G1 Block Research Articles

Calretinin is essential for mesothelioma cell growth/survival in vitro: A potential new target for malignant mesothelioma therapy?

Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epithelioid and mixed (biphasic) mesothelioma; however, little is known about CR's putative functions in tumorigenesis. CR protects against asbestos-induced acute cytotoxicity mediated by the AKT/PI3K pathway, and furthermore, SV40 early region genes are able to upregulate CR in mesothelial cells. However, the precise role of CR in mesothelioma is still unknown. Downregulation of CR via lentiviral-mediated short-hairpin RNA significantly decreased the viability and proliferation of mesothelioma cells in vitro. The effect was strong in epithelioid-dominated cell lines (ZL55 and MSTO-211H). A weaker and delayed effect was observed in mesothelioma cells with prevalent sarcomatoid morphology (SPC111, SPC212 and ZL34). The specificity of the effect was confirmed by stable enhanced green fluorescent protein-CR expression in mesothelioma cell lines and subsequent downregulation. Depletion of CR led these cancer cell lines to enter apoptosis within 72 hr postinfection via strong activation of the intrinsic caspase 9-dependent pathway. Downregulation of CR in immortalized mesothelial cells LP9/TERT-1 strongly blocked proliferation and caused a G1 block without decreasing viability or activating apoptosis pathways. These results demonstrate that downregulation of CR had a strong effect on the viability of MM cells and that CR is essential for cells derived from MM. The authors anticipate these findings to reveal CR as a highly interesting new putative therapeutic target for mesothelioma treatment of especially the epithelioid, as well as of the mixed and sarcomatoid type.

Influence of chronic hypoxia and radiation quality on cell survival

To investigate the influence of chronic hypoxia and anoxia on cell survival after low- and high-LET radiation, CHO-K1 cells were kept for 24 h under chronic hypoxia (94.5% N2; 5% CO2; 0.5% O2) or chronic anoxia (95% N2; 5% CO2). Irradiation was performed using 250 kVp X-rays or carbon ions with a dose average LET of 100 keV/μm either directly under the chronic oxygenation states, or at different time points after reoxygenation. Moreover, the cell cycle distribution for cells irradiated under different chronic oxic states was measured over 24 h during reoxygenation. The measurements showed a fairly uniform cell cycle distribution under chronic hypoxia, similar to normoxic conditions. Chronic anoxia induced a block in G1 and a strong reduction of S-phase cells. A distribution similar to normoxic conditions was reached after 12 h of reoxygenation. CHO cells had a similar survival under both acute and chronic hypoxia. In contrast, survival after irradiation under chronic anoxia was slightly reduced compared to that under acute anoxia. We conclude that, in hamster cells, chronic anoxia is less effective than acute anoxia in inducing radioresistance for both X-rays and carbon ions, whereas in hypoxia, acute and chronic exposures have a similar impact on cell killing.

MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation

Osteosarcoma is one of the most common tumors. The mechanisms of formation and development of osteosarcoma have been studied for a long time. Recently, more and more evidence showed that miRNAs play important roles in regulating tumor growth. In this study we found that miRNA-223 was downregulated in both osteosarcoma patients’ tumor tissues and osteosarcoma cell lines. Overexpression of miRNA-233 greatly inhibited the proliferation of Saos-2 cells. Cell cycle analysis by flow cytometry showed the arrest of cell cycle progression at the G1 phase. Further mechanistic study indicated that Ect2 was directly targeted by miR-223. Downregulation of Ect2 by miR-223 induces the expression of p21, p27 and the phospharylation of retinoblastoma, which are involved in the G1 block. We concluded that miR-223 functions as a tumor suppresser in osteosarcoma and miR-223/Ect2/p21 signaling is an important pathway that regulates the osteosarcoma cell cycle progression and proliferaion.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10–250nM for DPI, 0.5–2.5μM for DTI, and 155nM to 10μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

In vitro studies on radioprotective efficacy of silymarin against γ-irradiation

Purpose: Silymarin has been widely exploited for its hepatoprotective activities. This study aimed to evaluate the protective efficacy of silymarin against γ-radiation.Materials and methods: The radioprotective properties of silymarin were studied using different assays. Cytotoxicity of silymarin on Human embryonic kidney (HEK) cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by studying reduction in micronuclei frequency and free radical generation using 2′,7′-dichlorodihydroflurescin diacetate (H2DCFDA). Radiation-induced apoptosis was estimated by Annexin V-PI (propidium iodide) analysis and cell cycle analysis. γ-radiation induced changes in mitochondrial membrane potential (MMP) and DNA damage was estimated employing flow-cytometry and comet assay respectively.Results: MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from γ-irradiation. Significant reduction in apoptosis (76.36%) was observed. Silymarin also decreased the percentage of radiation-induced micronuclei (> 69%) (p < 0.05 ). Measurement of intracellular reactive oxygen species (ROS) by H2DCFDA revealed a reduction in ROS (21%) at 0.5 h. Cell cycle analysis revealed G1 block in the unirradiated control, which declined in the silymarin pretreated irradiated group (0.5 h). Silymarin treatment resulted in a significant increase in MMP (2 h) against the radiation control. Moreover, the presence of silymarin during irradiation significantly decreased the DNA damage (as measured by comet assay).Conclusions: Protection against radiation-induced cell-death and DNA damage by silymarin could be attributed to a reduction in ROS induced by γ-radiation. In vitro experiments on HEK cells explicitly prove that silymarin is a promising, effective and safe radiation countermeasure agent.

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice

To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice. The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting. Crotoxin (25, 50, and 100 μg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens. Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.

Givinostat and hydroxyurea synergize in vitro to induce apoptosis of cells from JAK2V617F myeloproliferative neoplasm patients

We investigated whether clinically achievable concentrations of the histone deacetylase (HDAC) inhibitors givinostat and hydroxyurea induce synergistic cytotoxicity in Jak2(V617F) cells invitro and through which possible mechanism. Givinostat and hydroxyurea at low doses potentiated the pro-apoptotic effects of each other in the Jak2(V617F) HEL and UKE1 cell lines. Givinostat induced 6.8%-20.8% and hydroxyurea (HU) 20.4%-42.4% cell death alone and 35.8%-75.3% in combination. The effect was statistically significant using the median effect Chou-Talalay method, resulting in a combination index less than 1, indicating synergy. Givinostat alone induced cell cycle arrest of the cell lines in G0/G1 and hydroxyurea in S phase, whereas both drugs together led to a G1 block. At the molecular level, hydroxyurea counteracted the induction of p21CDKN1A by Givinostat and potentiated caspase 3 activation, explaining at least in part the increased apoptosis observed in presence of both compounds. We also verified the effect of the same drugs in colony assays of freshly isolated Jak2(V617F) polycythemia vera cells. In this case, low doses of the compounds were additive to each other. These results suggest that combined treatment with givinostat and hydroxyurea is a potential strategy for the management of Jak2(V617F) myeloproliferative neoplasms.

Evidence for a cell cycle checkpoint that senses branched actin in the lamellipodium

Recent evidence indicates that branched actin might control cell progression through G1 in addition to lamellipodium protrusion.

Valproic acid (VPA), a class I and II histone deacetylase (HDAC) inhibitor and conjunctival melanoma

AbstractPurpose To investigate the expression of histone deacetylases (HDACs) in human conjunctival melanoma (CM) cell lines and primary melanocytes, and assess the effects of VPA, a broad Class I and II HDAC inhibitor, on cell viability and growth.Methods CM cell lines (CRMM1 &amp; 2; CM2005.1) and primary melanocytes were immunolabelled with antibodies to HDACs: Class I (HDAC1, 2 and 3), Class IIA and IIB (HDAC4 and HDAC6), and Class III (SIRT2). Antibody localisation was visualised with immunofluorescence and confocal microscopy. Dose‐response and proliferative potential following treatment with VPA was assessed for up to 72hrs using MTT and colony assays, respectively Cell cycle dynamics were also assessed.Results Differential HDAC expression was observed in CM cells and melanocytes, for both immunolocalisation (nuclear vs cytoplasmic) and cell type. CM2005.1 cells displayed lower level expression of HDACs compared to CRMM1 and 2 cells. VPA IC50 (72hrs) was 3.75mM, 5.42mM and 8.33mM for CRMM1, 2 and CM2005.1 cells, respectively. Colony assays showed similar patterns of response, with surviving fractions 0.2 and 0.5 for CRMM1 (VPA 0.3125mM) and CRMM2 (VPA 2.5mM) respectively. Cell cycle analysis showed dose‐related G1 block for CRMM1 and G2 block for CRMM2 cells at 24hrs (0mM to 1.25mM VPA).Conclusion Overall, CM2005.1 cells are more resistant to VPA compared to CRMM1 and 2 cells. VPA inhibition of CM cell growth and proliferation may be related to the observed differential expression of HDACs between cell lines. Combination therapies using VPA and other HDAC inhibitors may be potentially useful in managing the growth of primary CM. Supported by Sydney Foundation for Medical Research.

Cyclic X-Ray Responses in Mammalian Cellsin Vitro1

Various radiation responses in mammalian cells depend on the position of the cell within its generation cycle (that is, its age) at the time of irradiation. Studies have most often been made by irradiating synchronized populations of cells in vitro. Results in different cell lines are not easy to compare, but an attempt has been made here to point out similarities and differences with regard to cell killing and division delay. In general, survival data obtained so far show that, in cells with a short G(1), cells are most sensitive in mitosis and in G(2), less sensitive in G(1), and least sensitive during the latter part of the S period. In cells with a long G(1), in addition to the above, there is usually a resistant phase early in G(1) followed by a sensitive stage near its end. (The latter may be as sensitive as mitosis.) Exceptions to the above, especially in some L cell sublines, have been noted, and a possible explanation is given. In Chinese hamster cells, maximum survival after irradiation occurs during S, but it does not coincide with the time of the maximum rate of DNA synthesis or with the time of the maximum number of cells in DNA synthesis, and changes in survival also occur in cells inhibited from synthesizing DNA. Rather, survival depends on the position the cell has reached in the cycle at that time, which involves not only DNA synthesis but other processes as well. Survival is not completely correlated with DNA synthesis, since halting DNA synthesis just before or just after irradiation only slightly affects survival at its maximum. Division delay exhibits a pattern of response which is similar in most cell lines. Delay is considerable for cells irradiated in mitosis, is small for cells in G(1), increases to a maximum for cells during S, and declines for cells in G(2). L cells or human kidney cells may have a longer delay for cells irradiated in G(2) than for those irradiated in S. The results can be explained in terms of a two-component model of division delay. One component results from the prolongation of the S period due to the reduced rate of DNA synthesis, and the other, a block in G(2), is independent of DNA synthesis. The proportion of the two components may vary in different cell lines.

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-β-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3′UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

Selective Radiosensitization of Human Cervical Cancer Cells and Normal Cells by Artemisinin Through the Abrogation of Radiation-Induced G2 Block

Artemisinin has been shown to inhibit the growth of some human cancer cells. In this study, we investigated the radiosensitizing effects of artemisinin on cervical cancer cells and normal human fibroblast cells and also assessed some possible mechanisms for these effects. Two cervical cancer cell lines, HeLa and SiHa cells, and GM0639 normal human fibroblast cell line were treated with various concentrations of artemisinin plus radiation; the cell viability was tested using both 3-(4,5-dimethylthiazolyl-2-y1)-2, 5-diphenyltetrazolium bromide and clonogenic assays. Radiation dose-modifying factors were measured by clonogenic survival assay. Annexin V/propidium iodide assay for the evaluation of apoptosis and cell cycle phase were determined by flow cytometry, and the expression of the cell cycle-associated proteins Wee 1 and cyclin B1 were analyzed by Western blot analysis. Artemisinin showed higher cytotoxicity in cervical cancer cell lines, especially in SiHa cells, than in the normal cell line. In both clonogenic assay and apoptosis, artemisinin sensitized the HeLa cancer cells to the cytotoxicity of radiation, yielding a dose-modifying factor of 1.24, but not SiHa cancer cells and GM normal cells. At a dose of 110 nmol/L, artemisinin did not change the distribution of cell cycle in 3 tested cell lines, but artemisinin abrogated the radiation-induced G2 blockade. Analyses of G2-checkpoint-related proteins, the activation of Wee 1 and depression of cyclin B1 expression induced by radiation, could be restored to the control level by artemisinin. Given the unique cytotoxic profile of artemisinin on cancer cells and normal cells, artemisinin may be a potentially promising radiosensitizer through the regulation of the expression of G2 checkpoint-related proteins like Wee 1 and cyclin B1, and improve therapeutic ratios for the combination of artemisinin and ionizing irradiation in the treatment of patients with cervical cancer.

Anti‐leukemic effect of a synthetic compound, (±) trans‐dihydronarciclasine (HYU‐01) via cell‐cycle arrest and apoptosis in acute myeloid leukemia

(±) trans-Dihydronarciclasine, isolated from Chinese medicinal plant Zephyranthes candida, has been shown to possess quite potent anti-tumoral effect against selected human cancer cell lines. However, little is known about the anti-tumoral effect of (±) trans-dihydronarciclasine in acute myeloid leukemia (AML). This study was performed to investigate the effect of a novel synthetic (±) trans-dihydronarciclasine (code name; HYU-01) in AML. The HYU-01 inhibited the proliferation of various AML cell lines including HL-60 as well as primary leukemic blasts in a dose-dependent manner. To investigate the mechanism of the anti-proliferative effect of HYU-01, cell-cycle analysis was attempted in HL-60 cells, resulting in G1 arrest. The expression levels of CDK2, CDK4, CDK6, cyclin E, and cyclin A were decreased in a time-dependent manner. In addition, HYU-01 up-regulated the expression of the p27, and markedly enhanced the binding of p27 with CDK2, 4, and 6, ultimately resulting in the decrease of their kinase activities. Furthermore, HYU-01 induced the apoptosis through the induction of proapoptotic molecules and reduction of antiapoptotic molecules in association with the activation of caspase-3, -8, and -9. These results suggest that HYU-01 may inhibit the proliferation of HL-60 cells, via apoptosis, as well as G1 block in association with the induction of p27.

Suprainduction of p53 by disruption of 40S and 60S ribosome biogenesis leads to the activation of a novel G2/M checkpoint

Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.

Abstract 845: In vitro and in vivo pharmacology of SAR125844, a potent and selective intravenous MET kinase inhibitor undergoing Phase I clinical trial

Abstract SAR125844 is a potent MET kinase inhibitor for intravenous (IV) administration, with nanomolar activity against the wild-type enzyme (IC50 = 4.2 nM) and some kinase domain mutants, such as M1250T and Y1235D. It is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. In cellular assays, SAR125844 inhibits MET autophosphorylation at the nanomolar range (IC50 from 1.4 to 5.1 nM), translating into antiproliferative activity selectively in MET-driven cell lines such as MET-amplified cell lines, with IC50 values in the low nanomolar range and induction of apoptosis. SAR125844 induces a G1 block in the two MET-amplified tumor cell lines tested. Moreover, the compound is also able to inhibit HGF-induced cell migration in PC-3 prostate tumor cell line. In two MET-amplified human gastric tumor xenograft models tested, SNU-5 and Hs 746T, SAR125844 intravenous treatment leads to potent impact on the MET signaling pathway in a dose and time dependent manner, with potent inhibition of MET autophosphorylation, as well as a significant effect on downstream PI3K/AKT and RAS/MAPK pathways. As a single agent, this translates into a dose-dependent antitumor activity in these two xenograft models, with tumor stasis at the lowest active dose and tumor regression at the highly active doses. High loading single dose administration of SAR125844 using a nanosuspension formulation allowed a sustained P-MET inhibition in tumors up to 7 days. In all models, antitumor activity was achieved at well tolerated doses without significant effect on body weight. The observed correlation between P-MET inhibition in the tumor and antitumor activity in preclinical settings suggests that P-MET evaluation in tumor biopsies could be a direct pharmacodynamic biomarker of SAR125844 activity in patients. In order to investigate whether a non-invasive biomarker could also be used as pharmacodynamic read-out, we measured shed-MET (soluble extracellular domain of the MET receptor) in the plasma of tumor-bearing mice and showed that its level correlates with tumor burden in 2 MET-amplified xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 845. doi:1538-7445.AM2012-845

Mutation Burden in CDKN2C, CDK1 and E2F2 Is Associated with Differential Response to Targeting CDK4/CDK6 in Combination with Bortezomib in Mantle Cell Lymphoma,

Abstract 3738Dysregulation of the cell cycle is a hallmark of mantle cell lymphoma (MCL) in which cyclin D1 expression is constitutive due to the t (11:14) translocation and CDK4 levels are elevated. MCL remains incurable despite initial response to therapy. Our goal was to develop a mechanism- and genome-based therapy to both inhibit lymphoma cell proliferation and sensitize them for cytotoxic killing. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), the only known selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable, in combination with cytotoxic agents. We demonstrate, for the first time, that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs, and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant cytotoxic agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog cytarabine, in vitro and in a mouse model of MCL (Huang et al, submitted).In a completed phase I clinical study in MCL, PD potently and preferentially inhibited CDK4/CDK6 in lymphoma cells despite extensive chromosomal abnormalities, with an excellent toxicity profile and promising clinical response (Leonard et al, submitted). To advance targeting CDK4/CDK6 in MCL, we have now combined PD with escalating dose of bortezomib in an ongoing phase I clinical study (PD-B) in MCL. In this proof-of-concept study, PD is administered on days 1–12 of a 21-day cycle; bortezomib is administered first in prolonged G1 arrest concurrent with PD on days 8 and 11, and again after PD withdrawal in pG1-S on days 15 and 18. CD19+ MCL tumor cells were isolated at baseline, on day 8 and day 21 for analysis.To elucidate the mechanisms that underlie the progression of MCL and the differential response to this novel, cell-sensitizing therapy, we preformed 50×50 paired-end RNA-Sequencing on a HiSeq2000, using one lane for each sample of clinically responding and non-responding patients enrolled in this clinical trial. We generated an average of 76 million reads for each sample, then used the Burrow-Wheeler Aligner (BWA) to align the reads to the genome (Build 37), and SAMtools and the Genome Analysis Toolkit (GATK) to call non-reference variants. We focused on examining genes in the cell cycle and apoptotic pathways, and our data show 400 mutations in 16 genes including CDKN2C (p18), CDK1, E2F2, BBC3 (PUMA), BCL2L11(BIM), JUN and TP53, which are specific to each patient and whose expression changes dynamically during treatment. Moreover, we observe that the overall mutation burden is higher in a non-responding patient relative to the responding patient, and that certain genes (CDKN2C, CDK1, E2F2) show a highly significant (p=2.2×10–16) enrichment of mutations at baseline in the non-responder.By inhibiting CDK4/CDK6, p18 (CDKN2C) is essential for homeostatic cell cycle control of B cell activation and plasma cell differentiation in immunity. Conversely, mutations and deletions of CDKN2C are frequent in MCL, suggesting that loss of CDKN2C contributes to cell cycle dysregulation in this disease. Our RNA-Seq data reveal specific mutations in CDKN2C that are associated with compromised clinical response to PD, in line with cooperative inhibition of CDK4/CDK6 by p18 and PD in BCR-activated B cells as we reported previously. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that induction of prolonged G1 arrest by inhibition of CDK4/CDK6 with PD halts gene expression in early G1 and depletes the expression of those programmed for other phases of the cell cycle. This leads to a metabolic imbalance, which is not restored in pG1-S, thereby sensitizing MCL cells to cytotoxic killing. Mutations in E2F2, which promotes G1/S transition, and CDK1, which functions in G2/M, may therefore antagonize cell cycle sensitization to cytotoxic killing by CDK4/CDK6 inhibition. These data provide new mechanistic insight into therapeutic targeting of CDK4/CDK6 in MCL, and suggest novel molecular targets for personalizing and advancing cell cycle-based therapy in MCL. Disclosures:Martin:Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Leonard:Pfizer, Inc: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Bim Is Required to Sensitize Mantle Cell Lymphoma Cells for Killing by Bortezomib, but Not Ara-C, by Selective Inhibition of CDK4/CDK6

Abstract 1655Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing.To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1.pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim.Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures:Martin:Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Prolyl Hydroxylase PHD3 Enhances the Hypoxic Survival and G1 to S Transition of Carcinoma Cells

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.

MK-1775, a Novel Wee1 Kinase Inhibitor, Radiosensitizes p53-Defective Human Tumor Cells

Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G₂ block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G₂ block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.

Abstract 3791: MAPK p38 is the link between autophagy and lipid metabolism in colorectal cancer cells

Abstract Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Recent progress has demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, notably in cancer. We have showed previously, that the p38 pathway was involved in Irinotecan sensitivity of colon cancer cells p53WT. p38 being involved in the induction of autophagy we wanted to determine its role in the processes of autophagy in presence and in absence of p53 in colorectal cancer cells. Our results show that the overexpression of the constitutively active p38alpha isoform in HCT116 p53KO cells leads to a massive autophagy, accompanied with a block in G2 / M. The induction of autophagy in this context doesn't leads to the cellular death but rather to survival. The autophagy induction by p38 overexpression in HCT116 was weaker, indicating that the lack of p53 increase p38 role in autophagy induction. Furthermore, we have shown that autophagy induced by p38 comes along with an increase of the lipid metabolism by activating a key enzyme of the synthesis of fatty acids. We demonstrate here, that the role of p38 in the induction of autophagy depends on the presence of p53 and that p38 would be the link between the increase of the lipid metabolism and the induction of autophagy that lead to survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2011-3791