Cell Cycle Kinase Research Articles

Cellular Effects of Ultraviolet-Radiated Reduced-Titanium Dioxide Nanoparticles on Human Hypopharyngeal Adenocarcinoma Cells.

The cellular effects of ultraviolet (UV)-radiated reduced-titanium dioxide (TiO₂) nanoparticles were investigated on human hypopharyngeal adenocarcinoma cells (FaDu). In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the viability of FaDu cells exposed to UV (254 nm) for 10 minutes, in the presence of reduced-TiO₂ nanoparticles in rutile, was dose- and time-dependently decreased. The UV-radiated reduced-TiO₂ suppressed the cell proliferation by inhibiting the expression of cell cycle kinase, cyclin dependent kinase 2 (Cdk2), and its functional regulators Cyclin E and Cyclin B1 as well as proliferation-regulating proteins of p85 regulatory sub-unit of phosphoinositide3-kinases (PI3K p85), phosphorylated protein kinase B (p-AKT/p-PKB) and phosphorylated mammalian target of rapamycin (p-mTOR). In addition, the mitochondria disintegration and deoxyribonucleic acid (DNA) damage were confirmed by detecting the accumulated Bax in cytoplasm, phosphorylated-H2A histone family member X (γ-H2AX) in chromosomes and phosphorylated checkpoint 2 (p-Chk2). Our results support that UV-activated reduced-TiO₂ in rutile sensitized UV-induced proliferation suppression of FaDu cancer cells by the enhanced photocat-alytic activity.

Thermal proteome profiling identifies PIP4K2A and ZADH2 as off‐targets of Polo‐like kinase 1 inhibitor volasertib

Polo-like kinase 1 (PLK1) is an important cell cycle kinase and an attractive target for anticancer treatments. An ATP-competitive small molecular PLK1 inhibitor, volasertib, has reached phase III in clinical trials in patients with refractory acute myeloid leukemia as a combination treatment with cytarabine. However, severe side effects limited its use. The origin of the side effects is unclear and might be due to insufficient specificity of the drug. Thus, identifying potential off-targets to volasertib is important for future clinical trials and for the development of more specific drugs. In this study, we used thermal proteome profiling (TPP) to identify proteome-wide targets of volasertib. Apart from PLK1 and proteins regulated by PLK1, we identified about 200 potential volasertib off-targets. Comparison of this result with the mass-spectrometry analysis of volasertib-treated cells showed that phosphatidylinositol phosphate and prostaglandin metabolism pathways are affected by volasertib. We confirmed that PIP4K2A and ZADH2-marker proteins for these pathways-are, indeed, stabilized by volasertib. PIP4K2A, however, was not affected by another PLK1 inhibitor onvansertib, suggesting that PIP4K2A is a true off-target of volasertib. Inhibition of these proteins is known to impact both the immune response and fatty acid metabolism and could explain some of the side effects seen in volasertib-treated patients.

Acceleration or Brakes: Which Is Rational for Cell Cycle-Targeting Neuroblastoma Therapy?

Unrestrained proliferation is a common feature of malignant neoplasms. Targeting the cell cycle is a therapeutic strategy to prevent unlimited cell division. Recently developed rationales for these selective inhibitors can be subdivided into two categories with antithetical functionality. One applies a “brake” to the cell cycle to halt cell proliferation, such as with inhibitors of cell cycle kinases. The other “accelerates” the cell cycle to initiate replication/mitotic catastrophe, such as with inhibitors of cell cycle checkpoint kinases. The fate of cell cycle progression or arrest is tightly regulated by the presence of tolerable or excessive DNA damage, respectively. This suggests that there is compatibility between inhibitors of DNA repair kinases, such as PARP inhibitors, and inhibitors of cell cycle checkpoint kinases. In the present review, we explore alterations to the cell cycle that are concomitant with altered DNA damage repair machinery in unfavorable neuroblastomas, with respect to their unique genomic and molecular features. We highlight the vulnerabilities of these alterations that are attributable to the features of each. Based on the assessment, we offer possible therapeutic approaches for personalized medicine, which are seemingly antithetical, but both are promising strategies for targeting the altered cell cycle in unfavorable neuroblastomas.

Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer.

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

Transcriptomic and genomic heterogeneity in blastic plasmacytoid dendritic cell neoplasms: from ontogeny to oncogenesis

Oncogenesis and ontogeny of blastic plasmacytoid dendritic cell neoplasm (BPDCN) remain uncertain, between canonical plasmacytoid dendritic cells (pDCs) and AXL+ SIGLEC6+ DCs (AS-DCs). We compared 12 BPDCN to 164 acute leukemia by Affymetrix HG-U133 Plus 2.0 arrays: BPDCN were closer to B-cell acute lymphoblastic leukemia (ALL), with enrichment in pDC, B-cell signatures, vesicular transport, deubiquitination pathways, and AS-DC signatures, but only in some cases. Importantly, 1 T-cell ALL clustered with BPDCN, with compatible morphology, immunophenotype (cCD3+ sCD3− CD123+ cTCL1+ CD304+), and genetics. Many oncogenetic pathways are deregulated in BPDCN compared with normal pDC, such as cell-cycle kinases, and importantly, the transcription factor SOX4, involved in B ontogeny, pDC ontogeny, and cancer cell invasion. High-throughput sequencing (HaloPlex) showed myeloid mutations (TET2, 62%; ASXL1, 46%; ZRSR2, 31%) associated with lymphoid mutations (IKZF1), whereas single-nucleotide polymorphism (SNP) array (Affymetrix SNP array 6.0) revealed frequent losses (mean: 9 per patient) involving key hematological oncogenes (RB1, IKZF1/2/3, ETV6, NR3C1, CDKN2A/B, TP53) and immune response genes (IFNGR, TGFB, CLEC4C, IFNA cluster). Various markers suggest an AS-DC origin, but not in all patients, and some of these abnormalities are related to the leukemogenesis process, such as the 9p deletion, leading to decreased expression of genes encoding type I interferons. In addition, the AS-DC profile is only found in a subgroup of patients. Overall, the cellular ontogenic origin of BPDCN remains to be characterized, and these results highlight the heterogeneity of BPDCN, with a risk of a diagnostic trap.

Impact of COVID-19 pandemic on STIs in Greece

<h3>Abstract</h3> The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However,...

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes.

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell-specific conditional knockout strategy, we show that Polo-like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.

DMDRMR-Mediated Regulation of m6A-Modified CDK4 by m6A Reader IGF2BP3 Drives ccRCC Progression.

Aberrant N 6-methyladenosine (m6A) modification has emerged as a driver of tumor initiation and progression, yet how long noncoding RNAs (lncRNA) are involved in the regulation of m6A remains unknown. Here we utilize data from 12 cancer types from The Cancer Genome Atlas to comprehensively map lncRNAs that are potentially deregulated by DNA methylation. A novel DNA methylation-deregulated and RNA m6A reader-cooperating lncRNA (DMDRMR) facilitated tumor growth and metastasis in clear cell renal cell carcinoma (ccRCC). Mechanistically, DMDRMR bound insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) to stabilize target genes, including the cell-cycle kinase CDK4 and three extracellular matrix components (COL6A1, LAMA5, and FN1), by specifically enhancing IGF2BP3 activity on them in an m6A-dependent manner. Consequently, DMDRMR and IGF2BP3 enhanced the G1-S transition, thus promoting cell proliferation in ccRCC. In patients with ccRCC, high coexpression of DMDRMR and IGF2BP3 was associated with poor outcomes. Our findings reveal that DMDRMR cooperates with IGF2BP3 to regulate target genes in an m6A-dependent manner and may represent a potential diagnostic, prognostic, and therapeutic target in ccRCC. SIGNIFICANCE: This study demonstrates that the lncRNA DMDRMR acts as a cofactor for IGF2BP3 to stabilize target genes in an m6A-dependent manner, thus exerting essential oncogenic roles in ccRCC.

Baicalein Represses Cervical Cancer Cell Growth, Cell Cycle Progression and Promotes Apoptosis via Blocking AKT/mTOR Pathway by the Regulation of circHIAT1/miR-19a-3p Axis.

BackgroundBaicalein has a significant anti-cancerous function in the treatment of cervical cancer (CC). Its functional mechanism regarding circular RNA (circRNA) hippocampus abundant transcript 1 (circHIAT1) and microRNA-19a-3p (miR-19a-3p) was explored in this research.MethodsCC cell viability and colony formation were determined using Cell Counting Kit-8 (CCK-8) and colony formation assay. Cell cycle progression and apoptosis were analyzed via flow cytometry. Protein markers of cell cycle, apoptosis and protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway were detected by Western blot. CircHIAT1 and miR-19a-3p levels were assayed through the quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between circHIAT1 and miR-19a-3p was validated by dual-luciferase reporter and RNA pull-down assays. In vivo experiment was performed by xenograft model.ResultsCC cell growth and cell cycle progression were repressed while apoptosis was enhanced by baicalein. MiR-19a-3p was downregulated in baicalein-treated CC cells and miR-19a-3p overexpression lightened the baicalein-induced CC progression inhibition. Moreover, circHIAT1 was found to be a sponge of miR-19a-3p in CC cells. Baicalein-induced cell growth inhibition, cell cycle arrest and apoptosis promotion were neutralized by knockdown of circHIAT1 via targeting miR-19a-3p. Baicalein acted on the circHIAT1/miR-19a-3p to inactivate AKT/mTOR pathway. Baicalein also reduced CC tumor growth in vivo via regulating the levels of circHIAT1 and miR-19a-3p.ConclusionThese findings demonstrated that the inhibitory function of baicalein in CC progression was dependent on the repression of AKT/mTOR pathway by upregulating circHIAT1 to sponge miR-19a-3p, showing a specific mechanism for baicalein in CC.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi‐site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high‐affinity “gatekeeper” site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity.

Measuring kinase activity in different in vivo contexts is crucial for understanding the mechanism and functions of protein kinases, such as the cyclin-dependent kinases (Cdks) and other cell cycle kinases. Here, I present the rationale and the experimental framework for an alternative approach to measure kinase activity that is based on estimating substrate phosphorylation rates in vivo. The approach presented was first developed for experiments performed to measure Cdk1 activity at different stages of the fission yeast S. pombe's cell cycle [Swaffer et al., Cell 167:1750-1761, 2016]. However, it also affords a more generalizable framework that can be adaptable to other systems and kinases, as long as specific, rapid, and reversible kinase inhibition is possible. Briefly this involves transient and reversible kinase inhibition to dephosphorylate kinase substrates in vivo, followed by quantitative measurements of phosphorylation after inhibition is removed.

Grape Seed Proanthocyanidins Induce Apoptosis and Cell Cycle Arrest of HepG2 Cells Accompanied by Induction of the MAPK Pathway and NAG-1.

Hepatocellular carcinoma (HCC) is one of the common malignancies leading to death. Although radiotherapy and chemotherapy have certain effects, their side effects limit their therapeutic effect. Phytochemicals have recently been given more attention as promising resources for cancer chemoprevention or chemotherapy due to their safety. In this study, the effects of grape seed proanthocyanidins (GSPs) on the apoptosis, cell cycle, and mitogen-activated protein kinase (MAPK) pathway-related proteins and non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression of HepG2 cells were investigated. The results showed that GSPs inhibited the viability of HepG2 cells in a time- and dose-dependent manner, induced apoptosis and G2/M phase cell cycle arrest, and regulated cell cycle-related proteins, cyclin B1, cyclin-dependent kinase 1, and p21. GSPs also increased reactive oxygen species production and caspase-3 activity. In addition, GSPs also increased the expression of p-ERK, p-JNK, p-p38 MAPK and NAG-1, and GSPs-induced NAG-1 expression was related to the MAPK pathway-related proteins. These data suggest that GSPs may be promising phytochemicals for HCC chemoprevention or chemotherapy.

CDK4/6 and MAPK-Crosstalk as Opportunity for Cancer Treatment.

Despite the development of targeted therapies and novel inhibitors, cancer remains an undefeated disease. Resistance mechanisms arise quickly and alternative treatment options are urgently required, which may be partially met by drug combinations. Protein kinases as signaling switchboards are frequently deregulated in cancer and signify vulnerable nodes and potential therapeutic targets. We here focus on the cell cycle kinase CDK6 and on the MAPK pathway and on their interplay. We also provide an overview on clinical studies examining the effects of combinational treatments currently explored for several cancer types.

Cell-cycle phospho-regulation of the kinetochore.

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure.

Enhancing the Immune Surveillance in Multiple Myeloma Via CDK4/6 Inhibition

Deregulation of cyclin D genes is a uniform event in multiple myeloma (MM) and represent a striking addiction as observed in pan-cancer genome-wide CRISPR screening data. However, early stage Cyclin D and other cell cycle kinases inhibitors have shown a lack of single agent activity suggesting that targeting of cell cycle regulation is insufficient to produce a durable response in MM. Recent evidence recognizes the Cyclin D and CDK4 activities within the immune tumor microenvironment, supporting a previously unrecognized immunomodulatory functions of CDK4/6. This is particularly important in MM, a highly heterogeneous disease that resides in a complex ecosystem comprising of immune, endothelial, and stromal cells. We here evaluated the tumor intrinsic and extrinsic effects of CDK4/6 inhibition in MM with the goal to define rationally designed combination strategies to effectively impact MM growth. We have evaluated MM cell sensitivity to CDK4/6 inhibitors (both Palbociclib and Abemaciclib) in a panel of 32 MM cell lines and primary MM patients' samples. As expected, both inhibitors were mostly cytostatic with G0/G1 cell cycle arrest and significant impact on the pRB-E2F axis both in vitro as well as in vivo. In luminescence subcutaneous SCID models engrafted with H929 or MM1S MM cells expressing an E2F-driven luciferase reporter, treatment with low dose Palbociclib or Abemaciclib caused regression of bulky tumors, evidenced by a ~40% reduction in tumor volume at the 14-day end-point and a decreased E2F1 reporter activity. We next studied genome-wide transcriptional response to treatment using RNA-seq analysis in two MM cell lines using multiple doses and duration (24 and 72 hours) to evaluate the effect of short and long exposure to the drug. Gene set enrichment analysis (GSEA) of RNA-seq data confirmed significant downregulation of proliferation and E2F target genes at 24 hours post treatment. Interestingly, after longer exposure to both drugs we observed modulation of signatures for Ag presentation (including upregulation of HLA-A,B, and C; B2MG), innate immune response (ICAM-1 and 2; IL-8 and several CCLs) and interferon inducible genes IFN response (IRF1, IRF9, STAT1, STAT2, STAT4, OSA1, OSA2, MX1). To confirm genomic data and evaluate if CDK4/6i promotes the induction of the senescence-associated secretory phenotype (SASP) program in MM cells, we performed cytokine profile to assess the secretion of 174 soluble factors in the MM cell supernatant upon CDK4/6i. We confirmed a significant increase of chemokines involved in NK cell recruitment (CCL2, CCL4, CCL5), as well as cytokines that promote NK cell proliferation and activation. Moreover, we found that intercellular adhesion molecule-1 (ICAM-1) and the NKG2D ligands ULBP2 and MICA, required for activation of NK cell cytotoxicity and tumor cell targeting, were induced after CDK4/6i in MM cells. Although not secretory per se, these NK cell ligands are part of the transcriptional module linked to the SASP. Overall, these data suggest that, in addition to a more stable cell cycle arrest, CDK4/6i may promote MM cell immune surveillance through induction of the SASP program. To further confirm the immune effects, we tested freshly isolated NK cells from MM patients and healthy donors using in vitro MM-NK cell coculture assay and observed enhanced degranulation and cytokine production (intracellular IFN-γ and TNFα) by NK cells in response to MM cells. We finally investigated the potential of combining CDK4/6i with daratumumab in a standard 4-h ADCC assays using NK cells from MM patients as effector and MM cell lines as target. Daratumumab-mediated ADCC against MM cells was significantly augmented in the combination compared to single agent. In conclusion, we here report a novel anti-MM activity of CDK4/6i which is beyond the previously reported growth arrest. The observed ability to directly modulate the immune system along with decrease in the proliferative potential of MM cells may provide opportunities to develop unique combination approaches in MM. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Fulciniti:NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Legend: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; C4: Current equity holder in private company.

Mechanism of miRNA-based Aconitum leucostomum Worosch. Monomer inhibition of bone marrow-derived dendritic cell maturation

Delvestidine (DLTD) is a monomeric compound isolated from Aconitum leucostomum Worosch, a widely used medicine for local treatment of rheumatoid arthritis (RA). Studies have shown that Aconitum leucostomum Worosch. can inhibit maturation of bone marrow-derived dendritic cells (BMDCs). Further, microRNAs (miRNAs) have regulatory effects on DC maturity and function. However, the mechanism underlying DLTD effects on DC maturity and RA remains to be elucidated. This study investigated whether DLTD-mediated inhibition of DC maturation is regulated by miRNAs. LPS-induced mature BMDCs were treated with DLTD for 48 h. CD80 and CD86 expression on BMDCs was detected by flow cytometry, and levels of inflammatory factors IL-6, IL-23, IL-1β, and TNF-α were detected by ELISA and PCR. Further, gene expression and miRNA expression profiles were investigated by bioinformatics analysis and verified by PCR. DLTD was found to inhibit CD80 and CD86 expression on the surface of BMDCs and secretion of inflammatory factors IL-6, IL-23, IL-1β, and TNF-α. In total, 54 differentially expressed miRNAs were detected, including 29 up-regulated and 25 down-regulated miRNAs after DLTD treatment. Analysis of biological information revealed that the differentially expressed target genes mainly regulated biological processes, including cell differentiation, cell cycle, and protein kinase complexes. Additionally, miR-511-3p downstream targets Calcr, Fzd10, and Eps8, were closely related to BMDCs maturation. DLTD may induce BMDCs maturity through regulation of miRNAs that affect Calcr, Fzd10, and Eps8 gene signals.

Accelerated aging among childhood, adolescent, and young adult cancer survivors is evidenced by increased expression of p16INK4a and frailty.

BackgroundCellular senescence, measured by expression of the cell cycle kinase inhibitor p16INK4a, may contribute to accelerated aging in survivors of childhood, adolescent, and young adult cancer. The authors measured peripheral blood T‐lymphocyte p16INK4a expression among pediatric and young adult cancer survivors, hypothesizing that p16INK4a expression is higher after chemotherapy and among frail survivors.MethodsA cross‐sectional cohort of young adult survivors and age‐matched, cancer‐free controls were assessed for p16INK4a expression and frailty. Newly diagnosed pediatric patients underwent prospective measurements of p16INK4a expression before and after cancer therapy. Frailty was measured with a modified Fried frailty phenotype evaluating sarcopenia, weakness, slowness, energy expenditure, and exhaustion.ResultsThe cross‐sectional cohort enrolled 60 survivors and 29 age‐matched controls with a median age of 21 years (range, 17‐29 years). The prospective cohort enrolled 9 newly diagnosed patients (age range, 1‐18 years). Expression of p16INK4a was higher among survivors compared with controls (9.6 vs 8.9 log2 p16 units; 2‐sided P = .005, representing a 25‐year age acceleration in survivors) and increased among newly diagnosed patients from matched pretreatment to posttreatment samples (7.3‐8.9 log2 p16 units; 2‐sided P = .002). Nine survivors (16%) were frail and had higher p16INK4a expression compared with robust survivors (10.5 [frail] vs 9.5 [robust] log2 p16 units; 2‐sided P = .055), representing a 35‐year age acceleration among frail survivors.ConclusionsChemotherapy is associated with increased cellular senescence and molecular age in pediatric and young adult cancer survivors. Frail survivors, compared with robust survivors, exhibit higher levels of p16INK4a, suggesting that cellular senescence may be associated with early aging in survivors.

Abstract 6273: TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination

Abstract Background: Basal-like breast cancer (BC) has the highest rates of local recurrence despite the use of radiation therapy. Therefore, approaches for radiosensitization are critically needed for patients with this subtype of BC. Methods: Four independent datasets were used to correlate gene expression with local recurrence (LR) and Kaplan-Meier analysis validated the impact of TTK expression on LR. The METABRIC dataset was used to determine TTK expression in BC subtypes. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mouse models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using Rad51 foci formation assays. Rescue experiments were performed using wild-type (WT) and kinase-dead (KD) TTK plasmids in combination with siRNA targeting the UTR region of TTK. Results: Ten genes were found to significantly correlate with early LR (≤3 years) after surgery and radiation across 4 independent datasets (N=896 pts), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in multiple cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p&amp;lt;0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like BC. Using inducible shRNA, the combination of TTK knockdown and RT increases radiosensitivity in multiple basal-like BC cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in multiple cell lines (rER 1.10-2.27). In vivo, TTKi, using shRNA or B909, in combination with RT led to delayed tumor growth and a significant increase in time to tumor tripling (Placebo: 9 days vs. B909+RT: undefined [&amp;gt;35 days], p&amp;lt;0.0001) in both cell line and PDX models. Increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. The efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, Rad51 foci formation was reduced by TTKi after RT compared to RT alone. Reintroduction of WT TTK, after knockdown of endogenous TTK, rescued radioresistance and HR efficiency, however, reintroduction of kinase-dead (KD) TTK was unable to do so in multiple cell lines. WT TTK also rescued Rad51 foci formation after knockdown of endogenous TTK while KD TTK did not. Conclusion: These data support TTKi as a radiosensitizing strategy for clinical development in basal-like BC patients and that radiosensitization is mediated, at least in part, through impaired HR repair. Citation Format: Benjamin Chandler, Leah Moubadder, Cassandra Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea Pesch, Anna Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar Niknafs, Sofia Merajver, Dafydd Thomas, Powel Brown, Theodore Lawrence, Shyam Nyati, Lori Pierce, Arul Chinnaiyan, Corey Speers. TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6273.

Signaling from Plastid Genome Stability Modulates Endoreplication and Cell Cycle during Plant Development

Plastid-nucleus genome coordination is crucial for plastid activity, but the mechanisms remain unclear. By treating Arabidopsis plants with the organellar genome-damaging agent ciprofloxacin, we found that plastid genome instability can alter endoreplication and the cell cycle. Similar results are observed in the plastid genome instability mutants of reca1why1why3. Cell division and embryo development are disturbed in the reca1why1why3 mutant. Notably, SMR5 and SMR7 genes, which encode cell-cycle kinase inhibitors, are upregulated in plastid genome instability plants, and the mutation of SMR7 can restore the endoreplication and growth phenotype of reca1why1why3 plants. Furthermore, we establish that the DNA damage response transcription factor SOG1 mediates the alteration of endoreplication and cell cycle triggered by plastid genome instability. Finally, we demonstrate that reactive oxygen species produced in plastids are important for plastid-nucleus genome coordination. Our findings uncover a molecular mechanism for the coordination of plastid and nuclear genomes during plant growth and development.