Selective MEK Inhibitor Research Articles

Anti-proliferative effect in epithelial ovarian cancer cells by MEK Inhibitor AZD6244

목적: 상피성 난소암 세포에서 세포증식과 생존에 관여하는 ERK 경로의 기초 활성도를 조사하고, MEK 억제제인 AZD6244가 난소암 세포의 증식과 세포주기, 염증성 싸이토카인의 생성에 미치는 영향과 AZD6244를 파클리탁셀과 병합치료를 하였을 때 세포독성 효과의 증강이 있는 지를 알아보고자 하였다. 연구 방법: 난소암 환자의 복수에서 분리한 일차세포와 확립되어진 난소암 세포주를 각각 AZD6244 (0.1-10 μM)를 24, 48, 72시간동안 치료하였다. Cell viability는 CellTiter 96 AQueous One Solution Cell Proliferation Assay를 통해서, caspase 활성도는 Caspase-Glo assay를 이용하였다. 싸이토카인의 분석은 세포배양 후 배양액의 상층액을 채취하여 Luminex 200 system을 이용하여 측정하였고, flow cytometry를 이용해 세포주기를 분석하였으며, p-ERK와 cyclin D1은 western blot 분석을 하였다. 결과: AZD6244는 기초 p-ERK 활성도가 높은 세포나 낮은 세포 모두에서 p-ERK를 억제하였다. AZD6244는 p-ERK의 고발현 세포 (A2780, 01-28, R454)에서 치료농도와 치료기간에 비례하여 효과적으로 증식억제를 보였다. 하지만 p-ERK의 저발현 세포 (CP70, R182)에서는 큰 차이를 보이지 않았다. AZD6244에 민감하게 세포증식의 억제반응을 보인 A2780 세포에서도 caspase-3/7 활성도는 유의한 증가가 관찰되지 않았다. AZD6244는 IL-6, IL-8, MCP-1, GM-CSF를 감소시켰다. 파클리탁셀과 AZD6244의 병합요법은 파클리탁셀에 의한 ERK 경로의 활성화를 AZD6244에 의해 효과적으로 상쇄시키며, 세포독성 효과의 유의한 증강이 나타났다. 결론: AZD6244는 p-ERK의 고발현 세포에서 효과적으로 세포증식 억제효과를 보이며, 파클리탁셀과 AZD6244의 병합요법은 난소암 세포에서 유의한 세포고사를 유도하며 AZD6244에 의한 ERK 경로의 억제가 파클리탁셀의 세포독성 효과를 증강시키는 데에 관여함을 보여준다.

Magnolol Inhibits LPS-induced NF-κB/Rel Activation by Blocking p38 Kinase in Murine Macrophages.

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-κB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-κB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-κB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-κB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-κB/Rel and p38 kinase signaling.

The Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Single Oral Doses of CH4987655 in Healthy Volunteers: Target Suppression Using a Biomarker

CH4987655 (RO4987655) is an orally active and highly selective small-molecule MEK inhibitor. It potently inhibits mitogen-activated protein kinase signaling pathway activation and tumor cell growth, with an in vitro IC(50) of 5.2 nmol/L for inhibition of MEK1/2. Single-agent oral administration of CH4987655 resulted in complete tumor regressions in xenograft models. All 40 subjects received a single oral dose followed by 72 hrs of pharmacokinetic, pharmacodynamic, and safety/tolerability assessments. The pharmacodynamics were measured by changes in phosphorylated extracellular signal-regulated kinase (pERK) levels in a surrogate tissue peripheral blood mononuclear cells ex vivo stimulated by PMA. Doses of 0.5, 1, 2, 3, and 4 mg were safe and well tolerated. No clinically significant safety event was observed. A total of 26 adverse events (n = 15) were reported: 21 mild, 5 moderate, and none severe. Moderate adverse events were experienced by one subject at 1 mg (autonomic nervous system imbalance) and three subjects at 4 mg (diarrhea, abdominal pain, autonomic nervous system and acne). CH4987655 was rapidly absorbed with a t(max) of approximately 1 h. Exposures were dose proportional from 0.5 to 4 mg. The disposition was biphasic with a terminal t(1/2) of approximately 25 hr. Intersubject variability was low, 9% to 23% for C(max) and 14% to 25% for area-under-the-curve (AUC). pERK inhibition was exposure dependent and was greater than 80% inhibition at higher doses. The pharmacokinetic-pharmacodynamic relationship was characterized by an inhibitory E(max) model (E(max) approximately 100%; IC(50) 40.6 ng/mL) using nonlinear mixed-effect modeling. A significant extent of pERK inhibition was achieved for a single dose that was considered to be safe and well tolerated in healthy volunteers.

Treatment with a MEK Inhibitor Improves Myeloproliferation, Anemia and Survival in a Mouse Model of CMML and JMML.

Abstract 966Mutations that deregulate cellular signaling are a hallmark of myeloproliferative neoplasms (MPNs), and pharmacologic inhibitors of MPN-associated proteins have redefined therapy for some MPNs. However, this strategy cannot yet be applied to juvenile- and chronic myelomonocytic leukemias (JMML and CMML). These diseases are characterized by aberrant N-Ras, K-Ras, Cbl, and SHP-2 proteins that are not easily targeted by drugs. An attractive alternative approach is to inhibit downstream effector pathways, which include the Raf/MEK/ERK, phosphoinositide-3-OH kinase (PI3K)/Akt, and Ral-GDS/Ral-A cascades. However, it is not known which of these pathways are crucial for the aberrant growth and survival of JMML and CMML cells and might therefore provide the best targets for therapy. To address these questions, we developed an accurate mouse model of JMML and CMML by expressing a conditional “knock-in” KrasLSL-G12D oncogene in bone marrow. We administered PD0325901, a potent and selective MEK inhibitor, to Mx1-Cre, KrasG12D mutant mice to test the hypothesis that the Raf→MEK→ERK cascade is necessary for MPN initiated by KrasG12D expression. Oral administration of PD0325901 5 mg/kg caused deep and durable MEK inhibition in primary bone marrow progenitors. Mx1-Cre, KrasG12D mice with established MPN and wild-type (WT) littermates were randomly assigned to receive PD0325901 5 mg/kg/day or a control vehicle. Treated Mx1-Cre, KrasG12D mice demonstrated rapid correction of leukocytosis and anemia, and reduction in splenomegaly. Treatment was also associated with dramatic improvement in the survival of Mx1-Cre, KrasG12D mice (8.1 vs. 2.0 weeks after entry, p=0.003). Two of three Mx1-Cre, KrasG12D mice that were treated for 12 weeks ultimately died with KrasG12D T-lineage leukemia/lymphoma, but none succumbed with progressive MPN. Flow cytometry of bone marrow and peripheral populations showed that PD0325901 reversed the granulocyte/monocyte progenitor bias and ineffective erythropoiesis in KrasG12D mice. However, PD0325901 did not eliminate the rearranged mutant Kras allele in myeloid progenitors, and these cells remained hypersensitive to GM-CSF in methylcellulose cultures. Therefore, PD0325901 did not eliminate Kras mutant cells, but rather modified their behavior in vivo so as to restore a normal output of the hematopoietic system. To further address the biologic effects of PD0325901 on growth of primary progenitor cells in vitro, we examined colony growth over a range of GM-CSF concentrations. Importantly, whereas in vitro exposure to PD0325901 did not selectively abrogate colony growth from bone marrow of naïve Mx1-Cre, KrasG12D mice in the presence of saturating doses of GM-CSF, a low concentration of PD0325901 eliminated the growth of cytokine-independent progenitor colonies. Even more strikingly, this also restored a normal GM-CSF dose response curve in clonogenic progenitors, eliminating the hypersensitive growth pattern that is a hallmark of MPN. Finally, even at saturating doses of GM-CSF, a low concentration of PD0325901 was sufficient to normalize the numbers and types of cells within the colonies. Together, these data show that a low concentration of PD0325901 is sufficient to impart a normal program of proliferation and differentiation in KrasG12D myeloid progenitors. These findings are highly consistent with the in vivo data. Collectively, our data suggest that aberrant MEK activation mediates most aspects of the MPN phenotype in the progenitor compartment and support the development of clinical trials to evaluate MEK inhibitors in patients with JMML and CMML. Disclosures:No relevant conflicts of interest to declare.

Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Abstract 2757Poster Board II-733Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML. Disclosures:No relevant conflicts of interest to declare.

Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of (V600E)BRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of (V600E)BRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by (V600E)BRAF/MEK signalling in melanoma cells.

The Mitogen-activated Protein Kinase (MAPK)/Extracellular Signal-regulated Kinase (ERK) 1/2 Kinase Inhibitor, AZD6244 (ARRY-142886) Enhances Radiosensitivity by Interfering with Radiation-induced TGF-alpha Signaling

The MAPK/ERK pathway is important for cell proliferation, survival, and differentiation and is activated as a prosurvival pathway after irradiation (IR). AZD6244 is a potent, selective inhibitor of MEK 1/2 kinases. We previously reported that exposure to AZD6244 prior to IR enhanced radiosensitivity of cancer cell lines and xenografts, however the degree of sensitization varied among cell lines. We studied the role of soluble TGF-alpha in the degree of sensitization to IR with AZD6244. U251 (glioma) and A549 (non-small cell lung cancer) cell lines were used for all studies. Levels of soluble TGF-alpha in media from cells treated with AZD6244 +/- IR or vehicle +/- IR were assayed by ELISA. The importance of TGF-alpha on the radiation response was evaluated by treating cells with a TGF-alpha neutralizing antibody in clonogenic assays. Additional clonogenic assays were performed to determine the effects of AZD6244, recombinant human TGF-alpha, or the combination of AZD6244 and TGF-alpha on the in vitro radiosensitivity of the U251 and A549 cell lines. Phosphorylated Erk1/2 was evaluated after different treatments by immunoblotting. Mitotic catastrophe was measured using nuclear fragmentation. Basal and IR-induced TGF-alpha secretion was undetectable in U251 cells (not-sensitized with AZD6244). In A549 cells (sensitized), secretion of soluble TGF- alpha increased more than 2 fold at 24 h after IR compared to non-irradiated cells. Pretreatment of cell lines with AZD6244 reduced TGF-alpha production. The use of a neutralizing antibody to TGF-alpha reduced survival in A549 cells after IR. Conversely, the addition of TGF-alpha to AZD6244 treated A549 cells decreased the radiosensitizing effects of AZD6244. The addition of TGF-alpha to A549 cells treated with AZD6244 and IR resulted in similar levels of ERK phosphorylation and mitotic catastrophe as seen in cells treated with IR alone, in contrast to the elevated levels seen in cells treated with AZD6244 and IR. These results indicate that TGF-alpha is a survival factor in A549 cells after IR. AZD6244 enhanced radiosensitivity in vitro through an inhibition of TGF-alpha secretion leading to an increase in mitotic catastrophe.

Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction of long-term potentiation in rats

To explore the role of the extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway in the induction of long-term potentiation (LTP) in the anterior cingulate cortex (ACC) that may be implicated in pain-related negative emotion. LTP of field potential was recorded in ACC slice and the expressions of phospho-ERK (pERK) and phospho-CREB (pCREB) were examined using immunohistochemistry method. LTP could be induced stably in ACC slice by high frequency stimulation (2-train, 100 Hz, 1 s), while APv (an antagonist of NMDA receptor) could block the induction of LTP in the ACC, indicating that LTP in this experiment was NMDA receptor-dependent. Bath application of PD98059 (50 micromol/L), a selective MEK inhibitor, at 30 min before tetanic stimulation could completely block the induction of LTP. Moreover, the protein level of pERK in the ACC was transiently increased after LTP induction, starting at 5 min and returning to basal at 1 h after tetanic stimulation. The protein level of pCREB was also increased after LTP induction. The up-regulation in pERK and pCREB expressions could be blocked by pretreatment of PD98059. Double immunostaining showed that after LTP induction, most pERK was co-localized with pCREB. NMDA receptor and ERK-CREB pathway are necessary for the induction of LTP in rat ACC and may play important roles in pain emotion.

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser 473 induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr 389 in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

Involvement of PI3 kinase and MAP kinase in IGF-I- and insulin-induced oocyte maturation in Cyprinus carpio

Previously, we observed that in vitro germinal vesicle breakdown (GVBD) in Cyprinus carpio oocytes was induced by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) and this induction was steroid-independent. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during oocyte maturation was examined. IGF-I- and b-insulin-induced oocyte maturation was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase after 90 min of their treatment. Both IGF-I and b-insulin were found to activate cdc2 kinase at 21 h of treatment. We examined the relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in IGF-I- and b-insulin-induced oocyte maturation in C. carpio. MAP kinase was rapidly phosphorylated and activated (30–150 min) in response to exposure of the oocytes with IGF-I and b-insulin. This response preceded the phosphorylation and activation of cdc2 by several hours (almost 19 h). A potent and selective inhibitor of MEK, PD98059, the protein kinase that phosphorylates and activate MAP kinase, blocked the phosphorylation and activation of MAP kinase and cdc2 kinase and GVBD induction. Likewise, PI3 kinase inhibitors strongly inhibited phosphorylation and activation of MAP kinase, which was increased during oocyte maturation. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes MAP kinase, and MPF activation during IGF-I- and b-insulin-induced oocyte maturation in C. carpio.

A three-part, phase I, dose-escalation study of GSK1120212, a potent MEK inhibitor, administered orally to subjects with solid tumors or lymphoma

e14584 Background: GSK1120212 is a potent and highly selective inhibitor of MEK1, a component of the MAP kinase pathway. GSK1120212 demonstrates efficient inhibition of p-ERK which correlates with inhibition of cell proliferation and induction of apoptosis. PO administration of GSK1120212 achieved tumor regression in multiple mouse xenograft models. The objectives of this study are to define the maximum tolerated dose (MTD) and to evaluate the pharmacokinetics (PK) and pharmacodynamic (PD) effects of GSK1120212. Methods: In Part 1, patients (pts) with solid tumors or lymphoma are enrolled in successive cohorts and receive a single PO dose of GSK1120212 followed by QD doses on days 1 - 21 of each 28-day cycle. Tumor response is assessed Q 8 weeks. PK blood samples are collected from all pts. Ophthalmic exams are administered at baseline and as clinically warranted. Dose escalation occurs via an accelerated titration followed by a standard 3+3 escalation. In Part 2, pts with pancreatic or K-Ras mutant CRC will be enrolled at the MTD. In Part 3, pts with biopsiable tumors will enroll at MTD and sub-MTD doses. Tumor biopsies will be taken pre- and post-dose to measure pERK and other markers of cell proliferation. Results: Six pts with advanced malignancies (neuroendocrine, thyroid, colorectal (n=2), melanoma (n=2) have been treated with tablets at four dose levels: 0.125 (n=2), 0.25 (n=1), 0.5 (n=2), and 1.0 mg (n=1). No DLTs or grade 2 toxicities have been reported. One patient was on study for over 17 weeks. Based on mean AUC and Cmax, GSK1120212 exposures following 15 days of repeat-dose administration were approximately dose proportional across the dose range (0.125–0.5 mg) with a median Tmax of approximately 1.5 hours. Conclusions: GSK1120212 has been well tolerated to date. Dose escalation is ongoing. [Table: see text]

TGFβ inhibits GM‐CSF‐induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3‐kinase (PI3‐k)

Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFbeta has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-beta activates the ERK pathway and how TGFbeta communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells. TGFbeta dramatically suppressed proliferation of MV4-11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFbeta. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-alpha subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFbeta and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2. These studies thus indicate that TGFbeta does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human leukaemia cells.

Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Simultaneous Inhibition of the Raf/MEK/ERK and BCL-2 Pathways

The Raf/MEK/ERK pathway and the Bcl-2 family proteins are commonly overexpressed in hematologic malignancies, where they promote proliferation and survival of the neoplastic cells. We have previously demonstrated that selective MEK inhibitors (MEK-I) exert potent growth-inhibitory effects in preclinical models of both acute myeloid leukemia (AML) and multiple myeloma (MM) (Blood 2006, 108:254). More recently, we have obtained evidence that ABT-737, a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor (kindly provided by Abbott Laboratories), shows potent in vitro growth-inhibitory and pro-apoptotic activity on MM cell lines and primary CD138+ bone marrow cells from MM patients, regardless of the disease status (Libotte F, ASH 2008). Moreover, in AML models, we and others have reported a highly synergistic pro-apoptotic interaction between inhibitors of the Raf/MEK/ERK pathway and of the Bcl-2 family (Blood 2002, Cancer Cell 2006, Ricciardi ASH 2008). Here, we analyzed the impact of the simultaneous inhibition of these two pathways on cell proliferation and apoptosis in MM cell lines. To this purpose, we exposed different MM cell lines to increasing concentrations of MEK-I and ABT-737, alone and in combination. While single compounds dose-dependently inhibited cell growth, as assessed by trypan blue exclusion counting, we observed that their combination synergistically enhanced this effect with combination indexes (CI), as measured by isobologram analysis (Chou–Talalay method), of 0.28 and 0.12 for KMS18 and KMS27 cells, respectively. We next analyzed the effects of combined MEK and Bcl-2/Bcl-xL inhibition on apoptosis induction. Both MEK-I and ABT-737 induced apoptosis in MM cells at high concentrations, as determined by sub-G1 DNA peak and Annexin V staining. When used at concentrations that induced minimal apoptosis as single agents (7.55% and 6.8% net apoptosis induction in KMS18 cells after 72 hours of exposure to MEK-I and ABT-737, respectively), the combination of MEK-I and ABT-737 was able to induce substantial apoptosis (more than 50% net apoptosis induction after 72 hours in both KMS18 and KMS27 cell lines). Such pro-apoptotic interaction was highly synergistic in nature, with CI, as defined using isobologram analysis, of 0.2 and 0.17 for KMS18 and KMS27 cells, respectively. Mitochondrial membrane depolarization was similarly enhanced by the combination of MEK-I and ABT-737. Conversely, in the MEK-I resistant MM cell line ARH-77, ABT-737 was still able to induce apoptosis (up to 40% of the cells) but its effect was not significantly potentiated by MEK inhibition. Preliminary results on primary CD138+ MM cells exposed to both inhibitors confirmed the higher cell growth inhibition induced by combining MEK-I and Bcl-2/Bcl-xL inhibition. In conclusion, we demonstrated a striking synergistic pro-apoptotic activity with combined inhibition of Raf/MEK/ERK and Bcl-2 signaling in MM cell lines. Simultaneous disruption of these two pathways warrants further investigation as novel therapeutic strategy for this disease.

Upregulation of GADD153 by Butyrate: Involvement of MAPK

Butyrate inhibits the proliferation of cancer cells, but the early molecular events initiated by butyrate have not been fully identified. Herein, butyrate is shown to affect the growth arrest and DNA damage-inducible gene 153 (GADD153) in HCT-116 human colon adenocarcinoma cells. Despite absence of any detectable cellular DNA damage, the expression of GADD153 was upregulated before several features characteristic of apoptosis appeared. Butyrate-induced upregulation of GADD153 mRNA was attenuated by actinomycin D, but apparently not by cycloheximide. In investigating possible involvement of MAPK in mediating the effect of butyrate on GADD153 mRNA expression, the extracellular regulated kinase (ERK) inhibitor PD98059, but neither the JNK inhibitor SP600125 nor the p38 MAPK inhibitor SB203580, blunted the ability of butyrate to upregulate GADD153 mRNA expression. U0126, a selective inhibitor of upstream MEK, had a similar effect as PD98059 on butyrate-induced GADD153 mRNA upregulation. Collectively, these findings suggest that butyrate caused activation of the GADD153 gene at the level of transcription involving mainly the MEK/ERK branch of the MAPK signal transduction pathway. Moreover, these molecular events were not the result of any DNA damage and occurred before several features characteristic of apoptosis became evident.

Tumour cell survival signalling by the ERK1/2 pathway

Several advances in recent years have focused increasing attention on the role of the RAF-MEK-ERK1/2 pathway in promoting cell survival. The demonstration that BRAF is a human oncogene mutated at high frequency in melanoma, thyroid and colon cancer has provided a pathophysiological context, whilst the description of potent and highly selective inhibitors of BRAF or MEK has allowed a more informed and rational intervention in both normal and tumour cells. In addition, separate studies have uncovered new mechanisms by which the ERK1/2 pathway can control the activity or abundance of members of the BCL-2 protein family to promote cell survival. It is now apparent that various oncogenes co-opt ERK1/2 signalling to de-regulate these BCL-2 proteins and this contributes to, and even underpins, survival signalling in some tumours. New oncogene-targeted therapies allow direct or indirect inhibition of ERK1/2 signalling and can cause quite striking tumour cell death. In other cases, inhibition of the ERK1/2 pathway may be more effective in combination with other conventional and novel therapeutics. Here, we review recent advances in our understanding of how the ERK1/2 pathway regulates BCL-2 proteins to promote survival, how this is de-regulated in tumour cells and the opportunities this might afford with the use of new targeted therapies.

Persistent ERK activation maintains learning-induced long-lasting modulation of synaptic connectivity

Pyramidal neurons in the piriform cortex from olfactory-discrimination (OD) trained rats undergo synaptic modifications that last for days after learning. A particularly intriguing modification is reduced paired-pulse facilitation (PPF) in the synapses interconnecting these cells; a phenomenon thought to reflect enhanced synaptic release. The molecular machinery underlying this prolonged physiological modulation of synaptic connectivity is yet to be described. We have recently shown that extracellular regulated kinase (ERK) pathway and protein kinase C (PKC) are also required for learning-induced enhancement of intrinsic neuronal excitability. Here we examine whether these signal-transduction cascades are instrumental for the learning-induced, long-lasting PPF reduction. Days after learning completion, PD98059, a selective inhibitor of MEK, the upstream kinase of ERK, increased PPF in neurons from trained, but not in neurons from naïve and pseudo-trained rats. Consequently, the differences in PPF between neurons from trained rats and controls were abolished. The level of activated ERK in synaptoneurosomes was significantly higher in piriform cortex samples prepared from trained rats. Notably, ERK activation revealed that PPF reduction lags behind ERK activation by 2 d. Similarly, the PKC blocker, GF-109203X, enhanced PPF in neurons from trained rats only, thus abolishing the differences between groups. Interestingly, the PKC activator, OAG, had no effect, indicating that PKC activation is required, but not sufficient for long-lasting PPF reduction. Our data show that persistent ERK activation has a key role in maintaining learning-induced PPF reduction for days. This time frame of compartmental ERK-dependent synaptic modulation suggests a novel role for ERK in cortical function.

Rat Parathyroid Hormone 1 − 34 Signals through the MEK/ERK Pathway to Induce Cardiac Hypertrophy

This study aimed to characterize the role of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in cardiac hypertrophy induced by parathyroid hormone (PTH). Various concentrations of rat PTH1-34 were used to induce hypertrophy in neonatal rat ventricular cardiomyocytes, and the effects were compared with control cells and those treated with PD98059, a selective inhibitor of MEK1. Hypertrophy was assessed in terms of cell diameter, atrial natriuretic peptide (ANP) mRNA expression and protein synthesis; the MEK/ERK pathway was assessed by measuring levels of phosphorylated ERK1/2. Treatment with PTH1-34 at 100 nM for 24 h effectively induced cardiac hypertrophy (increased cell diameter, protein synthesis and ANP mRNA expression) and also increased levels of phosphorylated ERK1/2 compared with normal control cells. Treatment with PTH1-34 plus PD98059 significantly attenuated these changes. These results demonstrate that inhibition of the MEK/ERK pathway blocks PTH1-34-induced cardiac hypertrophy, suggesting that PTH1-34 might signal through the MAPK pathway to induce hypertrophy in cardiomyocytes.

577 POSTER Selective MEK Inhibitor RDEA119 exhibits efficacy in orthotopic hepatoma models and cytostatic potential in multiple cell based models of cancer

The present study was conducted to demonstrate of the immunohistochemical localization of vascular endothelial growth factor (VEGF) and its receptors (flt1/fms, flk1/KDR and flt4) as well as vascular endothelial growth inhibitor (VEGI) and to determine the correlation of VEGF and its receptors and VEGI with serum sex steroids (estrogen and progesterone) in the bovine uterus during the sexual cycle. The stage of the estrous cycle in 30 Holstein cattle was assessed based on the gross and histological appearance of the ovaries and uterus and on blood steroid hormone levels. Tissue samples obtained from the uterus were fixed in 10% formaldehyde for routine histological processing. During both follicular and luteal phases, positive cytoplasmic and membrane staining was achieved for VEGF and its receptors (flt1/fms, flk1/KDR and flt4) as well as VEGI in the luminal and glandular epithelial cells, the connective tissue and smooth muscle cells, and the vascular endothelial cells and smooth muscle cells in the uterus. The intensity, proportional and total scores determined for VEGF and its receptors (flt1/fms and flt4) as well as VEGI were greater in the luminal and glandular epithelial cells compared to the connective tissue and smooth muscle cells (P < 0.05). Furthermore, the number and intensity of the flk1/KDR positive cells were greater among the connective tissue cells compared to the luminal and glandular epithelial cells (P < 0.05). As a result, it was determined that the expression of VEGF and its receptors as well as VEGI in the bovine uterus during the follicular and luteal phases varied with different cell types. This suggests that depending on the stage of the sexual cycle, these factors may mediate the establishment of an appropriate environment for the nutritional supply and implantation of the embryo primarily due to the stimulation of angiogenesis but also through the increase in the secretory activity of the epithelial cells in the uterus. Furthermore, this indicates that ovarian steroid hormones play a significant role in regulating the expression of VEGF and its receptors as well as VEGI.

Complementary roles of tumor necrosis factor alpha and interferon gamma in inducible microglial nitric oxide generation

Proinflammatory cytokines and pathogen components activate microglia to release several substances such as nitric oxide (NO) produced after the induction of type II nitric oxide synthase (iNOS). The present study was designed to elucidate the interaction between the proinflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) on iNOS expression and NO production in microglial cells. In primary mouse microglial cells exposure to IFN-γ (5 and 10 ng/ml; 48 h) or TNF-α (20 ng/ml; 48 h) alone were unable to induce iNOS expression; however, when cells were exposed to both cytokines together, the expression of this enzyme and the NO production in culture media were found significantly increased. In the BV-2 microglial cell line, IFN-γ and TNF-α were shown to cooperate in nuclear factor κ B (NF-κB) activation, an essential transcription factor for iNOS gene transcription. Importantly, IFN-γ induced NF-κB binding to DNA was totally dependent on the endogenous TNF-α released via MEK/ERK signalling pathway. Thus, exposure of BV-2 cells to IFN-γ in the presence of the selective MEK inhibitor U0126 or a neutralizing anti-TNF-α antibody significantly reduced IFN-γ dependent NF-κB activation and iNOs expression. In addition, by activating the Jak/STAT pathway IFN-γ potentiated TNF-α induced NF-κB binding to DNA and activated additional transcription factors (i.e. IRF-1) known to be essential for iNOs gene expression. The present findings demonstrate that the proinflammatory cytokines IFN-γ and TNF-α have complementary roles on iNOS expression in microglial cells and this might be relevant to understand the molecular mechanisms of microglial activation associated with the pathogenesis of several neuroinflammatory disorders in which increased levels of IFN-γ and TNF-α have been reported.