G1 Arrest In Cells Research Articles

Inhibition of CDK4/CDK6 Sensitizes Myeloma to IMiD By Reducing the MEIS2 to Cereblon Ratio That Accelerates IKZF1 and IKZF3 Degradation

Lenalidomide (Len) and pomalidomide (Pom) are immunomodulatory drugs (IMiDs) effective in hematologic malignancies, in combination therapies for multiple myeloma (MM) in particular. Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Emerging evidence suggests that IMiDs bind CRBN and block an endogenous substrate MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN and their degradation. This then leads to loss of IRF4 necessary for myeloma survival. The clinical relevance of these novel findings, however, has not been defined.To address this question, we've investigated the mechanism of IMiD action and the functional consequences in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=31) in stromal co-culture ex vivo in the context of the clinical response to Len or Pom in vivo before or after biopsy. We showed by whole transcriptome sequencing, protein analysis and functional assays that 1) BMMCs are addicted to IKZF3-IRF4 for survival; 2) Len-mediated IRF4 loss leads to de-repression of IRF7, induction of interferon (IFN) response genes and TRAIL-mediated apoptosis; and 3) the magnitude of IFN induction is tightly associated with killing of BMMCs by Len. Importantly, the IMiD sensitivity in BMMCs ex vivo correlated with the prior or subsequent clinical response to IMiD-based therapies in individual myeloma patients, suggesting that the clinical response to IMiDs in myeloma is largely intrinsic to myeloma cells.IMiDs have been reported to cause cell cycle arrest. We found that before evidence of killing, Len and Pom induced late G1 arrest by both repressing CCNA2 (encoding cyclin A) mRNA synthesis and elevating p21 and p27 proteins independent of Rb and p53. This result suggests that IMiDs preferentially kill cells in G1 arrest, and that induction of p rolonged early G1 arrest (pG1) beyond the normal G1 transit time by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991, Ibrance) may sensitize MM cells to IMiD killing, as it does to killing by other agents. Indeed, induction of pG1 by palbociclib overrides cell cycle regulation by Len, and sensitizes BMMCs to Len-mediated apoptosis by augmenting the loss of IRF4 protein and the induction of IRF7, IFNb and TRAIL.Further investigation revealed that induction of pG1 by CDK4/CDK6 inhibition sensitizes primary myeloma cells to IMiD killing by rapid acceleration of Len-mediated loss of IKZF1 and IKZF3 proteins, within one hour of IMiD addition. Loss and gain of function studies demonstrates that MEIS2 opposes pG1 sensitization to Len killing; however, MEIS2 itself is regulated by the cell cycle. Induction of pG1 reduces the ratio of MEIS2 to CRBN by both reducing the MEIS2 protein rapidly and increasing the CRBN protein at a later time in cooperation with Len.In summary, our data provide the first evidence that induction of prolonged early G1 arrest by selective inhibition of CDK4/CDK6 amplifies IMiD killing of primary myeloma cells by both repressing MEIS2 and increasing CRBN protein in cooperation with Len. This leads to a profound reduction in the ratio of MEIS2 to CRBN that accelerates the loss of IKZF1, IKZF3 and IRF4, and enhances IFN and TRAIL induction. Reducing the MEIS2/CRBN ratio thus represents a novel mechanism by which CDK4/CDK6 inhibition sensitizes myeloma to IMiDs, and a means for developing mechanism-based IMiD therapy through cell cycle control. DisclosuresHuang:Celgene: Research Funding. Off Label Use: Palbociclib (PD 0332991) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Rossi:Calgene: Speakers Bureau. Pearse:Celegen: Consultancy. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Chen-Kiang:Celgene: Consultancy.

BCL-2 Inhibition By ABT-199 (Venetoclax/GDC-0199) and p53 Activation By RG7388 (Idasanutlin) Reciprocally Overcome Leukemia Apoptosis Resistance to Either Strategy Alone: Efficacy and Mechanisms

Acute myeloid leukemia (AML) is primarily treated with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel targeted therapy. We have reported elevated expression of BCL-2 in AML and that BCL-2 inhibition by ABT-199 (ABT, venetoclax) induced on-target apoptosis, which could be predicted by BH3 profiling (Pan, et al., Cancer Discovery, 2014). ABT also showed encouraging clinical activity in relapsed/refractory AML (Konopleva et al., ASH 2014), yet MCL-1-mediated resistance may limit its use as monotherapy.p53 mutations are relatively rare in AML, but its functions are often suppressed by overexpressed MDM2 protein. Since p53 and BCL-2 family proteins are central regulators of apoptosis, we asked whether concurrent BCL-2 inhibition and p53 activation (by MDM2 inhibitor) could overcome resistance to apoptosis and synergistically induce apoptosis in AML cells.The novel MDM2 inhibitor RG7388 (RG, Idasanutlin) robustly activated p53 and induced growth inhibition and apoptosis of AML cells in a p53-dependent manner. p53 activation by RG also synergized with BCL-2 inhibition in killing ABT-sensitive cell lines such as MOLM-13 or MV-4-11 (Fig. 1A). After long-term exposure to escalating doses of ABT, initially sensitive cells upregulated MCL-1 and acquired resistance. Nonetheless, the acquired resistance could be effectively abrogated by RG (Fig. 1B). OCI-AML3 cells express a high basal level of MCL-1, and are inherently resistant to ABT. Concurrent p53 activation and BCL-2 inhibition induced synergistic apoptosis and overcame the inherent ABT resistance (Fig. 1C).Next, we studied the underlying mechanisms. p53 activation by RG increased the expression of PUMA and BAX (but not NOXA, Fig. 1D), which are able to counteract MCL-1. In addition, p53 activation quickly dephosphorylated ERK2 and downregulated MCL-1 (Fig. 1E). Surprisingly, ABT increased ERK2 phosphorylation and upregulated MCL-1 (Fig. 1E). Because active ERK2 phosphorylates and stabilizes MCL-1, these results indicate that the observed changes in MCL-1 levels could be attributed to ERK2 phosphorylation status. Consistently, ERK2 dephosphorylation by MEK inhibitors quickly reduced MCL-1. Most importantly, ABT-induced ERK2 phosphorylation and MCL-1 upregulation could be reversed by p53 activation (Fig. 1F). These mechanistic studies provide insights into how p53 activation overcomes acquired/inherent resistance to BCL-2 inhibition.OCI-AML3 cells are relatively resistant to p53 activation by RG. We used concomitant Annexin V staining, EdU pulsing and PI staining to simultaneously analyze apoptosis induction and cell cycle distribution of live cells (Fig. 1G). p53 activation by RG induced cell accumulation in G1 phase, while little apoptosis occurred (Fig. 1H). Addition of ABT dramatically increased apoptosis, reduced G1-arrested cells, and boosted apoptotic hallmarks like the cleavage of caspase-9, -3 and PARP-1 (Fig. 1H-I). ABT did not affect p21 expression and cell cycle distribution, and p53 activation induced robust expression of p21 and G1 arrest. Furthermore, p21 knockdown significantly decreased G1-arrested cells and increased apoptosis following p53 activation, indicating that p21 upregulation and G1 arrest mediate apoptosis resistance to p53 activation. Nonetheless, addition of ABT effectively shifted cell response from G1 arrest to apoptosis, suggesting BCL-2 inhibition can reciprocally overcome apoptosis resistance to p53 activation.Next, we tested the combination in two AML mouse models. In an OCI-AML3-derived mouse model (with inherent resistance to ABT or RG), ABT or RG prolonged survival by 10 or 19 d, respectively, while the combination prolonged mouse survival by 61 d (Fig. 1J-K). Currently, we are also following the survival of mice in a MOLM-13 acquired resistance model. Early results indicate the tumor burden in combination group is <1/100 of that in control/ABT groups and ~1/20 of that in the RG group at day 14 (Fig. 1K-L).In summary, BCL-2 inhibition by ABT and p53 activation by RG can reciprocally overcome resistance to apoptosis encountered by using either treatment alone in vitro and in vivo. Since both BCL-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical impact in relapsed/refractory AML. [Display omitted] DisclosuresLeverson:AbbVie: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Nichols:Roche Pharma: Employment, Equity Ownership.

Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

A subset of cancer cell lines is acutely sensitive to the Chk1 inhibitor MK-8776 as monotherapy due to CDK2 activation in S phase.

DNA damage activates Checkpoint kinase 1 (Chk1) to halt cell cycle progression thereby preventing further DNA replication and mitosis until the damage has been repaired. Consequently, Chk1 inhibitors have emerged as promising anticancer therapeutics in combination with DNA damaging drugs, but their single agent activity also provides a novel approach that may be particularly effective in a subset of patients. From analysis of a large panel of cell lines, we demonstrate that 15% are very sensitive to the Chk1 inhibitor MK-8776. Upon inhibition of Chk1, sensitive cells rapidly accumulate DNA double-strand breaks in S phase in a CDK2- and cyclin A-dependent manner. In contrast, resistant cells can continue to grow for at least 7 days despite continued inhibition of Chk1. Resistance can be circumvented by inhibiting Wee1 kinase and thereby directly activating CDK2. Hence, sensitivity to Chk1 inhibition is regulated upstream of CDK2 and correlates with accumulation of CDC25A. We conclude that cells poorly tolerate CDK2 activity in S phase and that a major function of Chk1 is to ensure it remains inactive. Indeed, inhibitors of CDK1 and CDK2 arrest cells in G1 or G2, respectively, but do not prevent progression through S phase demonstrating that neither kinase is required for S phase progression. Inappropriate activation of CDK2 in S phase underlies the sensitivity of a subset of cell lines to Chk1 inhibitors, and this may provide a novel therapeutic opportunity for appropriately stratified patients.

Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection.

Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5′-3′ resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced ‘dirty’ DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids.

Abstract 2588: Anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene

Abstract HER2 is a member of the EGFR family of tyrosine kinase receptors and plays important roles in the pathogenesis of certain human cancers. In non-small cell lung cancer (NSCLC), HER2 amplification or mutations are reported. However, little is known about the benefit of HER2-targeted therapy for NSCLC harboring HER2 alteration. In this study, we investigated the anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alteration, including the novel HER2 mutations in transmembrane domain which we recently identified. Ectopically overexpressing HER2 wild-type or mutant (G776V,Cins, A775insYVMA, G660D, and V659E) in normal bronchial epithelial cells, BEAS2B, showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to EGFR-TKI (gefitinib). Furthermore, we examined anti-tumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alteration and sensitivity to afatinib treatment. Afatinib down-regulated the phosphorylation of HER2, EGFR, and their downstream signalings, and induced anti-proliferative effect through a G1 arrest and apoptotic cell death in HER2 altered cells (H2170, Calu-3, and H1781). Whereas, HER2 non-dependent NSCLC cells (A549, H1299, and H1993) were insensitive to afatinib. Our results strongly suggest that afatinib is a therapeutic option for NSCLC patients with HER2 alteration. Citation Format: Ken Suzawa, Shinichi Toyooka, Masakiyo Sakaguchi, Tomoaki Ohtsuka, Mototsugu Watanabe, Shinsuke Hashida, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. Anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene. [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 2588. doi:10.1158/1538-7445.AM2015-2588

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader.

The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

The Cell Cycle Timing of Human Papillomavirus DNA Replication.

Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

Phytochrome-mediated regulation of cell division and growth during regeneration and sporeling development in the liverwort Marchantia polymorpha.

Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.

The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin.

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results inthe sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.

RFPL4A Increases the G1 Population and Decreases Sensitivity to Chemotherapy in Human Colorectal Cancer Cells

Cell cycle-arrested cancer cells are resistant to conventional chemotherapy that acts on the mitotic phases of the cell cycle, although the molecular mechanisms involved in halting cell cycle progression remain unclear. Here, we demonstrated that RFPL4A, an uncharacterized ubiquitin ligase, induced G1 retention and thus conferred decreased sensitivity to chemotherapy in the human colorectal cancer cell line, HCT116. Long term time lapse observations in HCT116 cells bearing a "fluorescence ubiquitin-based cell cycle indicator" identified a characteristic population that is viable but remains in the G1 phase for an extended period of time (up to 56 h). Microarray analyses showed that expression of RFPL4A was significantly up-regulated in these G1-arrested cells, not only in HCT116 cells but also in other cancer cell lines, and overexpression of RFPL4A increased the G1 population and decreased sensitivity to chemotherapy. However, knockdown of RFPL4A expression caused the cells to resume mitosis and induced their susceptibility to anti-cancer drugs in vitro and in vivo. These results indicate that RFPL4A is a novel factor that increases the G1 population and decreases sensitivity to chemotherapy and thus may be a promising therapeutic target for refractory tumor conditions.

The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.

Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae.

Akacid medical formulation induces apoptosis in myeloid and lymphatic leukemic cell lines in vitro and in vivo.

Akacid medical formulation (AMF) is an oligoguanidine that exerts biocidal activity against airborne and surface microorganisms including bacteria, viruses, fungi, and molds, while showing relatively low toxicity to humans. We have previously shown that AMF exerts antiproliferative effects on a variety of solid tumor cell lines. In this study we raised the question whether AMF could also substantially inhibit cell growth or induce apoptosis in cell lines derived from hematologic malignancies such as leukemia or lymphoma. We found that AMF has antiproliferative effects on various hematologic cell lines derived from human leukemia and lymphoma. Additionally, we show that AMF induces apoptosis in leukemia cell lines not only via the extrinsic and intrinsic pathway, but also in a caspase-independent manner. This effect was found also in G0-arrested cells. Finally, in our animal experiments utilizing male nu/nu Balb/c mice we found a significant growth retardation, which was immunohistochemically associated with a significantly lower number of KI67-positive cells and caspase-3 induction in AMF-treated mice.

P53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress.

Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.

Abstract IA11: New insights into DNA double-strand break responses

Abstract Repair of damaged DNA in cells is complicated by the necessity to repair the damage in the context of complex chromatin structures. Elucidation of molecular mechanisms of DNA repair in intact cells has been challenging. We developed a system that can rapidly induce DNA double strand breaks (DSBs) at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs in viable cells. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Using chromatin immunoprecipitation, we found that nucleosomes are partially disassembled around DSBs during non-homologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its RGG domain and is rapidly recruited to DSBs in an MRE11-NBS1-RAD50 complex dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and repair of the DSB, demonstrating for the first time the functional importance of nucleosome disruption in DSB repair and identifying a chromatin remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for RPA recruitment to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair. Since inhibition of nucleolin function blunts the DSB repair process, it has become a potential therapeutic target for radiosensitization of tumors. Similarly, small molecule inhibitors of PIKK kinases like ATM and DNA-PK have been developed and are potent radiosensitizers. The potential clinical use of such compounds will be discussed. Citation Format: Michael B. Kastan. New insights into DNA double-strand break responses. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA11. doi:10.1158/1538-7445.CANSUSC14-IA11

Abstract 920: Secreted frizzled related protein 1 (SFRP1) as potential regulator of chemotherapy response for patients with triple negative breast cancer (TNBC)

Abstract Background: Triple negative breast cancer (TNBC) is associated with an unfavorable prognosis and increased response to chemotherapy. While proliferation rate and tumor grade are determining factors, we have recently suggested additional molecular structures to have significant impact. Based on differential gene expression analysis we identified SFRP1 (secreted frizzled related protein 1) to be a potential molecular marker for response to chemotherapy and potential prognostic marker. Methods: Differential gene expression analysis was performed between TNBC (n=27) versus non-TNBC (n=106) using AffymetrixU133A gene chips from breast cancers of patients treated with neoadjuvant taxane/anthracycline chemotherapy at MDACC (Hess et al. 2006). Differentially expressed genes were validated using profiles of 286 patients with breast cancer who did not receive systemic therapy (Wang et al. 2006). SFRP1 knockdown experiments with the TNBC cell line MDA-MB 468 were performed via siRNA transfection and carried out using assays for proliferation, chemo- and radioresistance. Results: SFRP1 was identified as differentially overexpressed gene in TNBC and successfully validated in the independent dataset. Protein and mRNA quantification of SFRP1 (TNBC vs. non-TNBC cell lines) confirm that. While in TNBC, SFRP1 expression showed no association with recurrence-free survival, it is significantly correlated with an increased sensitivity to neoadjuvant chemotherapy. siRNA-mediated knockdown of SFRP1 expression in MDA-MB 468 was associated with increased G2-arrested cells and importantly increased resistance to paclitaxel, doxorubicin and cis-platinum. Furthermore, radioresistance of MDA-MB 468 cells was significantly increased after SFRP1 knockdown. Interestingly, sensitivity to salinomycin was slightly increased after SFRP1 knockdown. Salinomycin is known to kill cancer stem cells (Gupta et al. 2009). Additionally, cancerous characteristics (invasion and migration) were increased after SFRP1 knockdown while target gene expression analysis revealed an association between loss of SFRP1 and activation of mTOR/PI3K signaling. Conclusion: We suggest SFRP1 as a novel predictive marker of chemotherapy sensitivity to taxane, anthracycline and platinum-containing chemotherapy independent of Ki67 expression. Further on, we have shown the influence of SFRP1 on cancerous characteristics thus, suggesting SFRP1 as potential prognostic marker. Molecular role of SFRP1 may be the influence on enrichment of cancer stem cell population which are known to be resistant against chemotherapeutics and radiation and are usually slow proliferating. Interestingly, the mTOR/PI3K signaling might be activated via loss of SFRP1 which could display an opportunity for patients with TNBC resistant to current chemotherapeutics. Citation Format: Carolin Huelsewig, Christof Bernemann, Christian Ruckert, Ludwig Kiesel, Martin Goette, Achim Rody, Lajos Pusztai, Georg Hempel, Cornelia Liedtke. Secreted frizzled related protein 1 (SFRP1) as potential regulator of chemotherapy response for patients with triple negative breast cancer (TNBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 920. doi:10.1158/1538-7445.AM2014-920

G1-arrested newborn cells are the predominant infectious form of the pathogen Brucella abortus

Several intracellular pathogens, such as Brucella abortus, display a biphasic infection process starting with a non-proliferative stage of unclear nature. Here, we study the cell cycle of B. abortus at the single-cell level, in culture and during infection of HeLa cells and macrophages. The localization of segregation and replication loci of the two bacterial chromosomes indicates that, immediately after being engulfed by host-cell endocytic vacuoles, most bacterial cells are newborn. These bacterial cells do not initiate DNA replication for the next 4 to 6 h, indicating a G1 arrest. Moreover, growth is completely stopped during that time, reflecting a global cell cycle block. Growth and DNA replication resume later, although bacteria still reside within endosomal-like compartments. We hypothesize that the predominance of G1-arrested bacteria in the infectious population, and the bacterial cell cycle arrest following internalization, may constitute a widespread strategy among intracellular pathogens to colonize new proliferation niches.

Real-time cell cycle imaging during melanoma growth, invasion, and drug response.

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination-based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1-arrested cells showed decreased invasion. Melanoma cells in a hypoxic environment or treated with mitogen-activated protein kinase pathway inhibitors remained G1-arrested for extended periods of time, with proliferation and invasion resuming after re-exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1-arrested subpopulation survives in the presence of targeted therapies.

Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing.

The G2 checkpoint monitors DNA damage, preventing mitotic entry until the damage can be resolved. The mechanisms controlling checkpoint recovery are unclear. Here, we identify non-genetic heterogeneity in the fidelity and timing of damage-induced G2 checkpoint enforcement in individual cells from the same population. Single-cell fluorescence imaging reveals that individual damaged cells experience varying durations of G2 arrest, and recover with varying levels of remaining checkpoint signal or DNA damage. A gating mechanism dependent on polo-like kinase-1 (PLK1) activity underlies this heterogeneity. PLK1 activity continually accumulates from initial levels in G2-arrested cells, at a rate inversely correlated to checkpoint activation, until it reaches a threshold allowing mitotic entry regardless of remaining checkpoint signal or DNA damage. Thus, homeostatic control of PLK1 by the dynamic opposition between checkpoint signalling and pro-mitotic activities heterogeneously enforces the G2 checkpoint in each individual cell, with implications for cancer pathogenesis and therapy.