Promotes Cell Cycle Arrest Research Articles

Abstract P030: Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer

Abstract The growing incidence of skin cancer (SC) is a global public health concern. Preventive approaches mostly embrace the sunscreen use against ultraviolet radiation (UVR), the main SC etiologic factor. Yet, the increasing SC incident have suggested that additional preventive agents could complement the sunscreen, helping to reverse this trend. Mutant p53 (mutp53) represents a promising target in SC prevention, since its occurrence is a key early event in UVR-induced SC. Thus, the inhibition of mutp53 formation by mutp53 reactivators, which are able to restore the wild-type-like function to p53, would be a valuable strategy for SC prevention. Recently, our group identified the tryptophanol-derived oxazoloisoindolinone SLMP53-2 as a new mutp53 reactivator. Herein, we aimed to study the potential of SLMP53-2 as a chemopreventive drug against UVR-induced SC. For that, assays with normal human keratinocyte HaCaT cells were performed. Cells were pre-treated with SLMP53-2 for 24h, followed by exposure to distinct UVB doses and further analysis. For in vivo studies, FVB/N mice were topically pre-treated for 1h prior to UVB radiation (180 mJ/cm2) and the skin was collected 24h after for immunohistochemistry/immunofluorescence analysis. Pre-treatment of HaCaT cells with SLMP53-2 enhanced cell survival in response to UVB with promotion of cell cycle arrest in G0/G1 phase, upregulation of p21 protein levels, reduction of UVB-induced apoptosis, and inhibition of JNK activity. SLMP53-2 also protected cells from UVB-induced ROS generation and from its consequent lipid peroxidation and protein carbonylation. It enhanced DNA repair through upregulation of the protein and mRNA levels of XPC (xeroderma pigmentosum complementation group C) and DDB-2 (DNA damaged binding protein 2) from the NER (nucleotide excision repair) pathway. In fact, SLMP53-2 depleted UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPDs) levels. SLMP53-2 also showed promising activity in suppressing UVB-induced inflammation, decreasing COX-2, IL-6 and TNF-α protein levels and reducing NF-kB nuclear translocation and DNA binding ability. Moreover, SLMP53-2 enhanced the expression of key players in keratinocytes differentiation, namely NOTCH1 and keratin 1. Consistently, pre-treatment with SLMP53-2 reduced mutp53 protein levels by potentially restoring its wild-type p53 form, which may underlie the increased resistance of HaCaT cells to UVB harmful effects. Importantly, in vivo studies supported that SLMP53-2 pre-treatment of UVB-irradiated mice promoted the survival of epidermal cells, reducing the DNA damage by enhancement of DNA repair and downregulating mutp53 expression levels. Further, it reduced the expression of inflammatory-related proteins and promoted cell differentiation. Collectively, these results support a promising chemopreventive activity for SLMP53-2 against UVB-induced skin damage. We thank FCT/MCTES for the projects UIDB/50006/2020, UIDP/04423/2020 and fellowship SFRH/BD/128673/2017 (J.B. Loureiro). Citation Format: Joana B. Loureiro, Rita Ribeiro, Nair Nazareth, Tiago Ferreira, Adelina Quaresma, Valentina Barcherini, Laura Marabini, Paula A. Oliveira, Maria M. M. Santos, Lucília Saraiva. Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P030.

Epigenetic silencing of UBXN8 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

The formation of the RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) translocation, is considered to be one of the initiating events of t(8;21) acute myeloid leukemia (AML). However, the mechanisms of the oncogenic mechanism of RUNX1-RUNX1T1 remain unclear. In this study, we found that RUNX1-RUNX1T1 triggers the heterochromatic silencing of UBXN8 by recognizing the RUNX1-binding sites and recruiting chromatin-remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in RUNX1-RUNX1T1+ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability of and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancing UBXN8 levels can significantly inhibit tumor proliferation and promote the differentiation of RUNX1-RUNX1T1+ cells in vivo. In conclusion, our results indicated that epigenetic silencing of UBXN8 via methylation of its promoter region mediated by the RUNX1-RUNX1T1 fusion protein contributes to the leukemogenesis of t(8;21) AML and that UBXN8 targeting may be a potential therapeutic strategy for t(8;21) AML.

MEOX1 suppresses the progression of lung cancer cells by inhibiting the cell-cycle checkpoint gene CCNB1.

The previous study has shown that transcriptional factor MEOX1 could promote proliferation and sphere formation ability of non-small cell lung cancer (NSCLC) cells, however, we found that MEOX1 mRNA was lowly expressed in lung cancer tissues compared to that in normal adjacent tissues, and MEOX1 mRNA expression was positively correlated with the survival of lung cancer patients, especially in lung adenocarcinoma patients. Functional experiments using in vitro and in vivo experiments revealed that stable overexpression of MEOX1 significantly suppressed the proliferation ability, promoted cell cycle arrest in G2 phase, and apoptotic ability of NSCLC cells. Additionally, it was identified that MEOX1 and CCNB1 mRNA expression exhibited a negative correlation in different lung cancer tissues. Mechanistically, we indicated that MEOX1 bound to the transcriptional initiation site of CCNB1 and thus suppressed CCNB1 expression. Notably, CCNB1 overexpression rescued the inhibition of MEOX1 overexpression on NSCLC progression. This study deciphers a novel MEOX1/CCNB1 axis suppressing NSCLC progression.

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

T-Cell Telomere Length As a Biomarker to Predict Outcome in Patients Receiving CAR-T Immunotherapy

Introduction: CAR T-cells remain in a quiescent or dormant state when unstimulated, showing no proliferative activity. In contrast, upon specific antigen stimulation (i.e., CD19) CAR T-cells divide both in-vitro and in-vivo, initiate immune responses and can kill their target cells in the body. However, one of the major physiological immune changes with increased age is the progressive impairment of T-cell responses. This process termed immunosenescence (which may be similar T-cell exhaustion) is associated with the shortening of telomeres, specific DNA repeated sequences that protect the end of linear chromosomes from degradation and fusion with neighbor chromosomes. We aim to investigate change in T-cell telomere length with CAR-T cell therapy and its potential impact on outcome in patients receiving CART immunotherapy.Methods: We enrolled adult patients (age range: 30-80 years old) receiving CART immunotherapy for diffuse large B cell lymphoma (DLBCL), multiple myeloma (MM), mantle cell lymphoma (MCL), or follicular lymphoma (FL). We collected peripheral blood at two time points: i) pre-lymphodepletion therapy and ii) two weeks post CAR-T cell infusion. Peripheral blood mononuclear cells were isolated from blood via density gradient and T-cells isolated from PBMC with magnetic beads (negative selection). Telomere lengths are quantified from T-cells by using a highly sensitive technique called TeSLA (Telomere Shortest Length Assay) that allows absolute quantification of both the average telomere length and the lengths of critically short telomeres, which are believed to play a major role in promoting cell cycle arrest and T-cell exhaustion.Results: We identified 7 patients receiving CAR T cell therapy for hematological malignancies at University of Texas Southwestern Medical Center. The cohort included 7 patients, 2 patients with DLBCL and 1 patient with MCL receiving CD19 CAR-T Cell therapy and 4 patients with MM receiving BCMA CAR-T cell therapy. Median age of patient was 65 yrs. Median follow up was 273 days post CAR T-cell therapy with all patients being alive at last follow-up. Two patients experienced Grade I Cytokine release syndrome (CRS), two patients with Grade 2 CRS and one patient with Grade 2 ICANS. Our initial analysis shows that patients telomere lengths changes pre and post CAR T-cell infusion. Regarding change in critically short telomere (<1.6kb); 6 out of 7 patients had reduction the shorter telomere from BL to post CAR-T. We are currently evaluating the effect of change in telomere length on outcomes.Conclusions: CAR T-cell therapy is a game-changer for hematological malignancies; however, disease still relapse. Understanding the mechanics of poor response or relapse after CAR T-cell therapy is critical in advancing the field. Initial results suggest T-cell telomere length are significantly affected during CAR T-cell manufacturing process and post infusion. These results are potentially important as telomere length can be utilized as a biomarker to predict CAR T-cell therapy outcomes. [Display omitted] DisclosuresAnderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan: Verastem: Consultancy; Incyte: Consultancy; Cardinal Health: Consultancy; Dava Oncology: Consultancy; BMS: Consultancy; ADCT therapeutics: Consultancy; Beigene: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; MEI Pharma: Consultancy; Merck: Consultancy; Kite pharma: Consultancy; Gilead sciences: Consultancy; Johnson and Johnson: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Astrazeneca: Consultancy; Genentech: Consultancy. Madanat: Onc Live: Honoraria; Blue Print Pharmaceutical: Honoraria; Geron Pharmaceutical: Consultancy; Stem line pharmaceutical: Honoraria. Patel: Celgene-BMS: Membership on an entity's Board of Directors or advisory committees; PVI: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees. Sweetenham: EMA Wellness: Membership on an entity's Board of Directors or advisory committees. Kansagra: Alynylam, Celgene/BMS, Cota Health, GSK, Janssen, Karyopharm, Oncopeptide, Pfizer, Takeda, Sanofi: Membership on an entity's Board of Directors or advisory committees.

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27.

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell modelsevaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.

Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.

Ethanol Extract of Croton Kongensis Promotes Cell Cycle Arrest and Consequently Inhibits Anchorage-Independent Growth of Cervical Cancer Hela Cells

Extracts from Croton kongenis present anticancer activities on various cancers. However, there is no research conducted to investigate the effects of Croton kongenis extracts on cervical cancer as well as on zebrafish. In this study, we demonstrated that Croton kongenis ethanol extract expressed high toxicity to cervical cancer Hela cells with an IC50 dose of 20.4 µg/mL and to zebrafish embryos with malformations, lethality and hatching inhibition at 72-hpf at effective dose of 125 µg/mL. Interestingly, treatment with Croton kongenis ethanol extract caused cell-cycle-arrest at the G2 phase. Particularly, percentages of Croton kongenis ethanol extract-treated cells in G1, S, G2/M were 70%, 6% and 23%, while percentages of control cells in G1, S, G2/M were 65%, 15% and 18%, respectively. Consistent with cell-cycle-arrest, the expressions of CDKN1A, CDNK2A and p53 in Croton kongenis ethanol extract-treated cells were up-regulated 2.0-, 1.65- and 1.8-fold, respectively. Significantly, treatment with Croton kongenis ethanol extract inhibited anchorage-independent growth of Hela cells; the number of colonies formed in soft-agar of Croton kongenis ethanol extract-treated cells was only one-fourth of that of control cells. In conclusion, we suggest that Croton kongenis ethanol extract could be able to use as a traditional medicine for treatment of cervical cancer.

CircATL2 enhances paclitaxel resistance of ovarian cancer via impacting miR-506-3p/NFIB axis.

Circular RNAs (circRNAs) play vital regulatory roles in the development of ovarian cancer (OC). However, the functions of circRNA Atlastin GTPase 2 (circATL2) in paclitaxel (PTX) resistance of OC are still unclear. As a result, circATL2 was upregulated in PTX-resistant OC tissues and cells. CircATL2 knockdown reduced IC50 of PTX, inhibited colony formation ability and promoted cell cycle arrest and apoptosis in PTX-resistant OC cells. Silencing of circATL2 restrained PTX resistance in vivo. Furthermore, miR-506-3p could be targeted by circATL2 and miR-506-3p inhibition reversed the impacts of circATL2 knockdown on PTX resistance and cell progression in PTX-resistant OC cells. NFIB was identified as the target of miR-506-3p. MiR-506-3p overexpression suppressed PTX resistance and malignant behaviors of PTX-resistant OC cells, with NFIB elevation rescued the impacts. To summarize, circATL2 promoted the resistance of OC to PTX by sponging miR-506-3p to upregulate NFIB expression, providing a new sight in chemoresistance of OC.

Chidamide Inhibits Cell Proliferation via the PI3K/AKT Pathway in K562 Cells Based on Network Pharmacology and Experimental Validation.

Chidamide, a novel benzamide-type histone deacetylase (HDAC) inhibitor, exerts antitumor effects on several types of cancer. However, the role of Chidamide in chronic myeloid leukemia (CML) remains elusive. Therefore, the present study aimed to investigate the effects of Chidamide on CML cell proliferation and explore its underlying mechanism. Cell proliferation was assessed by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry and the expression of related proteins was evaluated by western blot analysis. The potential mechanisms were systematically explored by the network-based pharmacological methods, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The results revealed that Chidamide inhibited the proliferation of K562 cells in a dose- and time-dependent manner. In addition, Chidamide blocked cells in the G0/G1 phase via downregulating cyclin-dependent kinase 4, and induced apoptosis via upregulating Bax and downregulating of Bcl-2. Additionally, using network- based pharmacological methods, we found that PI3K/AKT signaling pathway is involved and significantly related to cell proliferation in CML. Intriguingly, cell treatment with Chidamide suppressed the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway via decreasing the levels of phosphorylated (p)-PI3K and p-AKT. Moreover, insulin-like growth factor 1 (IGF-1), a PI3K/AKT activator, reversed the inhibitory effects of Chidamide on K562 cell proliferation. The study demonstrated that Chidamide may inhibit the proliferation of K562 cells by promoting cell cycle arrest and apoptosis via suppressing the PI3K/AKT pathway, suggesting that Chidamide could be a promising approach to the treatment of CML.

Mechanistic toxicity assessment of fine particulate matter emitted from fuel combustion via pathway-based approaches in human cells

Fuel exhaust particulate matter (FEPM) is an important source of air pollution worldwide. However, the comparative and mechanistic toxicity of FEPMs emitted from combustion of different fuels is still not fully understood. This study employed pathway-based approaches via human cells to evaluate mechanistic toxicity of FEPMs. The results showed that FEPMs caused concentration-dependent (0.1–200 μg/mL) cytotoxicity and oxidative stress. FEPMs at low concentration (10 μg/mL) induced cell cycle arrest in S and G2 phases, while high level of FEPMs (200 μg/mL) caused cell cycle arrest in G1 phase. Different FEPMs induced distinct expression profiles of toxicity-related genes, illustrating different toxic mechanisms. Furthermore, FEPMs inhibited the phosphorylation of protein kinase A (PKA), which related with reproductive toxicity. Spearman rank correlations among the chemicals carried by FEPMs and the toxic effects revealed that PAHs and metals promoted cell cycle arrest in the G1 phase and suppressed PKA activity. Furthermore, PAHs (Nap and Acy) and metals (Al and Pb) in FEPMs were highly and positively correlated with the expression of genes involved in apoptosis, ER stress, metal stress and inflammation. Our findings offered more mechanistic information of FEPMs at the level of subcellular toxicity and help to better understand their potential health effects.

Effects of PI3K inhibition in AI-resistant breast cancer cell lines: autophagy, apoptosis, and cell cycle progression.

Breast cancer is the leading cause of cancer death in women. The aromatase inhibitors (AIs), Anastrozole (Ana), Letrozole (Let), and Exemestane (Exe) are a first-line treatment option for estrogen receptor-positive (ER+) breast tumors, in postmenopausal women. Nevertheless, the development of acquired resistance to this therapy is a major drawback. The involvement of PI3K in resistance, through activation of the PI3K/AKT/mTOR survival pathway or through a cytoprotective autophagic process, is widely described. The involvement of autophagy in response to Ana and Let treatments and the effects of the combination of BYL-719, a PI3K inhibitor, with AIs were explored in AI-resistant breast cancer cell lines (LTEDaro, AnaR, LetR, and ExeR). We demonstrate that Ana and Let treatments do not promote autophagy in resistant breast cancer cells, contrary to Exe. Moreover, the combinations of BYL-719 with AIs decrease cell viability by different mechanisms by nonsteroidal vs. steroidal AIs. The combination of BYL-719 with Ana or Let induced cell cycle arrest while the combination with Exe promoted cell cycle arrest and apoptosis. In addition, BYL-719 decreased AnaR, LetR, and ExeR cell viability in a dose- and time-dependent manner, being more effective in the ExeR cell line. This decrease was further exacerbated by ICI 182,780. These results corroborate the lack of cross-resistance between AIs verified in the clinic, excluding autophagy as a mechanism of resistance to Ana or Let and supporting the ongoing clinical trials combining BYL-719 with AIs.

DNA Damage Response in Glioblastoma: Mechanism for Treatment Resistance and Emerging Therapeutic Strategies.

Glioblastoma (GBM) is an intrinsically treatment-resistant tumor and has been shown to upregulate DNA damage response (DDR) components after treatment. DNA damage response signaling mediates treatment resistance by promoting cell cycle arrest in order to allow for DNA damage repair and avoid mitotic catastrophe. Therefore, targeting the DDR pathway is an attractive strategy to combat treatment resistance in GBM. In this review, we discuss the different DDR pathways and then summarize the current preclinical evidence for DDR inhibitors in GBM, as well as completed and ongoing clinical trials.

P300/Sp1-Mediated High Expression of p16 Promotes Endothelial Progenitor Cell Senescence Leading to the Occurrence of Chronic Obstructive Pulmonary Disease

Objective Chronic obstructive pulmonary disease (COPD) is a common chronic disease and develops rapidly into a grave public health problem worldwide. However, what exactly causes the occurrence of COPD remains largely unclear. Here, we are trying to explore whether the high expression of p16 mediated by p300/Sp1 can cause chronic obstructive pulmonary disease through promoting the senescence of endothelial progenitor cells (EPCs). Methods Peripheral blood EPCs were isolated from nonsmoking non-COPD, smoking non-COPD, and smoking COPD patients. The expressions of p16, p300, and senescence-related genes were detected by RT-PCR and Western Blot. Then, we knocked down or overexpressed Sp1 and p300 and used the ChIP assay to detect the histone H4 acetylation level in the promoter region of p16, CCK8 to detect cell proliferation, flow cytometry to detect the cell cycle, and β-galactosidase staining to count the proportion of senescent cells. Results The high expression of p16 was found in peripheral blood EPCs of COPD patients; the cigarette smoke extract (CSE) led to the increase of p16. The high expression of p16 in EPCs promoted cell cycle arrest and apoptosis. The CSE-mediated high expression of p16 promoted cell senescence. The expression of p300 was increased in peripheral blood EPCs of COPD patients. Moreover, p300/Sp1 enhanced the histone H4 acetylation level in the promoter region of p16, thereby mediating the senescence of EPCs. And knockdown of p300/Sp1 could rescue CSE-mediated cell senescence. Conclusion p300/Sp1 enhanced the histone H4 acetylation level in the p16 promoter region to mediate the senescence of EPCs.

P53 induces a survival transcriptional response after nucleolar stress.

Accumulating evidence indicates that increased ribosome biogenesis is a hallmark of cancer. It is well established that inhibition of any steps of ribosome biogenesis induces nucleolar stress characterized by p53 activation and subsequent cell cycle arrest and/or cell death. However, cells derived from solid tumors have demonstrated different degrees of sensitivity to ribosome biogenesis inhibition, where cytostatic effects rather than apoptosis are observed. The reason for this is not clear, and the p53-specific transcriptional program induced after nucleolar stress has not been previously investigated. Here we demonstrate that blocking rRNA synthesis by depletion of essential rRNA processing factors such as LAS1L, PELP1, and NOP2 or by inhibition of RNA Pol I with the specific small molecule inhibitor CX-5461, mainly induce cell cycle arrest accompanied by autophagy in solid tumor–derived cell lines. Using gene expression analysis, we find that p53 orchestrates a transcriptional program involved in promoting metabolic remodeling and autophagy to help cells survive under nucleolar stress. Importantly, our study demonstrates that blocking autophagy significantly sensitizes cancer cells to RNA Pol I inhibition by CX-5461, suggesting that interfering with autophagy should be considered a strategy to heighten the responsiveness of ribosome biogenesis–targeted therapies in p53-positive tumors.

Synergistic inhibitory effects of the oxyresveratrol and dacarbazine combination against melanoma cells.

Various therapies have been developed to target malignant melanoma, which is associated with a high mortality rate worldwide. Although dacarbazine (DTIC) is employed for treating melanoma, it is associated with several side effects. Hence, patients with melanoma are co-treated with additional drugs to mitigate the side effects of DTIC. In the present study, synergistic therapeutic effects of the DTIC/oxyresveratrol (ORT) combination were examined using the human malignant melanoma WM-266-4 cell line. Treatment with ORT and DTIC inhibited the proliferation of WM-266-4 cells. Compared with those in the ORT- and DTIC-treated groups, the proportion of cells arrested at the S phase, as well as apoptotic rates, were increased in the ORT and DTIC co-treatment group. In WM-266-4 cells, synergistic proliferation-inhibitory activities of the ORT/DTIC combination were assessed based on cell viability and migration, antioxidant capacity, cytokine production, cell cycle arrest, apoptotic rate and protein expression through WST-1 assay, wound healing assay, flow cytometry and western blotting. Furthermore, the expression levels of proteins, including NOTCH, involved in the pathogenesis of solid cancers, such as melanoma, were examined. Overall, the ORT/DTIC combination synergistically promoted cell cycle arrest at the S phase and the apoptosis of WM-266-4 cells. Thus, this combination treatment may serve as a novel therapeutic strategy for treating malignant melanoma.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression. DisclosuresBruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.

Doxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells

BackgroundDoxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs.ResultsWe found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion.ConclusionsWe hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.