycle induction caused by the 4F. Methods and Results: We performed a drug screening of 30 chemical compounds with possible cell cycle regulatory activities using 60-day-old mature hiPS-CMs overexpressing the 4F and assessed the increase in EDU (DNA synthesis marker) and PHH3 (G2-M phase marker). The top hit was N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC). NDPPC showed a dose-dependent increase in the percentage of EDU and PHH3 positive nuclei in hiPS-CMs transduced with 4F. CMs transduced with 4F and treated with 1 μM NDPPC showed a significant increase in the percentage of EDU and PHH3 compared with vehicle (V)-treated CMs (PHH3; V: 20.41±2.02%, NDPPC: 33.75±3.9%, EDU; V: 18.24±1.7%, NDPPC: 30.81±2.1% n=6, p&lt;0.001). In Silico Drug-Target prediction showed that NDPPC could interact with p38 mitogen-activated protein kinase (P38), a critical negative regulator of mammalian cell cycle. Therefore, we overexpressed P38, in the presence and absence of NDPPC and 4F. As expected, overexpression of P38 in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect (in presence of 4F PHH3%; V: 20.38±0.97%, P38 overexpression+V: 11.02±1.3%, P38 overexpression+NDPPC: 23.62±2.79, n=4, P&lt;0.001). Knockdown of P38 showed a significant increase in the percentage of EDU and PHH3 positive nuclei in 4F treated CMs, and treatment with NDPPC did not show any further increase in cell cycle induction. These data indicate that NDPPC enhances the 4F cell cycle response in CMs through inhibition of P38. Conclusions: NDPPC is a novel inhibitor for P38. NDPPC is a promising drug to promote CM cell cycle response to the 4F. Further work in vivo is needed to test whether NDPPC could improve the 4F effect on cardiac function after ischemic injury.

Tubulin TUBB4B Is Involved in Spermatogonia Proliferation and Cell Cycle Processes.

Tubb4b (tubulin β-4b chain) is essential for cell growth and development as a microtubule network protein. Previous studies have shown that TUBB4B affects mouse pronucleus migration, but the gene function has yet to be elucidated. To study TUBB4B-related functions in mouse reproductive development, we designed a single sgRNA in chromosome 2 and generated a knockout spermatogonia cell line of the β-tubulin isoform Tubb4b by the CRISPR/Cas9 system. Tubb4b-KO spermatogonia recognized abnormal lysosomal membranes and cell morphology defects. Compared to control mouse spermatogonia, the proliferation rate was significantly slower and cycling stagnated in the G1/0 population. Although spermatogonia lacking TUBB4B have abnormal divisions, they are not lethal. We detected the mRNA levels of the cell-regulating cyclins CyclinsD1, CyclinsE, Cdk2, Cdk4, P21, Skp2 and the cell growth factors C/EBP α, C/EBP β, and G-CSF in the spermatogonia of Tubb4b-KO and found that the expressions of CyclinsD1, Skp2 and cell growth factors were significantly reduced. Further analysis revealed that 675 genes were expressed differently after Tubb4b deletion and were enriched in negative regulation of cell population proliferation (GO:0008285), negative regulation of cell cycle G2/M phase transition (GO:1902750), and positive regulation of cell death (GO: 0010942). We also found that there is a common gene Cdkn1a (P21) in these three GO pathways related to cell proliferation and cell cycle, and both quantitative analysis and transcriptome sequencing results showed that the expression of this gene was up-regulated in Tubb4b knockout cells. This implies that Tubb4b may be involved in the division of spermatogonia with multiple cell cycle regulatory proteins. Overall, these data indicate that Tubb4b has a specific role in regulating spermatogonia proliferation and cell cycle.

Abstract 946: Utility of intestinal organoids to model serrated colon cancer in vitro

Abstract Serrated colorectal cancer (CRC) is an aggressive and treatment-resistant form of colorectal carcinogenesis that accounts for one-fourth of the patients with CRC1. At the molecular level, serrated CRC is characterized by epigenetic modifications termed the CpG Island Methylator Phenotype (CIMP) and the presence of somatic mutations that activate the mitogen-activated protein kinase (MAPK) pathway1,2,3. The BRAFV600E is an early genetic change in serrated polyps, but it is not sufficient to drive tumor formation. Combinations of mutations and epigenetic modifications in P16INK4A, MLH1, ZNFR3, RNF43, and TGFBR2 are often seen in most patients4. Therefore, a combinatorial approach to develop BrafV600E-driven serrated CRC organoids harboring a variety of the above mutants will enable in vitro studies of the tumorigenic potential of each combination as well as allow engraftment into animals. In turn, the latter will allow for applying preclinical in-vivo pharmacology modalities in vitro, thereby bypassing the time required to create a specific knock-in/out strain for each genetic modification desired. Here, we describe a pipeline to directly genetically manipulate specific organoid systems in vitro. We first established organoid cultures from the small and large intestine of unique Tet-inducible RNAi mice where efficient gene knock-down can contribute to microsatellite instability. Using CRISPR/Cas9, we then edited these to generate CRC organoid models containing the BrafV600E mutation in combination with knock outs or knock-ins of cell cycle regulators and negative regulators of the Wnt pathway. These deficiencies mimic the progressive mutational changes upon acquisition BrafV600E. Our data lay the groundwork for future use of RNAi technology in combination with sequential gene editing for in vitro pharmacology and modeling diseases with complex genetic architecture both in vitro and in vivo. Citation Format: Prem K. Premsrirut, Huaien Wang, Eduardo J. Garcia Reino, Isabella Breen, Ana A. Vasileva. Utility of intestinal organoids to model serrated colon cancer in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 946.

Abstract 3987: Brain penetrant HDAC and PI3K/mTOR inhibitors synergize to induce DIPG cell death

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. We have previously shown that the brain penetrant class 1 HDAC inhibitor RG2833 (RGFP109) inhibits cell growth (MTS assay for HSJD007 p=0.0004, JHH-DIPG1 p=0.001, SF-7761 p=0.04, SU-DIPG13 p=0.01 by t-test), reduces proliferation (phospho-Rb Western blot, BrdU immunofluorescence HSJD007 p=0.008, JHH-DIPG1 p=0.0002 by t-test), and induces apoptosis (cPARP Western blot, cleaved caspase 3 immunofluorescence HSJD007 p&amp;lt;0.003, JHH-DIPG1 p=0.0026 by t-test) in multiple DIPG cell lines. We have also shown that treatment of a DIPG flank tumor mouse model with RG2833 alone for 1 week suppresses flank tumor growth (p&amp;lt; 0.005 by t-test) and induces apoptosis (Western blot for cPARP). In a DIPG orthotopic mouse model, we confirmed that RG2833 100mg/kg has on target activity in the mouse brain (Western blot for histone 3 acetylation) without causing toxicity. We next sought to identify a rational combination therapy to enhance RG2833 efficacy against DIPG. We found that RG2833 increases expression of negative cell cycle regulators p16, p21, and p27 and decreases expression of anti-apoptotic proteins BCL-2, BCL-XL, and XIAP (assessed by Western blot). Given that PI3K/mTOR pathway is upregulated in DIPG and plays an important role in cell cycle regulation via p21 and the FOXO signaling, we hypothesized that RG2833 would synergize with inhibitors of PI3K/mTOR. The brain penetrant PI3K/mTOR inhibitor Paxalisib is currently being evaluated in a phase 1 clinical trial of children with DIPG. We found that RG2833 has strong synergism with Paxalisib at clinically achievable doses (Loewe synergy scores by SynergyFinder for SF7761 and JHH-DIPG1 &amp;gt;20). Evaluation of combination treatment for apoptotic effects in vitro and in vivo are ongoing. These findings identify RG2833 and Paxalisib as a promising combination therapy for DIPG. Citation Format: Katherine Barnett, Hyuk Jean Kwon, Orlandi Novak, Charles Eberhart, Eric Raabe. Brain penetrant HDAC and PI3K/mTOR inhibitors synergize to induce DIPG cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3987.