Promote Cell Division Research Articles

Interplays between auxin and GA signaling coordinate early fruit development.

Phytohormones and their interactions are critical for fruit development and are key topics in horticulture research. Auxin, together with gibberellic acid (GA), promotes cell division and expansion, thereby regulating fruit development and enlargement after fertilization. Auxin- and GA-related mutants show parthenocarpy (fruit formation without fertilization of the ovule) in many plant species, indicating that these hormones and possibly their interactions play a key role in the regulation of fruit initiation and development. Recent studies have shown clear molecular and genetic evidence that ARF/IAA and DELLA proteins interact with one another and regulate both auxin and GA signaling pathways in response to auxin and GA during fruit growth in horticultural plants such as tomato (the most studied fleshy fruit) and strawberry (the model for Rosaceae). These recent findings provide new insights into the mechanisms by which the plant hormones auxin and GA regulate fruit development.

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.

Superior stemness of a rapidly growing subgroup of isolated human auricular chondrocytes and the potential for use in cartilage regenerative therapy.

IntroductionIn cartilage regenerative medicine, transplanted chondrocytes contain a mixture of populations, that complicates the regeneration of uniform cartilage tissue. Our group previously reported that chondrocytes with higher chondrogenic ability could be enriched by selection of rapidly growing cells. In this study, the detailed properties of rapidly growing chondrocytes were examined and compared to slowly growing cells.MethodsHuman auricular chondrocytes were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and analyzed using flow cytometry, focusing on division rates as indicated by fluorescence intensity and cell morphology according to the forward scatter and side scatter. Rapid and slow growing cell groups were harvested on days 2 and 4 after CFSE labeling, and their ability to produce cartilage matrix in vitro was examined. To compare the chondrogenic ability in vivo, the cells were seeded on poly-l-lactic acid scaffolds and transplanted into nude mice. Gene expression differences between the rapid and slow cell groups were investigated by microarray analysis.ResultsOn day 2 after CFSE labeling, the rapidly growing cell group showed the highest proliferation rate. The results of pellet culture showed that the rapid cell group produced more glycosaminoglycans per cell than the slow cell group. The amount of glycosaminoglycan production was highest in the rapid cell group on day 2 after CFSE labeling, indicating high chondrogenic ability. Furthermore, microarray, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed upregulation of genes that promote cell division such as origin recognition complex subunit 1 and downregulation of genes that inhibit cell division such as cyclin dependent kinase inhibitor 1A. Besides cell cycle-related genes, chondrocyte-related genes such as serpin family B member 2, clusterin, bone morphogenetic protein 2, and matrix metalloproteinase 3 were downregulated, while fibroblast growth factor 5 which is involved in stem cell maintenance, and coiled-coil and C2 domain containing 2A, which is required for cilia formation, were upregulated.ConclusionThe results showed that the rapid cell group proliferated well and had more undifferentiated properties, suggesting a higher stemness. The present findings provide a basis for the use of the rapid cell group in cartilage regeneration.

Transcriptomics reveals the molecular mechanism underlying kiwifruit expansion following forchlorfenuron (CPPU) treatment

Kiwifruit is an economically and nutritionally valuable fruit. Forchlorfenuron (CPPU) is widely used to improve fruit set and yield of kiwifruit. However, the mechanism through which CPPU regulates fruit enlargement remains unclear. Physiological and RNA-seq data revealed that the greatest contribution of CPPU treatment to fruit growth occurred at 28 to 42 d after bloom (DAB). CPPU application accelerated sugar metabolism by increasing the content of sucrose through increasing the transcripts of genes such as <i>SUSY</i>, <i>HK</i>, and <i>FK</i> in sucrose metabolism. In the early stages of fruit development, up-regulation of the starch synthesis gene <i>AGPase</i> and the decomposition gene <i>β-AMY</i> indicated the occurrence of starch turnover. After CPPU treatment, genes related to the synthesis and signal transduction of auxin and gibberellins were down-regulated. During the early fruit developmental stage, the cytokinin content and the expression of signal transduction genes were increased. In addition, the expression of cyclin genes, related to cell division, was up-regulated within three weeks after CPPU treatment. This indicates that CPPU treatment promoted cell division during the fastest growth stage, which would lead to an increase in cell number and further increases in fruit size.

Calmodulin and Its Interactive Proteins Participate in Regulating the Explosive Growth of Alexandrium pacificum (Dinoflagellate).

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.

The Active Phytohormone in Microalgae: The Characteristics, Efficient Detection, and Their Adversity Resistance Applications.

Phytohormones are a class of small organic molecules that are widely used in higher plants and microalgae as chemical messengers. Phytohormones play a regulatory role in the physiological metabolism of cells, including promoting cell division, increasing stress tolerance, and improving photosynthetic efficiency, and thereby increasing biomass, oil, chlorophyll, and protein content. However, traditional abiotic stress methods for inducing the accumulation of energy storage substances in microalgae, such as high light intensity, high salinity, and heavy metals, will affect the growth of microalgae and will ultimately limit the efficient accumulation of energy storage substances. Therefore, the addition of phytohormones not only helps to reduce production costs but also improves the efficiency of biofuel utilization. However, accurate and sensitive phytohormones determination and analytical methods are the basis for plant hormone research. In this study, the characteristics of phytohormones in microalgae and research progress for regulating the accumulation of energy storage substances in microalgae by exogenous phytohormones, combined with abiotic stress conditions at home and abroad, are summarized. The possible metabolic mechanism of phytohormones in microalgae is discussed, and possible future research directions are put forward, which provide a theoretical basis for the application of phytohormones in microalgae.

Abscisic acid mediates the reduction of petunia flower size at elevated temperatures due to reduced cell division.

Elevated temperatures suppress cell division in developing petunia buds leading to smaller flowers, mediated by ABA. Flower size is one of the most important showy traits in determining pollinator attraction, and a central factor determining the quality of floricultural products. Whereas the adverse effects of elevated temperatures on showy traits have been described in detail, its underlining mechanisms is poorly understood. Here, we investigated the physiological mechanism responsible for the reduction of flower size in petunia under elevated temperatures. We found that the early stages of flower-bud development were most sensitive to elevated temperatures, resulting in a drastic reduction of flower diameter that was almost independent of flower load. We demonstrated that the temperature-mediated flower size reduction occurred due to a shorter growth period, and a lower rate of corolla cell division. Consistently, local application of cytokinin, a phytohormone that promotes cell division, resulted in recovery of flower dimensions when grown under elevated temperatures. Hormone analysis of temperature-inhibited flower buds revealed no significant changes in levels of cytokinin, and a specific increase of abscisic acid (ABA) levels, known to inhibit cell division. Moreover, local application of ABA on flower buds caused a reduction of flower dimensions as a result of lower levels of cell division, suggesting that ABA mediates the reduction of flower size at elevated temperatures. Taken together, our results shed light on the mechanism by which elevated temperatures decrease petunia flower size, and show that temperature-mediated reduction of flower size can be alleviated by increasing the cytokinin/ABA ratio.

Overexpression of PvWOX3a in switchgrass promotes stem development and increases plant height

Switchgrass (Panicum virgatum L.) is an important perennial, noninvasive, tall ornamental grass that adds color and texture to gardens and landscapes. Moreover, switchgrass has been considered a forage and bioenergy crop because of its vigorous growth, low-input requirements, and broad geography. Here, we identified PvWOX3a from switchgrass, which encodes a WUSCHEL-related homeobox transcription factor. Transgenic overexpression of PvWOX3a in switchgrass increased stem length, internode diameter, and leaf blade length and width, all of which contributed to a 95% average increase in dry weight biomass compared with control plants. Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences. These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants, which promotes cell division, elongation, and vascular bundle development. We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers, thinner internodes, and narrower leaf blades. Double transgenic switchgrass plants displayed significant increases in internode length and diameter, leaf blade width, and plant height but retained a tiller number comparable to that of plants expressing miR156 alone. Ultimately, the double transgenic switchgrass plants produced 174% more dry-weight biomass and 162% more solubilized sugars on average than control plants. These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture, fodder, and biofuel crops.

Coordinating Carbon Metabolism and Cell Cycle of Chlamydomonasreinhardtii with Light Strategies under Nitrogen Recovery.

Nutrient supplementation is common in microalgae cultivation to enhance the accumulation of biomass and biofunctional products, while the recovery mechanism from nutrient starvation is less investigated. In this study, the influence of remodeled carbon metabolism on cell cycle progression was explored by using different light wavelengths under N-repletion and N-recovery. The results suggested that blue light enhanced cell enlargement and red light promoted cell division under N-repletion. On the contrary, blue light promoted cell division by stimulating cell cycle progression under N-recovery. This interesting phenomenon was ascribed to different carbon metabolisms under N-repletion and N-recovery. Blue light promoted the recovery of photosystem II and redirected carbon skeletons into proteins under N-recovery, which potentially accelerated cell recovery and cell cycle progression. Although red light also facilitated the recovery of photosystem II, it mitigated the degradation of polysaccharide and then arrested almost all the cells in the G1 phase. By converting light wavelengths at the 12 h of N-recovery with blue light, red and white lights were proved to increase biomass concentration better than continuous blue light. These results revealed different mechanisms of cell metabolism of Chlamydomonas reinhardtii during N-recovery and could be applied to enhance cell vitality of microalgae from nutrient starvation and boost biomass production.

Unraveling the Cytoprotective Effects of Valproic Acid: A Transcriptomics Meta-Analysis of Transfusion Strategies for Hemorrhagic Shock and Traumatic Brain Injury

Introduction: Traumatic brain injury (TBI) results in widespread impairment of hemostasis, fibrinolysis, coagulation, endothelial function, and immune function. While damage control resuscitation (DCR) is a well-established treatment strategy for life-threatening hemorrhage, alternative treatment strategies should be applied to patients with concurrent TBI. Commonly used resuscitation fluids such as crystalloids or colloids have several disadvantages and may even be harmful when administered in large quantities. In contrast, pharmacologic agents, such as the histone deacetylase inhibitor valproic acid (VPA), have shown promising results in animal studies of TBI and hemorrhagic shock (HS). We previously showed that VPA not only decreases platelet hyper-activation and improves clot dynamics in in-vitro experiments, but also decreases transfusion requirements and improves survival in a porcine DCR model. In those animal models, VPA was administered in conjunction with fluid resuscitation such as fresh frozen plasma (FFP) or hextend (HEX). However, we wondered whether VPA itself induces cytoprotective properties that may underlie the restoration of hemostasis, endothelial function, and immune function that we observed in our models. This meta-analysis used computational biology to identify changes in the brain transcriptome due to VPA treatment that occurred independent of the chosen transfusion fluid.Methods: Swine underwent TBI+HS, kept in shock for 2 hours, and resuscitated with normal saline (control), FFP, FFP+VPA, HEX, or HEX+VPA (n=5/group; all VPA doses 300 mg/kg). After 6 hours of observation, brain RNA was isolated and gene expression was analyzed using a microarray. Gene expression data were normalized to a normal saline control group. Transcriptomic data were imported into iPathwayGuide to identify significantly enriched genes and Gene Ontology (GO) terms. Genes were considered to be differentially expressed if they exhibited a log-fold change (logFC) > 1.0 (fold change > 2) and a p-value < 0.05. The differences in gene expression where then summarized in a Venn-diagram. GO terms identified the Biological Processes with the greatest modulation based on both significance and number of DE genes. GO term P-values were corrected using Elim-pruning.Results: A total of 673 differentially expressed genes were identified. The FFP+VPA group exhibited 206 uniquely expressed genes and the HEX+VPA group 121. We found a total of 113 genes that were expressed in both the FFP+VPA and HEX+VPA groups, but not in the FFP and HEX only groups (Figure). Table 1 summarizes the 10 most up- and down-regulated genes that are only expressed in VPA groups (i.e. FFP+VPA and HEX+VPA). Unregulated genes specifically associated with VPA were involved in promotion of cell division, neurogenesis, cytoskeleton, and ion-channels, while down-regulated genes were involved in metalloproteins, neurodegenerative diseases, and cell cycle arrest. Significantly modulated Biological Processes identified by GO terms include: erythrocyte maturation, macrophage activation, microglial cell proliferation, signal transduction by P53, fibrinolysis and plasminogen activation, fibroblast migration, and neurogenesis.Conclusion: Overall, this meta-analysis suggests that VPA altered the expression of approximately 1/6 th of all genes that were differentially expressed in our cohort. These genes are involved in a variety of biological processes such as cell division, neurogenesis, coagulation, cytoskeleton, and inflammation. These results suggest that VPA treatment may promote an environment that favors the restoration of hemostasis, production of new neurons, removal of damaged cells, and attenuation of inflammation. Such findings suggest that VPA treatment alone may be a promising therapeutic for the treatment of life-threatening hemorrhage and TBI. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Transcriptome Profiling Reveals Insights Into the Mechanisms Related to Explosive Growth of Alexandrium pacificum

Alexandrium pacificum is an organism that has an important impact on the aquaculture industry and human health. In this study, the digital gene expression approach was used to conduct a comparative analysis of differentially expressed genes (DEGs) that influence the explosive growth of A. pacificum following five treatment conditions: normal culture (C), high phosphorus and manganese (M), high irradiance (G), low phosphorus (P), and low nitrogen (N). Compared with the C conditions, a total of 265, 320, 185, and 150 DEGs were detected in the M, G, P, and N treatment groups, respectively. Clustering analysis suggested that A. pacificum acclimated to explosive growth using similar mechanisms in the M and G conditions. Analysis of DEGs showed that upregulation of genes associated with the pentose phosphate pathway and photosynthesis may contribute to explosive growth. Unigenes involved in the cell cycle were also found to be upregulated to promote cell division. The DEGs identified in this study may allow for the elucidation of molecular mechanisms responsible for the explosive growth of A. pacificum.

The Elite Alleles of OsSPL4 Regulate Grain Size and Increase Grain Yield in Rice

Grain weight and grain number, the two important yield traits, are mainly determined by grain size and panicle architecture in rice. Herein, we report the identification and functional analysis of OsSPL4 in panicle and grain development of rice. Using CRISPR/Cas9 system, two elite alleles of OsSPL4 were obtained, which exhibited an increasing number of grains per panicle and grain size, resulting in increase of rice yield. Cytological analysis showed that OsSPL4 could regulate spikelet development by promoting cell division. The results of RNA-seq and qRT-PCR validations also demonstrated that several MADS-box and cell-cycle genes were up-regulated in the mutation lines. Co-expression network revealed that many yield-related genes were involved in the regulation network of OsSPL4. In addition, OsSPL4 could be cleaved by the osa-miR156 in vivo, and the OsmiR156-OsSPL4 module might regulate the grain size in rice. Further analysis indicated that the large-grain allele of OsSPL4 in indica rice might introgress from aus varieties under artificial selection. Taken together, our findings suggested that OsSPL4 could be as a key regulator of grain size by acting on cell division control and provided a strategy for panicle architecture and grain size modification for yield improvement in rice.

Mechanism of exosomal miR-155 derived from bone marrow mesenchymal stem cells on stemness maintenance and drug resistance in myeloma cells

ObjectiveThis study was to explore the effect of exosomal miR-155 derived from bone marrow mesenchymal stem cells (BMSCs) on stemness maintenance and drug resistance in MPC-11 multiple myeloma cells.MethodsMPC-11 cells were transfected with mimics or inhibitors of miR-155. miR-155 expression was detected by qRT-PCR, cell condition was observed, and the expression of stemness maintenance markers OCT-4 and Nanog was observed by immunofluorescence. The expression of proteins associated with the Hedgehog signaling pathway and drug resistance was evaluated by western blot. To investigate whether exosomes affect cell behavior by horizontal delivery of miR-155, MPC-11 cells were co-cultured with exosomes isolated from BMSCs that were transfected with mimics or inhibitors of miR-155. Cell proliferation and the expression of proteins related to stemness maintenance protein and drug resistance were examined.ResultsIn function assays, after miR-155-mimics transfection, the expression levels of proteins related to stemness maintenance marker, Hedgehog signaling, and drug resistance were increased in MPC-11 cells. BMSC-derived exosomes carrying miR-155 inhibited apoptosis, promoted cell division, and upregulated the expression of protein associated with stemness maintenance, Hedgehog signaling, and drug resistance.ConclusionTherefore, our findings indicate that exosomal delivery of miR-155 exerted the same effect as transfection did on the stemness maintenance and drug resistance of multiple myeloma cells.

The Role and Clinical Interest of Extracellular Vesicles in Pregnancy and Ovarian Cancer.

Ovarian cancer and pregnancy are two states in which the host immune system is exposed to novel antigens. Indeed, both the tumor and placenta must invade tissues, remodel vasculature to establish a robust blood supply, and evade detection by the immune system. Interestingly, tumor and placenta tissue use similar mechanisms to induce these necessary changes. One mediator is emerging as a key player in invasion, vascular remodeling, and immune evasion: extracellular vesicles (EVs). Many studies have identified EVs as a key mediator of cell-to-cell communication. Specifically, the cargo carried by EVs, which includes proteins, nucleic acids, and lipids, can interact with cells to induce changes in the target cell ranging from gene expression to migration and metabolism. EVs can promote cell division and tissue invasion, immunosuppression, and angiogenesis which are essential for both cancer and pregnancy. In this review, we examine the role of EVs in ovarian cancer metastasis, chemoresistance, and immune modulation. We then focus on the role of EVs in pregnancy with special attention on the vascular remodeling and regulation of the maternal immune system. Lastly, we discuss the clinical utility of EVs as markers and therapeutics for ovarian cancer and pre-eclampsia.

Astaxanthin overproduction of Phaffia rhodozyma PR106 under titanium dioxide stress by transcriptomics and metabolic regulation analysis

In this study, astaxanthin yield of Phaffia rhodozyma PR106 increased significantly under titanium dioxide (TiO2) stress, and the yield of lycopene and β-carotene also increased significantly, as well as the yield of violaxanthin and lutein significantly decreased; in addition, TiO2 stress promoted cell division and changed cell morphology of PR106. Then, the mechanism of increasing astaxanthin yield was studied by transcriptomics and related metabolic regulation. The results showed that astaxanthin accumulation in PR106 was not directly related to mRNA transcription and post-translational modifications regulation under TiO2 stress; TiO2 stress accelerated glucose uptake of yeast, promoted reuse of ethanol, and increased the formation of acetyl-CoA and ATP. The more carbon flux was shifted to astaxanthin synthesis pathway and weakened carotenoids accumulation in astaxanthin branch pathway to improve the astaxanthin production of PR106. The metabolism regulation of ROS could continue in the PR106 strain.

Iron-Sulfur Cluster Biogenesis as a Critical Target in Cancer.

Cancer cells preferentially accumulate iron (Fe) relative to non-malignant cells; however, the underlying rationale remains elusive. Iron–sulfur (Fe–S) clusters are critical cofactors that aid in a wide variety of cellular functions (e.g., DNA metabolism and electron transport). In this article, we theorize that a differential need for Fe–S biogenesis in tumor versus non-malignant cells underlies the Fe-dependent cell growth demand of cancer cells to promote cell division and survival by promoting genomic stability via Fe–S containing DNA metabolic enzymes. In this review, we outline the complex Fe–S biogenesis process and its potential upregulation in cancer. We also discuss three therapeutic strategies to target Fe–S biogenesis: (i) redox manipulation, (ii) Fe chelation, and (iii) Fe mimicry.

Cytokinin Regulation of Source-Sink Relationships in Plant-Pathogen Interactions.

Cytokinins are plant hormones known for their role in mediating plant growth. First discovered for their ability to promote cell division, this class of hormones is now associated with many other cellular and physiological functions. One of these functions is the regulation of source-sink relationships, a tightly controlled process that is essential for proper plant growth and development. As discovered more recently, cytokinins are also important for the interaction of plants with pathogens, beneficial microbes and insects. Here, we review the importance of cytokinins in source-sink relationships in plants, with relation to both carbohydrates and amino acids, and highlight a possible function for this regulation in the context of plant biotic interactions.

A First-In-Class, Humanized Antibody Targeting Alternatively Spliced Tissue Factor: Preclinical Evaluation in an Orthotopic Model of Pancreatic Ductal Adenocarcinoma

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

Abstract LB119: VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient

Abstract Spindle assembly checkpoint (SAC) prevents mis-segregation of chromosome and allows accurate division in mitosis. Mps1 (also known as TTK) is one of the essential kinases for recruitment of SAC proteins after chromosome segregation. Failure of SAC induces chromosome instability, mitotic catastrophe, and ultimately apoptotic cell death and Mps1 is induced or activated in rapidly dividing tumor cells. Therefore, Mps1 has been considered an attractive anti-cancer drug target. VRN081569 is a potent type I kinase inhibitor of Mps1, which stabilizes Mps1 kinase domain in a closed conformation by forming a tight interface with its kinase domain. This structural stabilization appears to increase cellular potency for the induction of aneuploidy and apoptosis (~ 10 nM or less GI50 value). The loss of retinoblastoma tumor suppressor gene (Rb1), which is prevalent in advanced cancers, dysregulates cell cycle and promotes cell division. In particular, the resistance to CDK4/6 inhibitors has been reportedly mediated by loss of the Rb1 gene. We found that Rb1-negative cells express higher level of Mps1 than Rb1-positive cells and VRN081569 efficiently induce apoptosis of Rb1-negative Triple Negative Breast Cancer (TNBC) cell line MDA-MB-468 than Rb1-positive TNBC MDA-MB-231. Especially we confirmed that VRN081569 induces caspase 3/7 activity faster in Rb1-negative cells (~50-fold EC50 of caspase 3/7 activity compared to Rb1-positive cells), and cell cycle analysis has shown that our compound induces a dose-dependent dysregulation of mitosis, resulting in a decrease in the frequency of the G0/G1 phase, and also induces aneuploidy and apoptosis. Finally, we confirmed a combination effects with anti-microtubule agents BCL-2 inhibitor and PARP-1 inhibitor, suggesting that modulation of two different cellular processes may overcome the lack of single agent activity for TNBC. Citation Format: Hong Ryul Jung, Yeonsil Kim, Haelee Kim, Ji-Hye Kwon, Dong-hyuk Seo, Jung Beom Son, Nam Doo Kim, Daekwon Kim, Sunghwan Kim. VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB119.

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division.

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.