Changes In Cell Cycle Regulators Research Articles

Abstract PO2-01-08: Differential HOXB13 gene expression and promoter methylation analysis in breast cancer

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels: the Molecular Grade Index (MGI) and BCI (H/I), which is a ratio of the HOXB13 and IL17BR gene expression. Several studies have shown that BCI (H/I) is a predictive biomarker for extended adjuvant endocrine therapy benefit in hormone receptor-positive (HR+) early-stage breast cancer. Molecular mechanisms underlying endocrine responsiveness remain to be fully explored. The objective of this analysis is to evaluate the correlation between gene expression and methylation in breast cancer patients displaying different HOXB13 mRNA expression levels from The Cancer Genome Atlas (TCGA) project. Methods: Data from methylation microarrays and mRNA sequencing were examined in combination with clinical metadata from 1,095 TCGA breast cancer patients. After removing outliers, HR+ samples were ordered based on their HOXB13 expression levels and the top 10% of samples (HOXB13-high; n=95) and bottom 10% (HOXB13-low; n=95) were selected for further analysis. Differentially expressed genes (DEGs) were determined by comparing all protein-coding genes between the HOXB13-high and HOXB13-low sets, based on an absolute log-fold change above 2 and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Among the subset of samples with matching methylation data (n=60 for the HOXB13-high group and n=48 for the HOXB13-low group), hypo-methylated and hyper-methylated differentially methylated probes (DMPs) were identified based on at least 10% change of β (i.e., average promoter methylation) and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Motif and transcription factor (TF) enrichment analyses were identified at a minimum incidence of 10 and a lower boundary of 1.5 for the 95% confidence interval of the odds ratio. Results: Our analysis identified a total of 613 DEGs between the HOXB13-high and HOXB13-low groups. Gene ontology analysis of DEGs revealed a statistically significant enrichment of genes associated with PTK6 regulated cell cycle, oncogene-induced cellular senescence, and regulation of T cell activation. Analysis of protein-protein interactions of differentially expressed genes revealed differences in key biological processes including changes in cell cycle regulation, p53 pathway, and IL-17 signaling. A total of 5,295 hyper-methylated and 580 hypo-methylated DMPs were discovered when comparing HOXB13-high and HOXB13-low samples. Changes in global methylation patterns enabled classification of breast cancer samples with high or low HOXB13 expression with an accuracy of 0.90. Reconstruction of gene regulatory networks from DNA methylation and transcriptome profiles elucidated significant pairs of DMPs and differentially expressed genes. Specifically, a total of 197 differentially expressed genes were attributed to hyper-methylated DMPs, including genes involved in response to estrogen, activation of HOX genes during differentiation, and drug-mediated inhibition of CDK4/6 activity. Additionally, 8 differentially expressed genes were attributed to hypo-methylated DMPs and were associated with proteasomal protein catabolic processes. Lastly, motif analysis of transcriptional factors for the hyper-methylated DMPs revealed enrichment of binding motifs attributed to FOXA1, ESR1, XBR1, and FOXP1. Conclusion: A comprehensive and comparative analysis of mRNA expression levels and methylation patterns between TCGA breast cancer samples with high and low expression of HOXB13 revealed key signaling pathways and biological processes that may provide insights on the molecular mechanism of HOXB13 in the regulation of response to endocrine therapy in HR+ breast cancer. Citation Format: Natalia Siuliukina, Yi Zhang, Kai Treuner, Mark Pegram. Differential HOXB13 gene expression and promoter methylation analysis in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-08.

Abstract 447: Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition

Abstract The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia. Citation Format: Corey Fyfe, Gabriella Fiorentino, Michael J. Steinbaugh, Joana Campos, Hannah Lawson, Jozef Durko, Donal O'Carroll, Kamil Kranc, Darren Harvey, Shomir Ghosh, Robert M. Campbell. Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition [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 447.

Genetic and Pharmacological Inhibition of TUT4/TUT7 Sensitizes AML Cells to BCL-2 Inhibition

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.

Emerging Mechanisms of G1/S Cell Cycle Control by Human and Mouse Cytomegaloviruses.

ABSTRACTCytomegaloviruses (CMVs) are among the largest pathogenic viruses in mammals. To enable replication of their long double-stranded DNA genomes, CMVs induce profound changes in cell cycle regulation. A hallmark of CMV cell cycle control is the establishment of an unusual cell cycle arrest at the G1/S transition, which is characterized by the coexistence of cell cycle stimulatory and inhibitory activities. While CMVs interfere with cellular DNA synthesis and cell division, they activate S-phase-specific gene expression and nucleotide metabolism. This is facilitated by a set of CMV gene products that target master regulators of G1/S progression such as cyclin E and A kinases, Rb-E2F transcription factors, p53-p21 checkpoint proteins, the APC/C ubiquitin ligase, and the nucleotide hydrolase SAMHD1. While the major themes of cell cycle regulation are well conserved between human and murine CMVs (HCMV and MCMV), there are considerable differences at the level of viral cell cycle effectors and their mechanisms of action. Furthermore, both viruses have evolved unique mechanisms to sense the host cell cycle state and modulate the infection program accordingly. This review provides an overview of conserved and divergent features of G1/S control by MCMV and HCMV.

TAMI-50. PATHWAYS ACTIVATION IN GL261 MICE GLIOMA CELLS BY HIV-1 GLYCOPROTEIN GP120

Abstract Patients infected with human immunodeficiency virus type 1 (HIV-1) are more prone to developing cancers, including glioblastomas (GBMs). The median survival for GBM patients with HIV is significantly shorter than for HIV-negative GBM patients, even though they receive the same treatments. This difference indicates that HIV infection is associated with more aggressive tumor behavior and with treatment resistance. Earlier we demonstrated that gp120, a main glycoprotein in the HIV shell, stimulates glycolysis and protein synthesis in glioma cells. The purpose of this study was to evaluate the underlying gp120 dependent signaling mechanism in glioma cell. Mouse glioma cells GL-261 were continuously cultured for 7 days in medium with and without soluble gp120 Bal III (100ng/ml) and collected for Western blot and Cell cycle assays. Western blot analysis presented an increase in phosphorylation of Proline-rich tyrosine kinase (Pyk2(Y402)), p38(YT100/Y182) and p70s6(T421/S424), the key proteins of the Pyk2 pathway, along with the increased levels of Akt(S473) and Glycogen Synthase Kinase 3b (GSK3b (S9)) phosphorylation. Flow cytometry analysis of Cell Cycle revealed an increase of G2/M phase in cells cultured in gp120 Bal III when compared to control cells. Furthermore, GL-261 cells with knock-out of Pyk2 via CRISPR Cas 9 gene editing showed no significant change in cell cycle regulation when cultured with gp120 Bal III.Overall, these results demonstrate that gp120 triggers activation of Pyk2/MAPK and Akt/GSK3b pathways and alter cell cycle regulation in GBM. This research was made possible by NIH grant number 1SC1GM122691.

Cryptochromes and the Circadian Clock: The Story of a Very Complex Relationship in a Spinning World.

Cryptochromes are flavin-containing blue light photoreceptors, present in most kingdoms, including archaea, bacteria, plants, animals and fungi. They are structurally similar to photolyases, a class of flavoproteins involved in light-dependent repair of UV-damaged DNA. Cryptochromes were first discovered in Arabidopsis thaliana in which they control many light-regulated physiological processes like seed germination, de-etiolation, photoperiodic control of the flowering time, cotyledon opening and expansion, anthocyanin accumulation, chloroplast development and root growth. They also regulate the entrainment of plant circadian clock to the phase of light–dark daily cycles. Here, we review the molecular mechanisms by which plant cryptochromes control the synchronisation of the clock with the environmental light. Furthermore, we summarise the circadian clock-mediated changes in cell cycle regulation and chromatin organisation and, finally, we discuss a putative role for plant cryptochromes in the epigenetic regulation of genes.

DIPG-79. H3K27M INDUCES EPIGENETIC AND ONCOGENIC CHANGES THAT ARE PARTIALLY REVERSED BY SMALL MOLECULE AURORA KINASE B/C INHIBITION

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor with no curative treatments. Approximately 80% of DIPGs contain an H3K27M mutation. The implications of the mutation and how they may be targeted are not fully understood. We established an H3K27M effect-isolating model by transducing H3K27-wildtype lines (HSJD-GBM-001, normal human astrocytes) with lentiviral-packaged H3K27M. We characterized H3K27M-related changes through western blot, phenotypic assays, and RNA-seq. Drug screening of H3K27-wildtype and matched H3K27M-transduced lines was used to identify targets more effective with H3K27M present. Patient-derived pediatric glioblastoma and DIPG lines (BT-245, SU-DIPG-IV, HSJD-DIPG-007, SU-DIPG-XIII*, SF7761) were used for validation. We observed increased H3K27ac and decreased H3K27me3, as well as increased proliferative and migratory abilities, with the addition of H3K27M to H3K27-wildtype lines. RNA-seq showed downregulation of cell cycle regulation and upregulation of epithelial-mesenchymal transition. GSK1070916, an Aurora kinase B/C inhibitor, was isolated from a synthetic lethality screen with H3K27M. GSK1070916 showed strong efficacy in native H3K27M lines (IC50s=60nM-1250nM), superior to the Aurora kinase A inhibitor alisertib, to which all cell lines showed substantial resistance. Combination of both drugs was not synergistic. GSK1070916 treatment caused increased H3K27me3 and decreased H3S10ph and H3S28ph. GSK1070916 induced apoptosis and S-phase stall. The H3K27M mutation induces epigenetic, phenotypic, and cell cycle regulation changes resulting in relaxation of transcriptional controls and more aggressive growth. Aurora kinase B/C inhibition is a novel therapeutic modality for DIPG that appears capable of reversing some H3K27M-related epigenetic changes, inducing apoptosis, and repressing uncontrolled cellular division.

Next-generation sequencing analysis of circulating micro-RNA expression in response to parabolic flight as a spaceflight analogue

Understanding physiologic reactions to weightlessness is an indispensable requirement for safe human space missions. This study aims to analyse changes in the expression of circulating miRNAs following exposure to gravitational changes. Eight healthy volunteers (age: 24.5 years, male: 4, female: 4) were included. Each subject underwent 31 short-term phases of weightlessness and hypergravity induced by parabolic flight as a spaceflight analogue. At baseline, 1 and 24 h after parabolic flight, venous blood was withdrawn. Analysis of circulating miRNAs in serum was conducted by means of next generation sequencing. In total, 213 miRNAs were robustly detected (TPM > 5) by small RNA sequencing in all 24 samples. Four miRNAs evidenced a significant change in expression after adjusting for multiple testing. Only miR-223-3p showed a consistent significant decrease 24 h after parabolic flight compared to baseline values and values at 1 h after parabolic flight. miR-941 and miR-24-3p showed a significant decrease 24 h after parabolic flight compared to 1 h after parabolic flight but not to baseline values. miR-486-5p showed a significant increase 24 h after parabolic flight compared to 1 h after parabolic flight but not to baseline values. A target network analysis identified genes of the p53 signaling pathway and the cell cycle highly enriched among the targets of the four microRNAs. Our findings suggest cellular adaption to gravitational changes at the post-transcriptional level. Based on our results, we suggest a change in cell cycle regulation as potential explanation for adaptational changes observed in space missions.

Organising the cell cycle in the absence of transcriptional control: Dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally.

The cell division cycle of the unicellular eukaryote Trypanosome brucei is tightly regulated despite the paucity of transcriptional control that results from the arrangement of genes in polycistronic units and lack of dynamically regulated transcription factors. To identify the contribution of dynamic phosphorylation to T. brucei cell cycle control we have combined cell cycle synchronisation by centrifugal elutriation with quantitative phosphoproteomic analysis. Cell cycle regulated changes in phosphorylation site abundance (917 sites, average 5-fold change) were more widespread and of a larger magnitude than changes in protein abundance (443 proteins, average 2-fold change) and were mostly independent of each other. Hierarchical clustering of co-regulated phosphorylation sites according to their cell cycle profile revealed that a bulk increase in phosphorylation occurs across the cell cycle, with a significant enrichment of known cell cycle regulators and RNA binding proteins (RBPs) within the largest clusters. Cell cycle regulated changes in essential cell cycle kinases are temporally co-ordinated with differential phosphorylation of components of the kinetochore and eukaryotic initiation factors, along with many RBPs not previously linked to the cell cycle such as eight PSP1-C terminal domain containing proteins. The temporal profiles demonstrate the importance of dynamic phosphorylation in co-ordinating progression through the cell cycle, and provide evidence that RBPs play a central role in post-transcriptional regulation of the T. brucei cell cycle. Data are available via ProteomeXchange with identifier PXD013488.

Calcineurin-dependent Protein Phosphorylation Changes During Egg Activation in Drosophila melanogaster

In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca2+ in the oocyte's cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus,, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster,. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.

Snake venoms promote stress-induced senescence in human fibroblasts.

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.

KLLN-mediated DNA damage-induced apoptosis is associated with regulation of p53 phosphorylation and acetylation in breast cancer cells

KLLN is a target of p53 involved in S-phase cell cycle regulation deemed necessary and sufficient for p53-mediated apoptosis. Germline promoter hypermethylation of KLLN is associated with a cancer-predisposition syndrome, Cowden syndrome. KLLN’s DNA-binding ability is associated with transcription regulation and maintenance of genomic stability. Here, we report on KLLN’s role in DNA damage response (DDR) mediated through apoptosis in breast cells with and without a cancer phenotype. KLLN expression was upregulated after doxorubicin-induced DNA damage and this upregulation can be abrogated using RNAi-mediated gene silencing. Silencing KLLN after doxorubicin treatment effected DDR shown by decreased γ-H2AX foci and expression, and apoptosis assessed by decreased frequency of apoptotic nuclei and decreased expression of definitive markers of apoptosis. Contrary to expectations, there was no change in cell cycle regulation after KLLN silencing. These results were observed in breast cells with wildtype and mutant p53. At early timepoints after doxorubicin treatment, knocking down KLLN resulted in decreased Ser15-phosphorylation of p53 but not Thr68-phosphorylation of CHK2 or the phosphorylation of upstream regulators such as ATM and ATR. Interestingly, a second pathway for p53 activation was also affected by knockdown of KLLN. After doxorubicin treatment, Thr454-phosphorylation of DBC1, required to inhibit deacetylation of p53 by SIRT1, was decreased and therefore acetylation of p53 was also decreased with KLLN knockdown. Therefore, our observations suggest that KLLN’s role in DNA damage-induced apoptosis is likely independent of p53 and is associated with a two-pronged regulation of p53 activation.

Ammonium tetrathiomolybdate treatment targets the copper transporter ATP7A and enhances sensitivity of breast cancer to cisplatin.

Cisplatin is an effective breast cancer drug but resistance often develops over prolonged chemotherapy. Therefore, we performed a candidate approach RNAi screen in combination with cisplatin treatment to identify molecular pathways conferring survival advantages. The screen identified ATP7A as a therapeutic target. ATP7A is a copper ATPase transporter responsible for intercellular movement and sequestering of cisplatin. Pharmaceutical replacement for ATP7A by ammonium tetrathiomolybdate (TM) enhanced cisplatin treatment in breast cancer cells. Allograft and xenograft models in athymic nude mice treated with cisplatin/TM exhibited retarded tumor growth, reduced accumulation of cancer stem cells and decreased cell proliferation as compared to mono-treatment with cisplatin or TM. Cisplatin/TM treatment of cisplatin-resistant tumors reduced ATP7A protein levels, attenuated cisplatin sequestering by ATP7A, increased nuclear availability of cisplatin, and subsequently enhanced DNA damage and apoptosis. Microarray analysis of gene ontology pathways that responded uniquely to cisplatin/TM double treatment depicted changes in cell cycle regulation, specifically in the G1/S transition. These findings offer the potential to combat platinum-resistant tumors and sensitize patients to conventional breast cancer treatment by identifying and targeting the resistant tumors' unique molecular adaptations.

Age-related neurogenesis decline in the subventricular zone is associated with specific cell cycle regulation changes in activated neural stem cells.

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals, they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However, little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ), we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months, the proliferative status of activated NSCs was already altered by 6 months, with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures, as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications.

Expression of cyclin A in A549 cell line after treatment with arsenic trioxide.

Arsenic trioxide (ATO) is an effective drug used in acute promyelocytic leukemia (AML). Many reports suggest that ATO can also be applied as an anticancer agent for solid tumors in the future. The influence of arsenic trioxide on the expression of different cell cycle regulators is poorly recognized. The purpose of the current study is to investigate how arsenic trioxide affects cyclin A expression and localization in the A549 cell line. Morphological and ultrastructural changes in A549 cells were observed using light and transmission electron microscopes. Cyclin A localization was determined by immunofluorescence. Image-based cytometry was applied to evaluate the effect of arsenic trioxide on apoptosis and the cell cycle. Expression of cyclin A mRNA was quantified by real-time PCR. After treatment with arsenic trioxide, increased numbers of cells with cytoplasmic localization of cyclin A were observed. The doses of 10 and 15 μM ATO slightly reduced expression of cyclin A mRNA. The apoptotic phenotype of cells was poorly represented, and the Tali imagebased cytometry analysis showed low percentages of apoptotic cells. The A549 population displayed an enriched fraction of cells in G0/G1 phase in the presence of 5μM ATO, whereas starting from the higher concentrations of the drug, i.e. 10 and 15 μM ATO, the G2/M fraction was on the increase. Low expression of cyclin A in the A549 cell line may constitute a potential factor determining arsenic trioxide resistance. It could be hypothesized that the observed alterations in cyclin A expression/distribution may correlate well with changes in cell cycle regulation in our model, which in turn determines the outcome of the treatment.

Abstract 3376: Ras activation in aberrant crypt foci is associated with decreased expression of cell cycle regulators and disruption of stromal homeostasis

Abstract Aberrant crypt foci (ACF) are the earliest detectable premalignant lesion within the colon. A majority of ACF harbor somatically acquired mutations to cancer-related genes. ACF may be an indicator for a colonic mucosa at-risk for cancer development, however only a small subset of ACF have the potential to progress to malignancy. We believe that the majority of ACF activate growth inhibitory programs that ultimately result in their growth arrest and regression. In order to begin to define the gene expression changes that occur within these early microscopic lesions and their underlying stroma, we have developed a novel method that combines laser capture micro-dissection with targeted RNA-seq analysis. In the following study, gene expression profiles of thirteen human ACF biopsy specimens harboring known oncogenic mutations (K-ras, B-raf, N-ras, and APC) were compared to matched normal mucosa. Two gene panels were used to assess the status of important cell regulatory pathways: an apoptosis panel comprised of 267 genes and a customized cell senescence panel consisting of 20 genes. In the apoptosis panel, the expression of 81 genes were differentially expressed in ACF compared to matched normal mucosa. Expression of HRK, a potent activator of apoptosis, was the most significantly down-regulated gene (6.75-fold, q <0.01) in every ACF when compared to normal mucosa. Inactivation of HRK function occurs in a variety of tumor types, including prostate cancer, astrocytomas and lymphomas. ACF with mutations occurring within the MAP-K pathway (K-ras, B-raf, N-ras) showed strong activation of downstream ras-associated genes and down-regulation of cell cycle regulators (CDK4 (p = 0.01), CCNB1 (p = 0.04), CCNA2 (p = 0.01), and TP53 (p = 0.04)). Interestingly, these expression panels were distinct from ACF harboring somatic mutations to APC. Analysis of microdissected stroma directly underlying the aberrant crypt structures revealed 25 differentially expressed genes, with dysregulation of several inflammatory mediators (IL2, IL2RA, IL6, TGFB2, BMP4) and extracellular matrix remodeling targets (TIMP1, TIMP2). Our findings suggest that ras-activated ACF may cause significant changes in cell cycle regulation, as well as alterations in stromal extracellular matrix maintenance and inflammatory response. Citation Format: Allen Mo, Nicole A. Horelik, Stephen Jackson, Thomas J. Devers, Daniel W. Rosenberg. Ras activation in aberrant crypt foci is associated with decreased expression of cell cycle regulators and disruption of stromal homeostasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3376. doi:10.1158/1538-7445.AM2015-3376

Cyclin D1 Again Caught in the Act: Dyrk1a Links G1 and Neurogenesis in Down Syndrome

Cyclin D1 Again Caught in the Act: Dyrk1a Links G1 and Neurogenesis in Down Syndrome

Coordinating Cell Cycle Remodeling with Transcriptional Activation at the Drosophila MBT.

During the maternal-to-zygotic transition (MZT), major changes in cell cycle regulation coincide with large-scale zygotic genome activation. In this chapter, we discuss the current understanding of how the cell cycle is remodeled over the course of the Drosophila MZT, and how the temporal precision of this event is linked to contemporaneous alterations in genome-wide chromatin structure and transcriptional activity. The cell cycle is initially lengthened during the MZT by activation of the DNA replication checkpoint but, subsequently, zygotically supplied factors are essential for establishing lasting modifications to the cell cycle.

SUV39H1 downregulation induces deheterochromatinization of satellite regions and senescence after exposure to ionizing radiation.

While the majority of cancer patients are exposed to ionizing radiation during diagnostic and therapeutic procedures, age-dependent differences in radiation sensitivity are not yet well understood. Radiation sensitivity is characterized by the appearance of side effects to radiation therapy, such as secondary malignancies, developmental deficits, and compromised immune function. However, the knowledge of the molecular mechanisms that trigger these side effects is incomplete. Here we used an in vitro system and showed that low-senescent normal human diploid fibroblasts (WI-38) senesce in response to 5 Gy IR, while highly senescent cultures do not show changes in cell cycle regulation and only a slight increase in the percentage of senescent cells. Our study shows that this is associated with changes in the expression of genes responsible for cell cycle progression, apoptosis, DNA repair, and aging, as well as transcriptional and epigenetic regulators. Furthermore, we propose a role of the downregulation of SUV39H1 expression, a histone methyltransferase that specifically trimethylates H3K9, and the corresponding reduction in H3K9me3 levels in the establishment of IR-induced senescence.

Abstract A10: Functional characterization of Ewing's sarcoma susceptibility loci

Abstract Background: We previously identified in a GWAS common variants near the genes TARDBP (chr1), EGR2 and ADO (both chr10), and BMF (chr15) that are associated with the susceptibility of Caucasians toward Ewing's sarcoma (ES). These variants lie in intergenic regions that appear to harbor expression quantitative trait loci (eQTLs) as they show strong modulatory effects on the expression of these “candidate genes”. While TARDBP is an mRNA and miRNA processing protein, EGR2 is a conserved transcription factor involved proliferation and differentiation of osteoprogenitors and Schwann cells. ADO, which shows a highly significant co-expression with EGR2, is the key enzyme converting cysteamine into hypotaurine. The BCL2-modifying factor BMF is involved in pro- and anti-apoptotic processes depending on the cellular context. As most SNPs identified by GWAS are usually not the causal variants but are expected to be in strong linkage disequilibrium with such variants, this project aims to comprehensively sequence and epigenetically profile the three ES susceptibility regions and to functionally characterize the role of the four candidate genes in ES. Methods and Results: Fine-selection of the genomic regions (in total 1 Mb) was performed with own and HapMap CEU data. An initial pilot experiment (48 samples) using a custom Nextera DNA enrichment system followed by paired-end 150x150bp sequencing (Illumina HiSeq2500) yielded 95% coverage with at least 20 high-quality reads/nucleotide. Comparison with matched SNP arrays revealed that both the false positive and negative discovery rates were smaller than 1%. Currently, we sequence additional 576 cases and PCA-matched controls and perform a multilayered epigenetic profiling of ES cells with/without silencing of EWS/FLI1 for our susceptibility regions. Analysis of three comprehensive paired gene expression and genotype datasets for lymphoblastoid cell lines, medulloblastoma, and neuroblastoma, proved that none of our Ewing SNPs correlated with the expression the four candidate genes, suggesting a specific effect of these eQTLs in ES, possibly via direct or indirect interaction with EWS/FLI1. In accordance, siRNA-mediated knockdown of EWS/FLI1 in ES cell lines strongly down-regulates EGR2, while EWS/FLI1 expression in human mesenchymal stem cells (MSCs) induces EGR2. In contrast, only moderate modulatory effects of EWS/FLI1 were observed for TARDBP, ADO, and BMF. However, three independent ChIP-seq studies did not find evidence of EWS/FLI1 binding to the EGR2 promoter or nearby regions, arguing for an indirect mode of regulation. On a functional level, knockdown of TARDBP, EGR2, and BMF, but not ADO reduces proliferation and clonogenicity of ES cells by blocking cell cycle progression and inducing apoptosis, which is preceded by transcriptional changes in cell cycle regulators and pro-survival genes as measured by microarrays, RNA-seq and qRT-PCR. Interestingly, all candidate genes share a highly significant overlap in modulated genes with EWS/FLI1 and their transcriptional fingerprint is similar to that conferred by EWS/FLI1 to human MSCs, the most likely cells of origin of ES. Gene-set enrichment and Ingenuity pathway analyses revealed that the candidate genes are involved in cell growth and differentiation, albeit via diverse mechanisms. Additional experiments, including the generation of inducible shRNA expression systems, are ongoing to further characterize their role and their interplay with EWS/FLI1 in ES. Conclusions: In synopsis, our data indicate that the previously identified ES susceptibility regions and candidate genes may play a prominent role in ES pathobiology. Citation Format: Thomas Grunewald, Marie-Ming Aynaud, Franck Tirode, Eleni Tomazou, Didier Surdez, Thomas Rio Frio, Virginie Bernard, Virginie Raynal, Carlo Lucchesi, Gaelle Pierron, Pascale Gilardi-Hebenstreit, Patrick Charnay, Heinrich Kovar, Olivier Delattre. Functional characterization of Ewing's sarcoma susceptibility loci. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A10.