S-phase Cell Cycle Progression Research Articles

Abstract 1143: Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers

Abstract Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have been approved for treatment of metastatic ER+ HER2− breast cancer as monotherapy or in combination with endocrine therapy. However, many patients become resistant to CDK4/6 therapy due to various mechanisms such as loss of tumor suppressor Rb, hyperactivation of CDK4/6, and/or CDK2/Cyclin E1 expression and activity, resulting in a highly unmet clinical need. Triple-negative breast cancers (TNBC), owing to a lack of hormone receptor (HR) expression and extensive heterogeneity, remain notoriously aggressive and unresponsive to current therapies. CCNE1 overexpression or amplification has been shown to be an oncogenic driver associated with poor clinical outcomes in ovarian, gastric, endometrial, and HR+ breast cancers. CCNE1hi cancers are dependent on CDK2 for growth and survival. CDK2 in complex with CCNE1 phosphorylates Rb, resulting in E2F target gene expression and G1 to S-phase cell cycle progression. Using a potent and selective CDK2 inhibitor, INCB123667, we demonstrate the efficacy of CDK2 inhibition in CCNE1hi breast cancer models. Analysis of genome-wide CRISPR screen data identified a subset of CCNE1hi breast cancer models dependent on CDK2 comprised of basal and luminal breast cancer subtypes, including TNBC, ER+ HER2−, and HR− HER2+ models. Two CCNE1hi cell lines, HCC1569 and MDAMB157, were evaluated in cell assays monitoring pRb, S-phase, and growth inhibition, and found to be sensitive to treatment with INCB123667 and insensitive to multiple CDK4/6 inhibitors. Through extensive biochemical studies, INCB123667 was observed to be potently active against CDK2 at subnanomolar range and inactive against other CDKs (CDK1, CDK4, CDK6, CDK7, and CDK9). INCB123667 also selectively induced senescence in CCNE1hi HCC1569 cells. In contrast, CCNE1 nonamplified cell lines, MCF7 and T47D, were inactive in pRb, S-phase, and growth inhibition assays when treated with INCB123667. Significant tumor growth inhibition was observed in CCNE1hi PDX and xenograft models with minimal body weight loss. We also observed dose-dependent inhibition of CDK2 substrates, including pRb S780, both in vitro and in vivo. To further characterize downstream signaling and transcriptional changes with CDK2 inhibition, we identified a discrete gene signature associated with cell cycle regulation, which was notably distinct from CDK4/6 responsive target genes. In conclusion, we have demonstrated CDK2 activity in CCNE1hi breast cancers using INCB123667, a potent and selective small molecule inhibitor of CDK2 currently in clinical development. Treatment resulted in cell growth inhibition and antitumor activity in vitro and in vivo. Our data demonstrate the therapeutic potential of CDK2 inhibition in CCNE1hi HR+ HER2− and TNBC breast cancer models. Acknowledgments: R&D services provided by Pharmaron, Crown Bioscience, and Champions Oncology Citation Format: Saswati Chand, Michael Hansbury, Yvonne Lo, Pat Feldman, Justine Carl, Cynthia Timmers, Joshua Hummel, Susan Wee, Sunkyu Kim. Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1143.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.

Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (β-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kdapp119µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase.

Therapeutic Targeting of GLI1 in Acute Myeloid Leukemia

Hematopoietic stem and progenitor cell (HSPCs) proliferation are tightly regulated during normal homeostasis. Recent data suggests that aberrantly activated signaling pathways such as Hedgehog (Hh), Notch, and Wnt occur during early hematopoietic oncogenesis. Gli genes (Gli1-3), known as Gli-code, encode Cys2-His2 zinc finger transcription factors governing the expression of pro-survival genes at the distal end of the Hh pathway (Hh→PTCH1→SMO→GLI). These genes are tightly regulated in embryonic development. They are a common node of dysregulated activation through which many oncogenic signals, such as Hh, FLT3, PI3K-AKT, TGFβ, and RAS, converge. Hh-signaling critical for embryogenesis is mostly inactive in adult tissues; however, aberrant activation of the Hh/Gli has been implicated in cancer progression and chemotherapy resistance in AML. Activation of SMO triggers an intracellular signaling cascade leading to the formation of active Gli transcription factors which functionally converge on Gli1. In addition to canonical PTCH/SMO-dependent regulation of Gli1, we have shown that Gli-signaling axis can be activated independent of Hh-signaling (Fig.1).The specificity of the Gli pathway in leukemic cells suggests that Gli inhibition may have clinical utility. Most efforts have been devoted towards therapeutic targeting upstream of the Gli Hh-signaling through PTCH/SMO. These efforts remained mostly ineffective due to convergence of parallel oncogenic signals at Gli1 irrespective of Hh-signaling status.We have focused our structure-guided small molecule design efforts to develop potent Gli1-inhibitors, a nodal point of the convergence of various oncogenic signals (Fig.1). Our Gli1 inhibitor prevents both Gli1 binding to DNA and transcription of Gli-target genes, inducing cell death specifically in leukemic cells while sparing normal CD34+ hematopoietic cells. Our inhibitor binds on the Gli1-DNA interface between Zn-finger 2 and 3 (KD= 14nM) and is >200-fold more effective than the previously reported Gant61 Gli1 (KD=3.2µM) at inducing cell death of leukemic cells in semi-solid methylcellulose-based media. We performed a colony forming assay on CD34+ bone marrow cells derived from RAS mutant patients in the presence and abscence of our highly potent iGli1 inhibitor. iGLi1 demonstarted high potency (IC50, 50nM) in preventing colony formation in a dose dependent manner while no cytotoxic effect was observed on normal bone marrow cells. The LD50 for normal bone marrow cells was 10µM providing a therapeutic index of more than 200. Cellular mRNA sequencing analysis was also performed in MOLM13 and MV4-11 cells to confirm the downregulation of Gli1 target genes. Consistent with the Gli1 inhibition, iGli1 treatment of these AML cells led to the downregulation of Gli1 target genes Gli1, Gli2, PTCH1, Cyclin1/2, P21, P27, and IKK-β. iGli1 inhibited the binding of Gli to DNA and rapidly inhibits transcription of critical Gli-dependent target genes. Furthermore, inhibition of Gli1 resulted in the stalling of RNA Pol II, leading to redistribution of pause and pause-release factors DSIF/NELF and PTEFb, respectively. We also observed that iGli1 treatment of MOLM13 or THP1 cells arrests cell cycle progression in S-phase. Inhibition of Gli-dependent transcription by iGli1 leads to accumulation of R-loops (RNA:DNA hybrids) that cause a robust DNA damage, inhibition of DNA replication, S-phase progression, and subsequent apoptosis.The efficacy of iGli1 was also compared to standard care used in the treatment of AML (Cytarabine and Idarubicin under in vitro conditions against MOLM-13, THP-1, K562, and MV4-11). We observed that iGli1 was superior to cytarabine/idarubicin treatment in cell culture model system as a single agent. In addition, ex vivo exposure of MV4-11 cells with iGli1 completely shut down the tumor formation in NSG mice in a xenograft model.In summary, we have shown that Gli1 is not only a strong target to treat AML patients but also an excellent approach for developing a novel therapeutic for patients where Gli1 expression and activity is high, such as patients with RAS mutations. The Gli1 inhibitior is highly effctive as a single agent against highly aggressive form of AML with an excellent therapeutic index. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Histone deficiency and accelerated replication stress in T cell aging.

With increasing age, individuals are more vulnerable to viral infections such as with influenza or the SARS-CoV-2 virus. One age-associated defect in human T cells is the reduced expression of miR-181a. miR-181ab1 deficiency in peripheral murine T cells causes delayed viral clearance after infection, resembling human immune aging. Here we show that naive T cells from older individuals as well as miR-181ab1-deficient murine T cells develop excessive replication stress after activation, due to reduced histone expression and delayed S-phase cell cycle progression. Reduced histone expression was caused by the miR-181a target SIRT1 that directly repressed transcription of histone genes by binding to their promoters and reducing histone acetylation. Inhibition of SIRT1 activity or SIRT1 silencing increased histone expression, restored cell cycle progression, diminished the replication-stress response, and reduced the production of inflammatory mediators in replicating T cells from old individuals. Correspondingly, treatment with SIRT1 inhibitors improved viral clearance in mice with miR-181a-deficient T cells after LCMV infection. In conclusion, SIRT1 inhibition may be beneficial to treat systemic viral infection in older individuals by targeting antigen-specific T cells that develop replication stress due to miR-181a deficiency.

Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation.

Esophageal squamous cell carcinoma (ESCC) is a malignant epithelial cancer of the esophageal epithelium. Piezo-type mechanosensitive ion channel component 1 (Piezo1), an essential mechanosensitive protein, plays an important role in maintaining cell biological functions under the stimulation of physiological force. Immunohistochemical and bioinformatic analyses of ESCC tissue samples indicate that Piezo1 expression is higher in ESCC tissues than in paracancerous tissues. shRNA-mediated Piezo1 downregulation in the ESCC lines EC9706 and EC109 showed that proliferation, migration, and invasion were suppressed by Piezo1 knockdown. Piezo1 downregulation suppresses ESCC migration and invasion in both cells and tissues via the epithelial–mesenchymal transition pathway. Moreover, G0/G1 to S-phase cell cycle progression was inhibited, and cell apoptosis was induced by Piezo1 downregulation. Furthermore, we observed an interaction between Piezo1 and p53 using immunoprecipitation. The protein levels of p53, downstream factor Bax, apoptosis executioner cleaved-caspase3, and caspase3 were significantly upregulated by the downregulation of Piezo1. The inhibited growth rate and upregulated expression of these related factors were validated using tumor-bearing mice. Therefore, Piezo1 downregulation induces ESCC apoptosis via a Piezo1–p53–Bax–Caspase 3 axis. In conclusion, Piezo1 downregulation suppresses ESCC development, and mechanosensitive protein Piezo1 can be considered a new target for ESCC therapy.

A novel translation inhibitor, mersicarpine, inhibits S-phase progression and induces apoptosis in HL60 cells

Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.

The nucleolar protein SAHY1 is involved in pre-rRNA processing and normal plant growth.

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A3, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

Chk1-mediated phosphorylation of Cdh1 promotes the SCF\u03b2TRCP-dependent degradation of Cdh1 during S-phase and efficient cell-cycle progression

APC/CCdh1 is a ubiquitin ligase with roles in numerous diverse processes, including control of cellular proliferation and multiple aspects of the DNA damage response. Precise regulation of APC/CCdh1 activity is central to efficient cell-cycle progression and cellular homeostasis. Here, we have identified Cdh1 as a direct substrate of the replication stress checkpoint effector kinase Chk1 and demonstrate that Chk1-mediated phosphorylation of Cdh1 contributes to its recognition by the SCFβTRCP ubiquitin ligase, promotes efficient S-phase entry, and is important for cellular proliferation during otherwise unperturbed cell cycles. We also find that prolonged Chk1 activity in late S/G2 inhibits Cdh1 accumulation. In addition to promoting control of APC/CCdh1 activity by facilitating Cdh1 destruction, we find that Chk1 also antagonizes activity of the ligase by perturbing the interaction between Cdh1 and the APC/C. Overall, these data suggest that the rise and fall of Chk1 activity contributes to the regulation of APC/CCdh1 activity that enhances the replication process.

Bayesian Modeling Identifies PLAG1 as a Key Regulator of Proliferation and Survival in Rhabdomyosarcoma Cells.

We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified PLAG1 as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with PLAG1 gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation in vitro and as a xenograft and promoted G1 to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC50 of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor. IMPLICATIONS: Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.

The Cdk2-c-Myc-miR-571 Axis Regulates DNA Replication and Genomic Stability by Targeting Geminin.

DNA rereplication leads to genomic instability and has been implicated in the pathology of a variety of human cancers. Eukaryotic DNA replication is tightly controlled to ensure it occurs only once during each cell cycle. Geminin is a critical component of this control, it prevents DNA rereplication from occurring during S, G2, and early M phases by preventing MCM helicases from forming prereplication complexes. Geminin is targeted for degradation by the anaphase-promoting complex (APC/C) from anaphase through G1-phase, however, accumulating evidence indicates that Geminin is downregulated in late S-phase due to an unknown mechanism. Here, we used a high-throughput screen to identify miRNAs that can induce excess DNA replication and found that miR-571 could reduce the protein level of Geminin in late S-phase independent of the APC/C. Furthermore, miR-571 regulated efficient DNA replication and S-phase cell-cycle progression. Strikingly, c-Myc suppressed miR-571 expression by binding directly to the miR-571 promoter. At the beginning of S-phase, Cdk2 phosphorylated c-Myc at Serine 62, promoting its association with the miR-571 promoter region. Collectively, we identify miR-571 as the first miRNA that prevents aberrant DNA replication and the Cdk2-c-Myc-miR-571 axis as a new pathway for regulating DNA replication, cell cycle, and genomic stability in cancer cells. SIGNIFICANCE: These findings identify a novel regulatory mechanism that is critical for maintaining genome integrity by regulating DNA replication and cell-cycle progression.

MicroRNA-363-3p/p21(Cip1/Waf1) axis is regulated by HIF-2α in mediating stemness of melanoma cells.

Melanoma is a malignant tumor. The acquisition of stemness of melanoma cells aggravates the malignant transformation, which can be regulated by microRNAs (miRNAs, miR). MiR-363-3p is a key tumor-related miRNA, but its role in stemness and melanoma cells is still unknown. Presently, miR-363-3p, induced by hypoxia inducible factor (HIF)-2α, played a positive role in the stemness of melanoma cells. The levels of miR-363-3p and HIF-2α were upregulated in melanoma cell lines. Overexpression of HIF-2α significantly increased the levels of miR-363-3p. However, both HIF-2α knockdown and miR-363-3p inhibition inhibited the levels of the stemness markers (CD133, CD271, Jarid1B, and Nanog). Furthermore, the levels of miR-363-3p and HIF-2α were upregulated in fluorescence activated cell sorting (FACS)-sorted CD271high/+ cells. Whereas miR-363-3p depletion reduced the proportion and the spheroidization of the CD271high/+ cells, decreased the levels of CD133, CD271, Jarid1B and Nanog with restrained proliferative activity of CD271high/+ cells. Additionally, miR-363-3p was confirmed a key downstream of HIF-2α. Intriguingly, cyclin-dependent kinase inhibitor 1A [CDKN1A, p21(Cip1/Waf1)], a key inhibitor of S-phase DNA synthesis and cell cycle progression, was confirmed a target gene of miR-363-3p by luciferase reporter gene assay. The protein levels of CD133, CD271, Jarid1B and Nanog were upregulated with enhanced proliferative activity of CD271high/+ cells by inhibition of p21 in melanoma cells. In conclusion, miR-363-3p is induced by HIF-2α to promote the stemness of melanoma cells via inhibiting p21. The present study provides novel insights that HIF-2α/miR-363-3p/p21 signaling may be a potential target of research and therapy of melanoma.

Preparation, characterisation and biological evaluation of new N-phenyl amidobenzenesulfonates and N-phenyl ureidobenzenesulfonates inducing DNA double-strand breaks. Part 3. Modulation of ring A

N-Phenyl ureidobenzenesulfonates (PUB-SOs) are a new class of anticancer agents blocking the cell cycle progression in S-phase, inducing replicative stress and DNA double-strand breaks (DSBs). In this study, we evaluate the effect of modifying the nature and the position of different substituents on ring A of PUB-SOs on the antiproliferative activity, pharmacological activity as well as on calculated physicochemical, pharmacokinetics and drug-likeness properties. Modification of the urea group by an amide group led to new PUB-SO analogs designated as N-phenyl amidobenzenesulfonates (PAB-SOs). The 2-chloroethyl moiety on ring A was also substituted by different alkyl, cycloalkyl and chloroalkyl groups. The new PAB-SOs and PUB-SOs blocking the cell cycle progression in S-phase exhibit antiproliferative activity in the submicromolar to low micromolar range (0.14–27 μM) on four human cancer cell lines, namely HT-1080, HT-29, M21 and MCF7. Moreover, selected PUB-SO and PAB-SO derivatives induced the phosphorylation of H2AX in M21 cells and do not exhibit or only slightly alkylating activity as confirmed by the 4-(4-nitrobenzyl)pyridine (NBP) assay. Finally, our results show that structure modifications weakly affect the calculated physicochemical, pharmacokinetics and drug-likeness properties of PAB-SOs and PUB-SOs. Therefore, PAB-SOs and PUB-SOs are promising anticancer agents inducing replicative stress and DNA damage via a mechanism of action unrelated to DNA alkylation.

Cell-Based High Content Analysis of Cell Proliferation and Apoptosis.

High content imaging-based cell cycle analysis allows multiplexing of various parameters including DNA content, DNA synthesis, cell proliferation, and other cell cycle markers such as phosho-histone H3. 5'-Ethynyl-2'-deoxyuridine (EdU) incorporation is a thymidine analog that provides a sensitive method for the detection of DNA synthesis in proliferating cells that is a more convenient method than the traditional BrdU detection by antibody. Caspase 3 is activated in programmed cell death induced by both intrinsic (mitochondrial) and extrinsic factors (death ligand). Cell cycle and apoptosis are common parameters studied in the phenotypic analysis of compound toxicity and anti-cancer drugs. In this chapter, we describe methods for the detection of s-phase cell cycle progression by EdU incorporation, and caspase 3 activation using the CellEvent caspase 3/7 detection reagent.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost.

Background:The mechanism of the immunoregulatory activities of polysaccharide is still not clear.Materials and Methods:Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages.Results:These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups.Conclusion:An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways.SUMMARY Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide.

Downregulation of ZBTB24 hampers the G0/1- to S-phase cell-cycle transition via upregulating the expression of IRF-4 in human B cells.

It has been recently identified that loss-of-function mutations in the uncharacterized gene ZBTB24 (zinc finger and BTB domain-containing 24) cause ICF2 (immunodeficiency, centromeric instability and facial anomalies syndrome 2) with immunological characteristics of greatly reduced serum antibodies and circulating memory B cells. ZBTB24 belongs to the large ZBTB family of transcriptional repressors with members like B-cell lymphoma 6 (BCL-6; ZBTB27) playing critical roles in B-cell functions. Given the genotype-phenotype correlation analyses in ICF2 patients and the high expression of ZBTB24 in human B cells, we, in the present study, investigated the function of ZBTB24 in human B-cell line Raji cells. Knockdown of endogenous ZBTB24 by small hairpin RNAs results in a significantly reduced proliferation through blocking the G0/1- to S-phase cell-cycle progression, but not apoptosis induction. Moreover, downregulation of ZBTB24 increases the expression of IRF-4 (interferon regulatory factor 4) and Blimp-1 (B lymphocyte-induced maturation protein 1), two crucial factors involved in the proliferation and differentiation of B cells. Importantly, ZBTB24 exerts these functions independent of BCL-6 as it does not affect the expression and function of BCL-6. Our study thus not only provides a molecular explanation for the B-cell and antibody defects observed in ZBTB24-deficient ICF2 patients, but also indicates that ZBTB24 represents a novel transcriptional factor essentially involved in human B-cell functions.

Synthesis and biological evaluation of novel N-phenyl ureidobenzenesulfonate derivatives as potential anticancer agents. Part 2. Modulation of the ring B

DNA double strand-breaks (DSBs) are the most deleterious lesions that can affect the genome of living beings and are lethal if not quickly and properly repaired. Recently, we discovered a new family of anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs) that are blocking the cells cycle progression in S-phase and inducing DNA DSBs. Previously, we have studied the effect of several modifications on the molecular scaffold of PUB-SOs on their cytocidal properties. However, the effect of the nature and the position of substituents on the aromatic ring B is still poorly studied. In this study, we report the preparation and the biological evaluation of 45 new PUB-SO derivatives substituted by alkyl, alkoxy, halogen and nitro groups at different positions on the aromatic ring B. All PUB-SOs were active in the submicromolar to low micromolar range (0.24–20 μM). The cell cycle progression analysis showed that PUB-SOs substituted at position 2 by alkyl, halogen or nitro groups or substituted at position 4 by a hydroxyl group arrest the cell cycle progression in S-phase. Interestingly, all others PUB-SOs substituted at positions 3 and 4 arrested the cell cycle in G2/M-phase. PUB-SOs arresting the cell cycle progression in S-phase also induced the phosphorylation of H2AX (γH2AX) which is indicating the generation of DNA DSBs. We evidenced that few modifications on the ring B of PUB-SOs scaffold lead to cytocidal derivatives arresting the cell cycle in S-phase and inducing γH2AX and DSBs. In addition, this study shows that these new anticancer agents are promising and could be used as alternative to circumvent some of the biopharmaceutical complications that might be encountered during the development of PUB-SOs.

BTG/Tob family members Tob1 and Tob2 inhibit proliferation of mouse embryonic stem cells via Id3 mRNA degradation

The mammalian BTG/Tob family is a group of proteins with anti-proliferative ability, and there are six members including BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2. Among them, Tob subfamily members, specifically Tob1/Tob and Tob2, have the most extensive C-terminal regions. As previously reported, overexpression of BTG/Tob proteins is associated with the inhibition of G1 to S-phase cell cycle progression and decreased cell proliferation in a variety of cell types. Tob subfamily proteins have similar anti-proliferative effects on cell cycle progression in cultured tumor cells. An important unresolved question is whether or not they have function in rapidly proliferating cells, such as embryonic stem cells (ESCs). Tob1 and Tob2 were expressed ubiquitously in mouse ESCs (mESCs), suggesting a possible role in early embryonic development and mESCs. To address the above question and explore the possible functions of the Tob subfamily in ESCs, we established ESCs from different genotypic knockout inner cell mass (ICM). We found that Tob1−/−, Tob2−/−, and Tob1/2 double knockout (DKO, Tob1−/− & Tob2−/−) ESCs grew faster than wild type (WT) ESCs without losing pluripotency, and we provide a possible mechanistic explanation for these observations: Tob1 and Tob2 inhibit the cell cycle via degradation of Id3 mRNA, which is a set of directly targeted genes of BMP4 signaling in mESCs that play critical roles in the maintenance of ESC properties. Together, our data suggest that BTG/Tob family protein Tob1 and Tob2 regulation cell proliferation does not compromise the basic properties of mESCs.

Targeting mTORC1-mediated metabolic addiction overcomes fludarabine resistance in malignant B cells.

MTOR complex-1(mTORC1) activation occurs frequently in cancers, yet clinical efficacy of rapalogs is limited because of the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analogue and antimetabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K, also known as RPS6KB1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) (T389), an mTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR) derivatives, and primary human CLL cells. Consistent with the anabolic role of mTORC1, FluR cells had higher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells. Rapalogs (everolimus and rapamycin) induced moderate cell death in FluR and primary CLL cells, and everolimus significantly inhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphorylation in FluR cells was not coupled to higher ATP synthesis. Instead, it contributed primarily to an essential, dihydroorotate dehydrogenase catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotes pyrimidine biosynthesis by p70S6 kinase-mediated phosphorylation of CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; Ser1859) and favors S-phase cell-cycle progression. We found increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacological inhibition of de novo pyrimidine biosynthesis using N-phosphonacetyl-l-aspartate and leflunomide, RNAi-mediated knockdown of p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic to FluR, but not FluS, cells. These results reveal a novel link between mTORC1-mediated metabolic reprogramming and Flu resistance identifying mitochondrial respiration and de novo pyrimidine biosynthesis as potential therapeutic targets. This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.

ATAD5 deficiency decreases B cell division and Igh recombination (IRM8P.700)

Abstract Mammalian ATAD5 and its yeast homolog ELG1 are responsible for unloading PCNA from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound PCNA and slower cell division. We used B cells from heterozygous (Atad5+/m) mice to examine the effects of PCNA buildup and decreased cell proliferation on antibody diversity. ATAD5 haploinsufficiency did not affect the frequency or spectrum of somatic hypermutation in antibody genes from Peyer’s patch B cells, indicating that PCNA accumulation did not alter DNA repair or DNA polymerase η usage at the Igh locus. However, following ex vivo stimulation, Atad5+/m B cells were delayed during S-phase cell cycle progression and had reduced cell proliferation compared to wild type cells. These cells had a significant decline in activation-induced deaminase expression, resulting in decreased double-strand breaks in the switch region and inter-chromosomal translocations. Class switch recombination was also reduced, although the types of end-joining pathways were not affected. Our results describe a defect in DNA replication that directly affects Igh recombination via reduced cell division.