Cell Cycle Arrest In Response Research Articles

Abstract 2077A: MM141, a novel bispecific antibody co-inhibitor of IGF-1R and ErbB3, inhibits the proliferation of melanoma cells.

Abstract Identification of druggable targets along with new therapies in metastatic melanoma is a main concern given the bad prognosis of this disease in the metastatic setting. The development of targeted therapies for cancer has fundamentally changed the ways in which we develop new drugs, select therapy for patients, design and conduct clinical trials and assess treatment outcomes. The problems can be most effectively attacked by an integrated approach that leads from discovery through preclinical and clinical development. Our therapeutic focus is the development of targeted therapeutics and associated predictive biomarkers that can be used to treat melanoma. Here we present data indicating that the IGF-1R and ErbB3 growth factor pathways are co-active in preclinical models of melanoma and propose that MM-141, a novel bispecific antibody co-inhibitor of IGF-1R and ErbB3, has the potential to provide therapeutic benefit to melanoma patients. Our approach is based on a set of 48 melanoma cell lines that have already been extensively characterized both in terms of genetic background and pharmacologic response. We have quantitatively measured basal expression and activation of 40 total and phospho receptor tyrosine kinases expressed by melanoma cell lines. This characterization was placed in the context of the genetic and pharmacologic data that has already been generated in order to develop pathophysiological hypotheses and points of intervention in melanoma. We have also determined anti-proliferative response and mechanism of action for the MM-141 activity in this cell line panel. Proteomic data analyses showed that melanoma cells have lower basal phospho-protein levels relative to other solid tumor cell line panels. The data showed that EGFR and ErbB2 were not overexpressed. ErbB3 and IGF-1R were the most widely expressed RTKs on melanoma cells, and c-MET and FGFR1 were also prevalent. Genomic and proteomic analyses indicated that ErbB3 and IGF-1R mRNA and protein expression were positively correlated. Upon this preliminary analysis of the data we have tested the activity of MM-141 on 48 human melanoma cell lines with well-established genotypes. Taking 15% growth inhibition as a cutoff for response in cell-based assays after 100μg/ml (500nM) MM-141 treatment, 24 out of 48 cell lines resulted in inhibition of cell proliferation 5 days post-treatment. The anti-proliferative response to MM-141 was independent of genotype. We chose 6 cell lines, 3 from the responder population and 3 from the non-responder population to further investigate the mechanism of action of MM-141. No evidence of apoptosis was found after 5 days of MM-141 treatment. All the sensitive cell lines showed a G0/G1 cell cycle arrest in response to MM-141. These combined data provide the rationale for further preclinical and clinical evaluation of MM-141 as a melanoma therapeutic. Citation Format: Erika M. Von Euw, Emily Pace, Karina Covarrubias, Abhi Jairam, Diana Chai, Veerauo Konkankit, Ke-Wei Gong, Bryan Johnson, Birgit Schoeberl, Alexey Lugovskoy, Finn Richard, Dennis Slamon. MM141, a novel bispecific antibody co-inhibitor of IGF-1R and ErbB3, inhibits the proliferation of melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2077A. doi:10.1158/1538-7445.AM2013-2077A

Abstract 4404: Multicellular tumor spheroid models to evaluate drugs targeting cell cycle checkpoints in 3D.

Abstract MultiCellular Tumor Spheroid (MCTS) mimic the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. We report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization, the dynamics of cell cycle checkpoints and the effects of anticancer drugs in 3D. Spheroids expressing fluorescent reporters were engineered and used to explore cell cycle distribution and regionalization during growth. In these models we monitored cell cycle arrest in response to checkpoint activation after growth factor starvation, DNA injury or treatment with chemotherapeutic agents. The kinetics and regionalized aspects of the response were investigated both on fixed sections and on live spheroids using light sheet microscopy. All together, our data demonstrate the power of the combination of spheroids made of genetically modified cells expressing cell cycle reporters with both classical antibody-based detection and the use of innovative 3D imaging strategy. This study paves the way for the investigation of the molecular aspects of checkpoint response in 3D models and the dynamic studies of the 3D response to novel antiproliferative agents. Citation Format: Bernard Ducommun, Valérie Lobjois, Jennifer Laurent, Céline Frongia, Odile Mondesert, Martine Cazales. Multicellular tumor spheroid models to evaluate drugs targeting cell cycle checkpoints in 3D. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2013-4404

Abstract 782: The Six1 homeoprotein downregulates p53, via a novel, MDM2 independent mechanism.

Abstract The p53 tumor suppressor is a critical regulator of genomic stability. P53 is known as “the guardian of the genome” because it plays a crucial role in inducing apoptosis and cell cycle arrest in response to DNA damage. P53 is mutated in approximately 50% of all human tumors and is downregulated in a large majority of the remainder. An important regulator of p53 is the E3 ubiquitin ligase, MDM2, which acts to ubiquitinate p53 and target it for degradation through the proteasome. There is currently a large effort in the field directed toward developing targeted therapies that inhibit MDM2′s ability to interact with p53, thus allowing p53 to be stabilized. One such drug that is already undergoing clinical trials is Nutlin-3a. Our preliminary data demonstrate that the homeobox containing transcription factor, Six1, decreases the p53 response to DNA damage in a proteasome independent manner, suggesting that it may downregulate the p53 response even in the presence of Nutlins. Six1, is an important developmental regulator as it increases cell migration, invasion, and proliferation, however, it is not expressed in most normal adult tissues. It is, however, re-expressed in cancerous cells, including human breast lesions. As we are currently developing drugs to target Six1, it is essential to understand mechanistically how Six1 downregulates p53 and whether or not inhibition of Six1 may re-sensitize cells to Nutlin therapies. Here we show that Six1 downregulates p53, not on the mRNA level, but on the protein level, and that this downregulation leads to a decrease in p53 function. We have shown that Six1 can target p53 in a variety of different cell culture systems, including both breast and colon cancer cell lines, as well as normal breast and embryonic kidney cell lines. We have begun to examine the mechanism by which Six1 downregulates p53, and have shown that it is able to target p53 independent of MDM2, as siRNA knock down of MDM2 is not able to rescue p53 levels in the presence of Six1. Furthermore, Six1 does not affect the half life of p53, and congruently Six1 is able to downregulte p53 even in the presence of a proteosome inhibitor. Together, these results suggest that MDM2’s ability to target p53 for proteasomal degradation is dispensable for the ability of Six1 to decrease p53 levels. Furthermore, our preliminary data suggests that Six1 regulates p53 in a post-transcriptional mechanism, potentially through the use of the p53 3’UTR region. Six1 overexpressing lines are more resistant to Nutlin therapies in-vitro, demonstrating a significant increase in viability in the presence of Nutlins when compared to control cell lines. Thus, high levels of Six1 may correlate with decreased response to Nutlins clinically, and targeting of Six1 may be a novel approach to re-sensitize tumors expressing high levels of Six1 to Nutlin therapy. Citation Format: Christina N. Garlington, Douglas S. Micalizzi, Heide L. Ford. The Six1 homeoprotein downregulates p53, via a novel, MDM2 independent mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2013-782

Resveratrol abrogates the Temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the Temozolomide-induced senescence in glioma cells

BackgroundTemozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects.MethodsGlioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity.ResultsAs expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence.ConclusionThe presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.

The BH3 mimetic ABT-263 synergizes with the MEK1/2 inhibitor selumetinib/AZD6244 to promote BIM-dependent tumour cell death and inhibit acquired resistance

Tumour cells typically exhibit a G(1) cell cycle arrest in response to the MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitor selumetinib, but do not die, and thus they acquire resistance. In the present study we examined the effect of combining selumetinib with the BH3 [BCL2 (B-cell lymphoma 2) homology domain 3]-mimetic BCL2 inhibitor ABT-263. Although either drug alone caused little tumour cell death, the two agents combined to cause substantial caspase-dependent cell death and inhibit long-term clonogenic survival of colorectal cancer and melanoma cell lines with BRAF(V600E) or RAS mutations. This cell death absolutely required BAX (BCL2-associated X protein) and was inhibited by RNAi (RNA interference)-mediated knockdown of BIM (BCL2-interacting mediator of cell death) in the BRAF(V600E)-positive COLO205 cell line. When colorectal cancer cell lines were treated with selumetinib plus ABT-263 we observed a striking reduction in the incidence of cells emerging with acquired resistance to selumetinib. Similar results were observed when we combined ABT-263 with the BRAF(V600E)-selective inhibitor PLX4720, but only in cells expressing BRAF(V600E). Finally, cancer cells in which acquired resistance to selumetinib arises through BRAF(V600E) amplification remained sensitive to ABT-263, whereas selumetinib-resistant HCT116 cells (KRAS(G13D) amplification) were cross-resistant to ABT-263. Thus the combination of a BCL2 inhibitor and an ERK1/2 pathway inhibitor is synthetic lethal in ERK1/2-addicted tumour cells, delays the onset of acquired resistance and in some cases overcomes acquired resistance to selumetinib.

A nontranscriptional role for HIF-1α as a direct inhibitor of DNA replication.

Cell cycle arrest in response to hypoxia is a fundamental physiological mechanism to maintain a balance between O(2) supply and demand. Many of the cellular responses to reduced O(2) availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the minichromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. Although the interaction between Cdc6 and the MCM complex increased the association of the MCM proteins with chromatin, the binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. These findings establish a transcription-independent mechanism by which the stabilization of HIF-1α leads to cell cycle arrest in response to hypoxia.

Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

BackgroundMultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D.MethodsCell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters.ResultsWe describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage.ConclusionOur data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models.

Plasma membrane proteomics identifies Notch1 as a potential regulator of ras-induced senescence

BackgroundOncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism leading to stable cell-cycle arrest in response to oncogene activation. OIS is a heterogeneous phenotype of multiple effector mechanisms; understanding of in-vivo OIS is lacking because of the difficulty in identifying senescence. We wished to establish a cell-surface phenotype of OIS. MethodsWe used ER:Ras IMR90 human diploid fibroblasts, which undergo OIS after 6 days with 4-hydroxytamoxifen (4-OHT). We used SILAC (stable isotope labelling with amino acids in cell culture)-based proteomics and three labelling conditions (light, IMR90 plus 4-OHT; medium [lysine, K+4Da, arginine, R+6Da] ER:ras IMR90; heavy [K+8Da, R+10Da] ER:Ras IMR90 plus 4-OHT), combined with cell surface aminooxybiotinylation before streptavidin pulldown. Tryptic peptides were fractionated by high pH reversed-phase high performance liquid chromatogrpahy and then subjected to liquid chromatograph mass spectrometry. Data were processed by the analytical packages MaxQuant and MASCOT. Hits were validated by fluorescence-activated cell sorting (FACS), quantitative PCR, and immunoblotting. shRNA-mediated knockdown of protein expression used the murine stem cell virus-miR30 system. Findings899 proteins were identified, of which 73% were present at the cell surface by Gene Ontology annotation. Notch1 was significantly upregulated in OIS compared with both control conditions (3·1–3·4 fold). Upregulation was confirmed by both FACS and immunoblotting. Downstream Notch target-genes were also upregulated in OIS. Treatment with the γ-secretase inhibitor DAPT increased both senescence-associated heterochromatin foci (SAHF) and senescence-associated β galactosidase (SA-β-gal) activity, features of OIS. However, Notch1 knockdown reduced both SAHF and SA-β-gal. The effect of DAPT was not mediated through canonical Notch1 signalling since RBP-j knockdown had no effect upon DAPT-mediated SAHF and SA β-gal upregulation. Numb, a canonical Notch pathway inhibitor, is upregulated in OIS. Numb may underpin a transition switch from canonical to non-canonical Notch signaling in OIS. InterpretationPlasma membrane proteomics has identified a cell-surface phenotype of OIS. Notch 1 cell surface expression and downstream target-genes are upregulated in OIS. The transition to OIS is correlated with a transition from canonical to non-canonical Notch1 signalling that may be driven by Numb expression. FundingCancer Research UK.

Gene Expression Profiling from a Prostate Cancer PC-3 Cell Line Treated with Salinomycin Predicts Cell Cycle Arrest and Endoplasmic Reticulum Stress

Abstract Previously, we reported that salinomycin induces apoptosis of human prostate cancer cells through accumulated reactive oxygen species and mitochondrial membrane depolarization. To extend our understanding for the genomewide expression pattern, we performed cDNA microarray analysis for gene expression profiles in prostate PC-3 cells treated with salinomycin. We found a couple of differences from gene expression profiles. First of them, cyclins and cyclin-dependent kinases were down-regulated, whereas cyclin dependent kinase inhibitors were upregulated, implicating inhibition of cell cycle progression. Second, HSPA5/Bip, DDIT3/CHOP, TRIB3, ATF4 and ATF6 regarding endoplasmic reticulum (ER) stress and unfolded protein response (UPR) were increased at mRNA and protein levels, indicating salinomycin-induced growth inhibition of PC-3 cells seem to be mediated through induction of ER stress and activation of the UPR pathway. Our finding should be useful for understanding genomewide expression patterns of salinomycin-mediated cell cycle arrest and ER stress response toward induction of apoptosis and be helpful for finding future cancer therapeutic targets in prostate cancer cells.

Human rpL3 induces G₁/S arrest or apoptosis by modulating p21waf1/cip1 levels in a p53-independent manner

It is now largely accepted that ribosomal proteins may be implicated in a variety of biological functions besides that of components of the translation machinery. Many evidences show that a subset of ribosomal proteins are involved in the regulation of the cell cycle and apoptosis through modulation of p53 activity. In addition, p53-independent mechanisms of cell cycle arrest in response to alterations of ribosomal proteins availability have been described. Here, we identify human rpL3 as a new regulator of cell cycle and apoptosis through positive regulation of p21 expression in a p53-independent system. We demonstrate that the rpL3-mediated p21 upregulation requires the specific interaction between rpL3 and Sp1. Furthermore, in our experimental system, p21 overexpression leads to a dual outcome, activating the G₁/S arrest of the cell cycle or the apoptotic pathway through mitochondria, depending on its intracellular levels. It is noteworthy that depletion of p21 abrogates both effects. Taken together, our findings unravel a novel extraribosomal function of rpL3 and reinforce the proapoptotic role of p21 in addition to its widely reported ability as an inhibitor of cell proliferation.

The CCAAT/enhancer-binding protein δ (C/EBP-δ) transcription factor is heat inducible in the cold-adapted antarctic fish Trematomus bernacchii

Notothenioids dominate the Antarctic ichthyofauna, which for millions of years has evolved in a thermally stable environment near the freezing point of seawater. These species are extreme stenotherms and at least some have lost the ability to up-regulate heat shock proteins during exposure to thermal stress. A recent cDNA microarray study revealed the capacity to mount a broad-scale genomic response to heat in the common notothenioid Trematomus bernacchii. Two of the heat-sensitive genes identified were of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. One of these, C/EBP-δ, mediates inflammatory and pro-apoptotic processes, though it is best known for functioning as a brake on cell division. Because apoptosis and cell-cycle arrest are two understudied facets of the cellular stress response, we used western blotting to examine C/EBP-δ expression in T. bernacchii under normal and heat-stressed conditions. A highly tissue-specific pattern of constitutive levels of C/EBP-δ was detected in field-acclimated individuals. Highest levels were found in white muscle followed by spleen, brain, and gill. C/EBP-δ was not detected in heart or liver. Individuals acclimated for 1 month at 4.0 °C possessed more C/EBP-δ in their white muscle compared with field-acclimated fish. Acute heat stress of 2.0 or 4.0 °C caused a transient increase in white muscle C/EBP-δ after 6 h when compared with controls (−1.0 ± 0.5 °C). This up-regulation appears to be reversible upon return to non-stressful conditions. We posit that in unstressed T. bernacchii, C/EBP-δ performs housekeeping functions and that it mediates inflammation, apoptosis, and/or cell-cycle arrest in response to sub-lethal heat stress.

Doxorubicin promotes transcriptional upregulation of Cdc25B in cancer cells by releasing Sp1 from the promoter

Cdc25B phosphatases have a key role in G2/M cell-cycle progression by activating the CDK1-cyclinB1 complexes and functioning as important targets of checkpoints. Overexpression of Cdc25B results in a bypass of the G2/M checkpoint and illegitimate entry into mitosis. It can also cause replicative stress, which leads to genomic instability. Thus, fine-tuning of the Cdc25B expression level is critical for correct cell-cycle arrest in response to DNA damage. In response to genotoxic stress, Cdc25B is mainly regulated by post-transcriptional mechanisms affecting either Cdc25B protein stability or translation. Here, we show that upon DNA damage Cdc25B can be regulated at the transcriptional level. Although ionizing radiation downregulates Cdc25B in a p53-dependent pathway, doxorubicin transcriptionally upregulates Cdc25B in p53-proficient cancer cells. We show that in the presence of wild-type p53, doxorubicin activates the Cdc25B promoter by preventing the binding of Sp1 and increasing the binding of NF-Y on the Cdc25B promoter, thus preventing p53 from downregulating this promoter. Our results highlight the mechanistically distinct regulation of the three Cdc25 phosphatases by checkpoint signalling following doxorubicin treatment.

Methylation of the Proximal Promoter of GADD45A Is Common in Acute Myeloid Leukemia and Is Associated with Poor Survival.

AbstractAbstract 2396 Background:Despite recent advances in understanding the key molecular mechanisms of leukemogenesis, the outcome for patients with Acute Myeloid Leukemia, particularly with a normal karyotype, remains poor. For this large group of patients, genetic alterations in genes such as FLT3, NPM1, CEBPA, IDH1/2, and DNMT3A provide useful prognostic information. However, risk stratification of this group remains only partially resolved and markers of response that can be therapeutically targeted would likely improve outcome for these patients. GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses (Cancer Ther. 2009;7:268). Methylation of four discrete CpG residues in the proximal promoter of GADD45A is a hallmark of many solid tumours and has been associated with impaired cell stress signalling and reduced drug response (Cancer Res. 2009;69:1527; Oncogene. 2005;24:2705). In AML, GADD45A expression is broadly down-regulated both in normal karyotype and other cytogenetic classes. Down-regulation of GADD45A in AML has been associated with FLT3-ITD (Leukemia. 2009;23:729) and RUNX1 mutations (Satoh et al, Leukemia. 2011;Epub). For those patients without these mutations, the mechanism of GADD45A down-regulation and its prognostic significance remains unknown. We hypothesised that the promoter of GADD45A is methylated in AML and that this methylation is functionally important in patient response. Methods:Using the Sequenom MassARRAY methodology we screened for methylation of four GADD45A promoter CpG dinucleotides (CpG1–4) previously shown to be associated with silencing of GADD45A in breast and prostate cancer, in a retrospective cohort of 222 AML patients collected at diagnosis from the Royal Adelaide Hospital. We then determined association of CpG methylation with outcome and mutation status in our treated patient cohort of 167 patients. In AML cell lines and in primary patient samples we also determined the response to cytotoxic agents in vitro in the presence and absence of demethylating agents. Results:We observed hypermethylation of the CpG1–4 in the proximal promoter of GADD45A in 93 of 222 (42%) of AML patients and in 6 AML cell lines. In the 167 patients treated with standard induction chemotherapy regimes, 61 patients showed methylation of the GADD45A proximal promoter. Of the four CpG residues, methylation of CpG1 was associated with poor overall and event-free survival, in AML overall (Figure 1A) and in normal karyotype AML (Figure 1B). GADD45A CpG1 methylation was significantly associated with IDH1 and IDH2 mutations (p<0.001), but was not associated with FLT3-ITD or other high risk cytogenetic groups. Multivariate analysis (including age, wcc, FLT3-ITD, IDH1/2) revealed that methylation of GADD45A CpG1 is an independent predictor of poor survival in AML, overall (OS; HR 2.17, p=0.006: EFS; HR 2.43, p=0.001), and in normal karyotype AML (OS; HR 2.86, p=0.014: EFS; 5.25, p<0.001). Additionally, treatment of AML cell lines and patient blasts with decitabine (5-Aza-deoxycitidine) resulted in induction of GADD45A mRNA selectively in samples with GADD45A hyper-methylation, and this was associated with increased sensitivity to daunorubicin. Conclusions:DNA methylation of the GADD45A proximal promoter marks a large percentage AML patients, including those with with IDH1/2 mutations, and is an independent predictor of poor outcome, particularly in AML patients with normal karyotype. Our biological data shows that induction of GADD45A mRNA expression with decitabine in methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A proximal promoter methylation represents a new biomarker that may provide prognostic information in the heterogeneous normal karyotype AML group. Given that recent clinical trials are combining Azacitidine with chemotherapy and other agents for induction therapy in AML (Blood;118:1472), this may represent a marker to help define patients that will benefit from this approach. [Display omitted] [Display omitted] Disclosures:Wei:Celgene: Research Funding.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

AbstractAbstract 2417Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011).BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown.To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels.Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C).Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Distinct Drug Responses of CD8+CD4+ and CD8+CD4−leukemic Cell Subsets in Mice with Notch1-Induced T-ALL

AbstractAbstract 1504Leukemic cells are heterogeneous, so defining the differences of leukemic cell sub-populations in response to drug treatment may ultimately help design better strategies to maximize the efficacy of chemotherapeutic agents for patients. We have begun to study this issue with Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in mice. The Notch1 signaling pathway plays an essential role in cell growth and differentiation, and activated mutations in the Notch1 gene are frequently observed in human T-ALL. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-ALL in mice. In our current study, we examined T-ALL cells, specifically the CD8 and CD4 double positive (CD8+CD4+) and CD8 single positive (CD8+CD4−) cell sub-populations, for their distinct sensitivities to drug treatment.We first treated T-ALL mice with a cell-cycle nonspecific chemical drug, cyclophosphamide (CTX), or a mTORC1 inhibitor, rapamycin, when the overall T-ALL cell population rose to more than 10% in mouse peripheral blood. We found that CTX treatment effectively reduced the T-ALL cells and led to an increase in the ratio of CD8+CD4+ versus CD8+CD4− T-ALL, indicating that CD8+CD4− T-ALL cells were more sensitive to CTX treatment. In contrast, rapamycin had the opposite effect. When compared with mice treated with rapamycin or CTX alone, the combination of the two drugs led to a significant improvement for T-ALL bearing mice with respect to leukemia initiation, progress and overall survival. We then set out to investigate the mechanism of different sensitivities of CD8+CD4+ and CD8+CD4− T-ALL cells upon CTX or rapamycin treatments. We found that CD8+CD4+ and CD8+CD4−T-ALL cell groups responded similarly to CTX treatment after 12 hours of treatment. However, CD8+CD4−T-ALL cells continued to undergo apoptosis 24 hours after CTX treatment. The expression of the anti-apoptosis gene Bcl-2 in CD8+CD4+ T-ALL cells was significantly higher than that in CD8+CD4− T-ALL cells, while the expression of the pro-apoptosis genes Bax, p53 and Noxa in CD8+CD4+ T-ALL cells was lower than that of CD8+CD4−T-ALL cells after 24 hour CTX treatment. This suggests that CD8+CD4+ and CD8+CD4− T-ALL cells activate apoptosis differently after CTX treatment. Interestingly, rapamycin treatment did not affect apoptosis in the same manner as CTX treatment, but instead, it arrested more T-ALL cells in the G0 phase. More importantly, only CD8+CD4+, but not CD8+CD4−T-ALL cells were sensitive to rapamycin treatment in arresting cell cycle at different time points. The expression levels of CDK2 and CDK4 were significantly lower in CD8+CD4+ T-ALL cells when compared with CD8+CD4− T-ALL cells, while p27 expression in CD8+CD4+ T-ALL cells was higher than that of CD8+CD4− T-ALL cells after rapamycin treatment for 3 or 10 days. These data provide a molecular basis for the distinct apoptotic and cell cycle arrest responses of CD8+CD4+ and CD8+CD4− T-ALL cell subsets to CTX or rapamycin treatment.Taken together, our study documents previously unappreciated, yet distinct properties of CD8+CD4+ and CD8+CD4−Notch1-induced T-ALL cells in the context of specific drug treatment and suggests a therapeutic potential of combining CTX and rapamycin treatment for T-ALL patients. This strategy may also help design better chemotherapeutical regimes for other types of leukemia and cancer. Disclosures:No relevant conflicts of interest to declare.

SUMOylation of hnRNP-K is required for p53-mediated cell-cycle arrest in response to DNA damage

Heterogeneous ribonucleoprotein-K (hnRNP-K) is normally ubiquitinated by HDM2 for proteasome-mediated degradation. Under DNA-damage conditions, hnRNP-K is transiently stabilized and serves as a transcriptional co-activator of p53 for cell-cycle arrest. However, how the stability and function of hnRNP-K is regulated remained unknown. Here, we demonstrated that UV-induced SUMOylation of hnRNP-K prevents its ubiquitination for stabilization. Using SUMOylation-defective mutant and purified SUMOylated hnRNP-K, SUMOylation was shown to reduce hnRNP-K's affinity to HDM2 with an increase in that to p53 for p21-mediated cell-cycle arrest. PIAS3 served as a small ubiquitin-related modifier (SUMO) E3 ligase for hnRNP-K in an ATR-dependent manner. During later periods after UV exposure, however, SENP2 removed SUMO from hnRNP-K for its destabilization and in turn for release from cell-cycle arrest. Consistent with the rise-and-fall of both SUMOylation and stability of hnRNP-K, its ability to interact with PIAS3 was inversely correlated to that with SENP2 during the time course after UV exposure. These findings indicate that SUMO modification plays a crucial role in the control of hnRNP-K's function as a p53 co-activator in response to DNA damage by UV.

Nek4 Regulates Entry into Replicative Senescence and the Response to DNA Damage in Human Fibroblasts

When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage.

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents

DNA-damaging agents (DDAs) constitute the backbone of treatment for most human tumors. Here we used the National Cancer Institute Antitumor Cell Line Panel (the NCI-60) to identify predictors of cancer cell response to topoisomerase I (Top1) inhibitors, a widely used class of DDAs. We assessed the NCI-60 transcriptome using Affymetrix Human Exon 1.0 ST microarrays and correlated the in vitro activity of four Top1 inhibitors with gene expression in the 60 cell lines. A single gene, Schlafen-11 (SLFN11), showed an extremely significant positive correlation with the response not only to Top1 inhibitors, but also to Top2 inhibitors, alkylating agents, and DNA synthesis inhibitors. Using cells with endogenously high and low SLFN11 expression and siRNA-mediated silencing, we show that SLFN11 is causative in determining cell death and cell cycle arrest in response to DDAs in cancer cells from different tissues of origin. We next analyzed SLFN11 expression in ovarian and colorectal cancers and normal corresponding tissues from The Cancer Genome Atlas database and observed that SLFN11 has a wide expression range. We also observed that high SLFN11 expression independently predicts overall survival in a group of ovarian cancer patients treated with cisplatin-containing regimens. We conclude that SLFN11 expression is causally associated with the activity of DDAs in cancer cells, has a broad expression range in colon and ovarian adenocarcinomas, and may behave as a biomarker for prediction of response to DDAs in the clinical setting.

E2F7, a novel target, is up-regulated by p53 and mediates DNA damage-dependent transcriptional repression

The p53 tumor suppressor protein is a transcription factor that exerts its effects on the cell cycle via regulation of gene expression. Although the mechanism of p53-dependent transcriptional activation has been well-studied, the molecular basis for p53-mediated repression has been elusive. The E2F family of transcription factors has been implicated in regulation of cell cycle-related genes, with E2F6, E2F7, and E2F8 playing key roles in repression. In response to cellular DNA damage, E2F7, but not E2F6 or E2F8, is up-regulated in a p53-dependent manner, with p53 being sufficient to increase expression of E2F7. Indeed, p53 occupies the promoter of the E2F7 gene after genotoxic stress, consistent with E2F7 being a novel p53 target. Ablation of E2F7 expression abrogates p53-dependent repression of a subset of its targets, including E2F1 and DHFR, in response to DNA damage. Furthermore, E2F7 occupancy of the E2F1 and DHFR promoters is detected, and expression of E2F7 is sufficient to inhibit cell proliferation. Taken together, these results show that p53-dependent transcriptional up-regulation of its target, E2F7, leads to repression of relevant gene expression. In turn, this E2F7-dependent mechanism contributes to p53-dependent cell cycle arrest in response to DNA damage.