Ras Kinase Research Articles

Abstract 4258: Targeting MGAT5B-mediated glycosylation of kinase receptors for the treatment of malignant peripheral nerve sheath tumors and RAS-related malignancies.

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that occur either sporadically or in patients with Neurofibromatosis type I. Surgery is the main stay of treatment, however, due to the invasive growth, propensity to metastasis, limited sensitivity to chemotherapy and radiation, 5-year survival rates is only 20%-50%. Recent work has identified several altered intracellular signal transduction cascades and deregulated kinase receptors, posing the possibility of personalized, targeted therapeutics. Kinase receptors depend on the glycosylation for stabilization, maturation, transportation onto the cell surface, phosphorylation and activation, and aberrant glycosylation to stabilize the expression of kinase receptors on the cell surface is associated with cancers. The appropriate and accurate modification of glycan to proteins mainly depends on the action of highly specific and precisely located enzymes known as glycosyltransferases and glycosidases in different tissues and cells. In this report, glycoproteins were highly expressed in MPNST cell lines compared with normal Schwann cells (NSC). Human Glycosylation PCR Array was used to profile the expression of 84 key genes encoding enzymes to regulate the glycosylation of proteins. Compared with NSC, we did not find any change in the mRNA expression levels of glycosidases, however, multiple glycosyltransferases (B4GALT5, FUT8, MGAT3, MGAT4A, MGAT5, MGAT5B, GALNT13, GALNT14, POMT1, ST8SIA2) were overexpressed in some MPNST cell lines, especially MGAT5B, one of glycosyltransferases to promote N-linked or O-linked glycosylation of proteins was highly expressed in MPNST cell lines and clinical specimens, and colon and pancreatic cancer cell lines with K-ras mutations. Exogenous expressing K-ras (G12V) significantly upregulated the expression of MGAT5B. The expression levels of MGAT5B were correlated with RAS kinase activity. We also found AKT interacted with MGAT5B and phosphorylation of S192 of MGAT5B promoted transportation from the cytoplasm into the Golgi apparatus and mutated MGAT5BS192A expression was distributed in the ER and cytoplasm. Experimental tumor lung metastasis was significantly inhibited in MPNST cells expressing MGAT5BS192A. Furthermore, we found that transient or stable knockdown of MGAT5B significantly attenuated the glycosylation and phosphorylation of kinase receptors to block their signaling, arrested cells in G1 phase and altered the morphology of MPNST cell lines. Taken together, these data suggest that RAS-driving the expression of MGAT5B plays a critical role in promoting glycosylation of kinase receptors to mediate their intracellular signaling and targeting MGAT5B-mediated glycosylation of kinase receptors may open a new therapeutic window for MPNSTs and RAS-related malignancies Citation Format: Quansheng Zhu, Yechun Song, Hui Shang, Thresa Nguyen, Svetlana Bolshakov, Xiaoyan Ma, Alexander Lazar, Raphael Pollock, Dina Chelouche Lev. Targeting MGAT5B-mediated glycosylation of kinase receptors for the treatment of malignant peripheral nerve sheath tumors and RAS-related malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4258. doi:10.1158/1538-7445.AM2013-4258

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Abstract 3571 Purpose:Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children.Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design:A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results:Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions:In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures:No relevant conflicts of interest to declare.

Glucocorticoids suppress selected components of the senescence‐associated secretory phenotype

Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors, and proteases that can alter the tissue microenvironment and may promote age-related pathology. To identify small molecules that suppress the senescence-associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are approved for human use. Among the promising library constituents was the glucocorticoid corticosterone. Both corticosterone and the related glucocorticoid cortisol decreased the production and secretion of selected SASP components, including several pro-inflammatory cytokines. Importantly, the glucocorticoids suppressed the SASP without reverting the tumor suppressive growth arrest and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Suppression of the prototypical SASP component IL-6 required the glucocorticoid receptor, which, in the presence of ligand, inhibited IL-1α signaling and NF-κB transactivation activity. Accordingly, co-treatments combining glucocorticoids with the glucocorticoid antagonist RU-486 or recombinant IL-1α efficiently reestablished NF-κB transcriptional activity and IL-6 secretion. Our findings demonstrate feasibility of screening for compounds that inhibit the effects of senescent cells. They further show that glucocorticoids inhibit selected components of the SASP and suggest that corticosterone and cortisol, two FDA-approved drugs, might exert their effects in part by suppressing senescence-associated inflammation.

Abstract 2826: Preclinical development of a Pim kinase inhibitor for cancer treatment

Abstract Pim kinases were identified as the crucial downstream effectors various important oncogenes like Jak, FLT3 or Ras kinases. Multiple targets phosphorylated by Pim kinases, such as 4EBP1, p21Waf, p27KIP1, c-Myc or CXCR4 play important roles in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Moreover, the significance and relevance of Pim kinases as valid therapeutic targets is further confirmed by their expression levels in variety of cancer types, especially in various types of lymphomas, leukemias or myelomas. For example Pim kinase overexpression contributes to the development of diffuse large B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia and FLT3-mediated acute myelogenous leukemia. As members of the family, Pim-1, Pim-2 and Pim-3 were shown to be prevent apoptosis, promote protein translation and cell survival thereby enhancing proliferation of malignant cells, Pim kinases emerged as a novel and interesting target with significant potential for therapeutic intervention in cancer. In the current study we are reporting the results of a second generation small molecule Pim kinase inhibitors that were developed by Selvita and results for the best characterized potent Pim kinase inhibitor developed so far. The small molecule inhibitors exert high potency in vitro both on all three Pim kinases as well as on a large panel of cancer cell lines. Mechanism of action for selected compounds was confirmed both in vitro in variety of cell lines and in vivo in xenograft models by downregulation of c-Myc and 4EBP1 phosphorylation inhibition. Selected compounds were tested in a range of combinations with standard anti-cancer therapeutics in both leukemias and solid tumor cell lines and showed potent synergistic effects. Strong synergies were observed particularly in combination with targeted therapies like the PI3K/Akt pathway inhibitors or Jak kinase inhibitors. Data will be presented on in vivo efficacy of lead compounds examined in xenograft models. Additionally, the results of safety assessment of the lead compound will be presented revealing preferable safety profile with no effect on ion channel mediated cardiotoxicity and favorable pharmacokinetic profile. Taken together, presented data will further support the rationale of using Pim kinase inhibition as a novel approach to standalone cancer therapy, and in combination with other targeted and cytotoxic therapies, especially to overcome developing drug resistance. 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 2826. doi:1538-7445.AM2012-2826

Abstract A243: Novel Pim kinase inhibitors for treatment of hematological malignancies and solid tumors.

Abstract The family of Pim kinases emerged as a novel and interesting target with significant potential for therapeutic intervention in cancer. All three members of the family Pim-1, Pim-2 and Pim-3 were shown to be prevent apoptosis, promote protein translation and cell survival thereby enhancing proliferation of malignant cells. Pim kinases seem to be crucial downstream effectors various important oncogenes like Jak, Flt3 or Ras kinases. Multiple targets phosphorylated by Pim kinases play important roles in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Moreover, the significance and relevance of Pim kinases as valid therapeutic targets is further confirmed by their expression levels in variety of cancer types of both hematological origin. For example Pim kinase overexpression contributes to the development of diffuse B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia and Flt3-mediated acute myelogenous leukemia. Interestingly, accumulating literature data point towards emerging role of Pim kinases also in development of malignancies that originate from epithelial cells, especially gastric, colon and hepatocellular cancers, but also prostate and pancreatic cancers. In this study we are reporting the results for novel small molecule Pim kinase inhibitors that were developed by Selvita. in Currently, we are performing a lead optimization program of novel, small molecule pan-Pim kinase inhibitors. Compounds developed within the SEL24 series exert high potency in vitro both on all three Pim kinases as well as on a large panel of cancer cell lines. Among synthesized compounds we have identified most selective Pim kinase inhibitors described so far with selectivity scores on a panel of 290 kinases inhibited over 90% at 1 μM concentration was equal to 1.8%. Mechanism of action for selected compounds was confirmed both in vitro in variety of cell lines and in vivo in xenograft models by downregulation of c-Myc and 4EBP1 phosphorylation inhibition. Further experiments indicated that our compounds induced both apoptotic cell death and cell cycle arrest at G0/G1, consistently with the predicted Pim kinase inhibition phenotype. SEL24 compounds showed mostly synergistic effects in a range of combinations with standard anti-cancer therapeutics in both leukemias and solid tumor cell lines. Strong synergies were observed not only for cytotoxic agents like docetaxel, gemcitabine or cyratabine, but also for targeted therapies like the PI3K/Akt pathway inhibitors or Jak kinase inhibitors. Data will be presented on in vivo efficacy of SEL24 lead compounds examined in xenograft models of leukemias, lymphomas and solid tumors (lung and colon cancer). Apart from excellent efficacy in in vivo xenograft models, SEL24 compounds show preferable safety profile with no effect on ion channel mediated cardiotoxicity and favorable pharmacokinetic profile. Taken together, presented data supports the rationale of using Pim kinase inhibition as a novel approach to standalone cancer therapy, and in combination with other targeted and cytotoxic therapies, especially to overcome developing drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A243.

Evolving Molecular Mechanism-Based Strategies for Control of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide. Unresectable or metastatic HCC has a poor prognosis, and systemic cytotoxic chemotherapy has failed to show a substantial benefit for patients with HCC. However, there has been increasing interest in developing novel molecularly targeted agents in HCC due to the accumulation of knowledge of cell signaling and molecular carcinogenesis. Furthermore, some of these agents have proven to be efficacious in other traditionally challenging carcinomas, such as renal cell carcinoma. Recently, a phase III, randomized, placebo-controlled trial demonstrated that sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor and Ras kinase, improves overall survival (OS) in patients with advanced HCC. This seminal study described the first agent to improve OS in HCC and began a new era of molecule-targeted cancer therapies. Currently, many novel molecularly targeted agents are under evaluation in clinical trials. In this review, we comprehensively summarize the molecular pathogenesis, targets, and signal transduction pathways involved in HCC. We also detail the current status of molecularly targeted agents that are under clinical development in advanced HCC, including the mechanisms of action of these agents.

Abstract 4394: Identification of molecular determinants of response to GSK1120212B, a potent and selective MEK inhibitor, as a single agent and in combination in RAS/RAF mutant non-small cell lung carcinoma cells

Abstract MEK, a downstream effector for RAS, RAF and receptor tyrosine kinases, represents a promising target for treatment of RAS/RAF mutated Non Small Cell Lung Carcinoma (NSCLC). GSK1120212B is a highly potent and selective oral MEK inhibitor currently in phase II clinical trials. To identify markers of response to GSK1120212B and to generate hypotheses for effective combination therapies, we characterized baseline signaling pathways and gene expression profiles in a panel of 29 NSCLC lines with RAS/RAF mutations and determined their sensitivity to GSK1120212B, erlotinib (EGFR inhibitor), everolimus (mTOR inhibitor), docetaxel and the combination of GSK1120212B with these agents. Inhibition of cell growth and induction of apoptosis were determined after 72 and 24 hours of treatment, respectively. GSK1120212B potently inhibited cell growth (IC50≤ 50 nM) in 13/29 (45%) of the lines and induced apoptosis in 8/29 (28%) of cell lines. We also identified genes such as TGFA, EREG, LGALS3, CTNND1, ARHGEF3, KLF4, RPH3AL, STN2, TWIST2 and FOXC1 whose expression levels were highly associated with the response to GSK1120212B. Cell signaling analyzed at baseline demonstrated that sensitivity to GSK1120212B was associated with high levels of pMEK, low levels of pAKT/pFOXO3a and wild-type PTEN, and resistance was associated with high levels of pAKT/pFOXO3a or mutations of PTEN/PIK3CA/CTNNB1 in RAS/RAF mutant lines. These results suggest combination approaches for cell lines resistant to single agent GSK1120212B. Erlotinib or everolimus alone showed little activity in RAS/RAF mutant lines. The combination of GSK1120212B and erlotinib was synergistic and/or enhanced cell growth inhibition in 6 out of 10 RAS/RAF mutant lines tested, three of which had HER1 amplification or over-express TGFA and EREG. The combination of GSK1120212B and everolimus enhanced cell growth inhibition in 25/29 (86%) of cell lines and increased apoptosis in 12/28 (43%) of RAS/RAF mutant lines. Intriguingly, the RAS/RAF mutated lines that were sensitive to GSK1120212B alone as measured by induction of apoptosis were less responsive to docetaxel. Further, the combination of GSK1120212B and docetaxel was synergistic or increased apoptosis in 20/28 RAS/RAF mutated lines. Our results provide a strong rationale for testing GSK1120212B and these combinations in the clinic and warrant further investigation of the gene expression signatures and genetic mutations in patients to ultimately improve treatment outcome in RAS/RAF mutated NSCLC. 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 4394. doi:10.1158/1538-7445.AM2011-4394

Nanoscale approaches to define biologic signatures and measure proteomic response to targeted therapies in hematologic and solid tumors

Abstract The ability to quantify changes in protein activation in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. A barrier to analyzing proteomic response in patients is the large amount of specimen required to make measurements at each time point. To overcome this barrier, we have developed the use of a highly sensitive microfluidic nano-immunoassay system (NIA) to quantify unphosphorylated, single- and multiple phosphorylated isoforms of proteins in patient specimens collected by minimally invasive blood draws or fine needle aspirates. We first developed tissue collection, handling and processing protocols to produce reliable NIA results using as little as 4 nanoliters of lysate. We next developed assays to measure over 40 proteomic parameters, including changes in proteins that mediate RAS and MAP Kinase signaling, cell cycle and apoptosis. To investigate the diagnostic potential of NIA, we have collected over 300 specimens from normal controls and patients with hematopoietic and solid tumors. We found that phosphoprotein profiles in tumor cells could distinguish tumor from normal cells. Further, patient tumors could be grouped based upon different patterns of percent ERK and MEK phosphorylation. In a clinical trial setting, we have now used NIA in conjunction with multicolor intracellular flow cytometry (FACS) to monitor changes in phospho-profiles in patients with receiving novel agents for hematopoietic cancers. In a prospective study of a novel cell cycle inhibitor in myelodysplastic syndrome (MDS), we have used NIA to measure changes in MAPK and cell cycle proteins in patient leukocytes sampled throughout serial time points during the study. In a prospective study of atorvastatin in patients with non-Hodgkin's lymphoma, we have used NIA and FACS to distinguish three patterns of signaling changes in patient tumor cells, monocytes, and T cells. First, pathways that were initially activated in tumor cells decreased upon treatment with atorvastatin, with up to a 70% decrease in phospho-STAT3 and phospho-STAT5. Second, pathways that were initially suppressed in tumor cells (including phospho-PLC), normalized upon treatment. Third, expression levels of proteins in apoptotic pathways, including p38, increased upon atorvastatin treatment. Finally, changes in signaling associated with atorvastatin were found to be cell specific: effects on tumor cells were distinct from effects on non-tumor T cells and monocytes. Our studies demonstrate that NIA and FACS are complementary proteomic methods that can be used to quantify changes in cell signaling and distinguish responses in different cellular populations from very small clinical specimens sampled during therapeutic interventions. Nanoscale analysis of patient specimens is a feasible and promising approach for the development of new diagnostic markers and biomarkers of therapeutic activity. This talk is also presented as Poster A21.

Protein tyrosine kinase pathway-derived ROS/NO productions contribute to G2/M cell cycle arrest in evodiamine-treated human cervix carcinoma HeLa cells

A previous study indicated that reactive oxygen species (ROS) and nitric oxide (NO) played pivotal roles in mediating cytotoxicity of evodiamine in human cervix carcinoma HeLa cells. This study suggested that G2/M cell cycle arrest was triggered by ROS/NO productions with regulations of p53, p21, cell division cycle 25C (Cdc25C), Cdc2 and cyclin B1, which were able to be prevented by protein tyrosine kinase (PTK) activity inhibitor genistein or JNK inhibitor SP600125. The decreased JNK phosphorylation by addition of Ras or Raf inhibitor, as well as the increased cell viability by addition of insulin-like growth factor-1 receptor (IGF-1R), Ras, Raf or c-Jun N-terminal kinase (JNK) inhibitor, further demonstrated that the Ras-Raf-JNK pathway was responsible for this PTK-mediated signalling. These observations provide a distinct look at PTK pathway for its suppressive effect on G2/M transition by inductions of ROS/NO generations.

Molecular targets for liver cancer therapy: From screening of target genes to clinical trials

Cancer arises from the accumulation of genetic alterations, and the inactivation of oncogenes, or recovery of suppressor genes, are promising strategies for cancer treatment. Genome-based drug research starts with identification of target genes and is accomplished by exploitation of target-based drugs such as monoclonal antibodies, small molecules and antisense drugs. Recently, clinical trials for treatment of advanced hepatocellular carcinoma (HCC) have been performed, and the effectiveness of sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor and Ras kinase, has been demonstrated. In addition to known target genes, microarray technology has enabled us to constitute novel therapeutic targets, and many researchers have applied this technology in studies of HCC and have identified candidate target genes, validated to affect cell growth. In addition, promoter arrays for whole-genome epigenetic aberration analysis, ChIP-chip analysis using tiling arrays, and high-throughput sequencing systems have been applied to drug discovery. To elucidate the status of therapeutic target genes in vivo, development of diagnostic markers for stratification of patients is a pressing need. Here, we review recent advances in microarray technology for liver cancer, discuss the innovations and approaches to therapeutic target discovery, and present data regarding the outcome of gene target therapy using monoclonal antibodies and molecular diagnostic markers in our laboratory.

Abstract A250: Preclinical combination efficacy and identification of biomarkers of MEK and PI3K inhibitors in phase I

Abstract Introduction: The Raf/MEK/ERK and PI3K/Akt/mTOR pathways are two major effector pathways downstream of activated Ras and receptor tyrosine kinases. While Ras is an attractive cancer target, direct pharmacological targeting has so far been unsuccessful. We describe preclinical studies combining GDC-0973, an inhibitor of MEK, and GDC-0941, an inhibitor of PI3K, both of which are in Phase I trials. Summary of Results: GDC-0973 is a selective allosteric orally bioavailable small molecule inhibitor of the MEK1/2 dual specificity kinases. It has nanomolar potency against MEK1/2 in cells, and has potency across a broad range of cancer cell lines, most notably in those that are mutated for B-Raf and K-Ras. GDC-0941 is a selective orally bioavailable small molecule inhibitor of Class I PI3 kinases that has been described previously. Given the widespread and frequently concurrent activation of the Ras/Raf/MEK and Ras/PI3K/Akt pathways in tumors such as melanoma, colorectal, non-small cell lung cancer, and pancreatic cancer, we carried out cellular and in vivo xenograft studies to evaluate the efficacy of GDC-0973 and GDC-0941, both individually and in combination. We demonstrate strong combination efficacy in B-raf or K-ras mutant melanoma, colon and NSCLC models, driven primarily by increased apoptosis. The combination resulted in modulation of markers of both the MEK and PI3K pathways, and we discovered several markers whose modulation correlated with the induction of apoptosis. Continuous suppression of the two pathways is not required for combination efficacy. Conclusions: The preclinical data indicate that combined use of MEK and PI3K inhibitors can produce greater efficacy than either agent alone at tolerated doses. Additionally, biomarkers have been identified which correlate with induction of apoptosis in the combination regimens. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A250.

Polycythemia vera erythroid precursors exhibit increased proliferation and apoptosis resistance associated with abnormal RAS and PI3K pathway activation

Polycythemia vera (PV) is characterized by erythrocytosis associated with the presence of the activating JAK2(V617F) mutation in a variable proportion of hematopoietic cells. JAK2(V617F) is detected in other myeloproliferative neoplasms, does not appear to be the PV-initiating event, and its specific role in deregulated erythropoiesis in PV is incompletely understood. We investigated the pathogenesis of PV to characterize abnormal proliferation and apoptosis responses and aberrant oncogenic pathway activation in primary PV erythroid precursors. Peripheral blood CD34(+) cells isolated from PV patients and healthy controls were grown in liquid culture to expand a population of primary erythroblasts for experiments designed to analyze cellular proliferation, apoptosis, JAK2(V617F) mutation status, cytokine-dependent protein phosphorylation and gene expression profiling using Affymetrix microarrays. The survival and proliferation of PV erythroblasts were growth factor-dependent under strict serum-free conditions requiring both erythropoietin (EPO) and stem cell factor. PV erythroblasts exhibited EPO hypersensitivity and enhanced cellular proliferation associated with increased EPO-mediated extracellular signal-regulated kinases 1 and 2 phosphorylation. EPO-induced AKT phosphorylation was observed in PV but not normal erythroblasts, an effect associated with apoptosis resistance in PV erythroblasts. Analysis of gene expression and oncogenic pathway activation signatures revealed increased RAS (p<0.01) and phosphoinositide-3 kinase (p<0.05) pathway activation in PV erythroblasts. Deregulated erythropoiesis in PV involves EPO hypersensitivity and apoptosis resistance of erythroid precursor cells associated with abnormally increased activation of RAS-ERK and phosphoinositide-3 kinase-AKT pathways. These data suggest that investigation of the mechanisms of abnormal RAS and phosphoinositide-3 kinase pathway activation in erythroblasts may contribute to our understanding of the molecular pathogenesis of PV.

Dose- and Time-Dependent Changes in Gene Expression in Human Glioma Cells after Low Radiation Doses

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2-2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.

Solid-State 31P NMR Spectroscopy of Precipitated Guanine Nucleotide-Binding Protein Ras in Complexes with Its Effector Molecules Raf Kinase and RalGDS

Liquid-state 31P NMR spectroscopy is a well-established method for the study of guanine nucleotide-binding proteins (GNB proteins) such as the proto-oncogene Ras. Solid-state 31P NMR spectroscopy could meanwhile also be used to study microcrystalline samples of Ras as well as its partial loss-of-function mutants Ras(T35S) and Ras(T35A). However, solid-state NMR studies of the latter mutants in complex with effector molecules such as RalGDS or Raf kinase were so far prevented, since it has been impossible to crystallize these complexes yet. The aim of the present contribution is to make such complexes accessible to solid-state 31P NMR spectroscopy by the application of precipitation methods. The complex formed by Ras(T35S) and Raf kinase is preserved during precipitation. In contrast, the weakly bound complex of Ras(T35S) with RalGDS is dissociated or at least perturbed by the precipitation procedure. Solid-state 31P NMR experiments on precipitates of these complexes deliver spectra of high resolution and signal-to-noise ratio which allows the application of two-dimensional techniques. Precipitates prepared using polyethylene glycol 6000 (PEG) as precipitant were found to exhibit spectra of maximum resolution and signal-to-noise ratio. Interestingly, the 31P signal due to the alpha-phosphate of GppNHp bound to Ras(T35S) in crystalline samples or aged precipitates has a significantly different isotropic chemical shift than in the liquid state or in freshly prepared precipitates. This directly indicates that the crystal structure differs from the equilibrium solution structure at least in the neighborhood of the alpha-phosphate group.

Genome-Wide Analysis and Experimentation of Plant Serine/ Threonine/Tyrosine-Specific Protein Kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia

Activating mutations in RAS and receptor tyrosine kinases such as KIT and FLT3 are hypothesized to cooperate with chimeric transcription factors in the pathogenesis of acute myeloid leukemia (AML). To test this hypothesis, we genotyped 150 pediatric AML samples for mutations in KIT (exons 8, 17), NRAS and KRAS (exons 1, 2) and FLT3/ITD. This is the largest cohort of pediatric AML patients reported thus far screened for all four mutations. Of the children with AML, 40% had a mutation in KIT (11.3%), RAS (18%) or FLT3/ITD (11.1%), and 70% of cases of core-binding factor (CBF) leukemia were associated with a mutation of KIT or RAS. Mutations in RAS or FLT3/ITD were frequently found in association with a normal karyotype. Patients with a FLT3/ITD mutation had a significantly worse clinical outcome. However, the presence of a KIT or RAS mutation did not significantly influence clinical outcome. We demonstrate that KIT exon 8 mutations result in constitutive ligand-independent kinase activation that can be inhibited by clinically relevant concentrations of imatinib. Our results demonstrate that abnormalities of signal transduction pathways are frequent in pediatric AML. Future clinical studies are needed to determine whether selective targeting of these abnormalities will improve treatment results.

Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

BackgroundG protein-coupled receptors (GPCRs) interact with heterotrimeric GTP-binding proteins (G proteins) to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP) kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways.ResultsHere we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK) inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1) on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin.ConclusionThese results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

From VEGF to Ras Through Sphingosine Kinase

Many growth factor receptors activate the mitogen-activated protein kinase (MAPK) pathway producing activated, phosphorylated extracellular-regulated kinases 1 and 2 (ERK1 and ERK2), which regulate gene expression. Shu et al. used pharmacological manipulation and expression of dominant-negative constructs in cultured human umbilical vein endothelial cells and T24 bladder cell carcinoma cells. They demonstrated that the pathway activated by vascular endothelial growth factor (VEGF) that produces activated ERK1 and ERK2 involves activation of sphingosine kinase (SPK) by protein kinase C (PKC) and the production of sphingosine-1-phosphate. Inhibition of the SPK blocked the formation of the activated form of Ras [Ras-guanosine triphosphate (GTP)], however the VEGF-stimulated phosphorylation of ERK1 and ERK2 was not blocked by expression of the dominant-negative Ras mutant RasN17, which prevents the activation of Ras by guanine exchange factors. Thus, the authors propose that VEGF stimulates PKC, which activates SPK, which then promotes Ras activation, possibly by inhibiting the function of a Ras GTPase-activating protein (Ras-GAP) to activate the MAPK pathway. X. Shu, W. Wu, R. D. Mosteller, D. Broek, Sphingosine kinase mediates vascular endothelial growth factor-induced activation of Ras and mitogen-activated protein kinases. Mol. Cell. Biol. 22 , 7758-7768 (2002). [Abstract] [Full Text]

Internalized EGF-EGFR Complex Continues to Signal

Epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) induces receptor dimerization and tyrosine autophosphorylation, initiating a complex cascade of events leading to cell proliferation. In addition to activating various signaling pathways, EGF binding triggers endocytosis of the EGF-EGFR complex. Most internalized EGF-EGFR is degraded in lysosomes; some, however, may contribute to the cellular response. Wang et al. used selective activation of endosomal EGFR to address the possible role of endosomal EGF-EGFR in intracellular signaling. After inactivation of EGFR with the EGFR tyrosine kinase inhibitor AG-1478, BT20 or MDCK cells were treated with EGF, to stimulate endocytosis, and with monensin, to inhibit EGFR recycling to the plasma membrane. Immunoblot analysis of cells fractionated through a sucrose-density gradient, as well as immunofluorescent analysis of fixed cells, demonstrated internalization of nonphosphorylated EGFR together with labeled EGF. Removal of AG-1478 and monensin led to reactivation of endosomal EGFR (phosphorylation) without its reappearance at the plasma membrane. Similar results were obtained without monensin by washing cells after AG-1478/EGF treatment. Western analysis of immunoprecipitates indicated that activated endosomal EGFR associated with growth receptor-binding protein 2, src homologous and collagen-like protein, and phospholipase C-γ1. Activation of endosomal EGFR led to phosphorylation of Ras and extracellular signal-regulated kinase 1 and 2, as well as Akt. Activated endosomal EGFR suppressed apoptosis in response to serum withdrawal. This work indicates that endocytosed EGFR does participate in intracellular signaling, and describes a system applicable to analyses of signaling by other endocytosed receptor tyrosine kinases. Y. Wang, S. Pennock, X. Chen, Z. Wang, Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol. Cell. Biol. 22 , 7279-7290 (2002). [Abstract] [Full Text]

Soluble E-selectin Induces Monocyte Chemotaxis through Src Family Tyrosine Kinases

Cellular adhesion molecules such as E-selectin function to recruit leukocytes into the inflammatory lesions of diseases such as rheumatoid arthritis (RA) and atherosclerosis. Monocytes are the key components of the cellular infiltrates present in these disorders. We hypothesized that soluble E-selectin (sE-selectin) might mediate the chemotaxis of monocytes. In this report, we show that sE-selectin induced normal human peripheral blood monocyte migration in the nanomolar range in a concentration-dependent manner. Neutralization studies using RA human joint synovial fluids and anti-E-selectin antibody showed a mean 31% reduction in RA synovial fluid-mediated monocyte chemotaxis (p < 0.05), indicating that sE-selectin is a major monocyte recruiter in RA. Next, we investigated the role of tyrosine phosphorylation pathways in sE-selectin-induced monocyte chemotaxis. Human peripheral blood monocytes stimulated with sE-selectin showed a time-dependent increase in the tyrosine phosphorylation of a broad range of cellular proteins, predominantly in the molecular size range of Src family kinases (50-60 kDa) and mitogen-activated protein kinases (MAPKs). Western blot analysis of Src family kinases showed a time-dependent increase in Src, Hck, and Lyn phosphorylation. The pretreatment of monocytes with the Src inhibitor AG1879: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) prior to stimulation with sE-selectin markedly inhibited Hck and Lyn phosphorylation, whereas the phosphorylation of Src was partially inhibited. In addition, the sE-selectin stimulation of monocytes resulted in the increased phosphorylation of extracellular signal-related kinase (ERK1/2) and p38 MAPK. The pretreatment of monocytes with PP2 showed 89 and 83% inhibition of ERK1/2 and p38 MAPK phosphorylation, respectively. sE-selectin also showed a time-dependent activation of Ras kinase. Furthermore, the pretreatment of monocytes with PP2 completely inhibited sE-selectin-mediated monocyte chemotaxis. Taken together, our data demonstrate a novel function for sE-selectin as a monocyte chemotactic agent and suggest that sE-selectin might be mediating its biological functions through the Src-MAPK pathway.