Constitutive Phosphorylation Of ERK Research Articles

Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

Abstract 2656: Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma

Abstract Cholangiocarcinoma (CC) responds poorly to chemo- and immunotherapy and is nearly always fatal within one year. In recent years we have gained important insight into the signaling pathways that drive this cancer. These cancers are characterized by deregulated mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and a dependence on the IL-6 axis of signal transduction. Importantly, we previously reported clinical responses for CC patients receiving single agent MEK inhibitors, indicating these agents have activity in this disease. We hypothesized that dual targeting of these pathways in CC using MEK162 and buparlisib would lead to potent antitumor and immunomodulatory activity, possibly circumventing resistance to single agent MEK162. In a panel of n = 7 human CC cell lines with diverse genetic profiles, constitutive phosphorylation of ERK (7/7) and Akt (2/7) was observed. Human CC cell lines displayed variable sensitivity to the growth inhibitory and pro-apoptotic effects of single agent MEK162 or buparlisib. CC cell lines with basal AKT phosphorylation (WITT, Mz-Cha-1) showed greater sensitivity to growth inhibition by buparlisib (IC50 10-20 μM), as compared to CC lines lacking pAKT (HuCCT1, HuH28, IC50 > 20μM). Immunoblot analysis confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 and SNU-478 CC cell lines following treatment with MEK162. Culture supernatants from four separate human CC cell lines displayed significant reductions in IL-6, VEGF, and GM CSF in a concentration-dependent manner after treatment with MEK162. Immunomodulatory effects of MEK162 were also evident, independent of its tumor-intrinsic effects upon CC cell lines. Namely, it significantly reduced IL 6/GM-CSF driven MDSC differentiation (HLA-DRlo CD11b+ CD33+) from healthy normal donor PBMC in vitro. These observations were not due to cytotoxic activity as treatment with MEK162 for 72 hours did not reduce viability of bulk human PBMCs. Finally, to evaluate the effect of this treatment combination, in vivo studies were conducted in athymic mice bearing Mz-Cha1 or SNU-478 xenografts. Tumor bearing mice received daily oral administration of MEK162 (30 mg/kg), buparlisib (25 mg/kg), or both agents combined. Vehicle treated animals served as negative controls. Inhibition of tumor growth was observed following administration of single agent MEK162 or buparlisib as compared to control animals. This effect was further enhanced in the combination treated animals. Body weight of animals indicated this regimen was well-tolerated. Together, these data suggest that dual PI3K and MEK inhibition can target CC with varying genotypes and represents a promising therapeutic regimen with potential for direct antitumor activity and immunomodulation in CC. Citation Format: Jennifer Yang, Omar Elnaggar, Thomas Mace, Matthew Farren, Gregory Young, Patrice Lee, Kaitlin Keenan, Zheng Che, Jacob Kaufman, Denis Guttridge, David Carbone, Cynthia Timmers, Tanios Bekaii-Saab, Gregory Lesinski. Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2015-2656

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

A5.1 Abnormal Calcium Influx in T and B Lymphocytes from Systemic Lupus Erythematosus Patients is Related to STIM-1 Over-Expression

Background and Objectives Recently described, the molecule STIM1 (stromal interaction molecule 1) acts as a key mediator of calcium influx by controlling cell proliferation after antigen stimulation, Erk phosphorylation, cytokine production and apoptosis. Although STIM1 mutations have been associated with severe immunodeficiency, no study has focused on the STIM1 molecule in autoimmune diseases. Materials and Methods T and B lymphocytes, purified by negative selection from peripheral blood of patients with systemic lupus erythematosus (SLE, n = 11), rheumatoid arthritis (RA, n = 7), primary Sjogren’s syndrome (pSS, n = 11) and healthy controls (HC, n = 12) were tested by flow cytometry and Western blotting to determine the expression of STIM1. Video microscopy using specific probes has been used to assess intracellular calcium levels. Results T cells from peripheral blood of HC express more STIM1 molecules (mean fluorescence intensity (MFI) 3.42 ± 0.13) than B cells (MFI 2.18 ± 0.20, P high CD38 high transitional B cells (MFI 4.83 ± 0.63) compared with CD24 low CD38 low mature B cells (MFI 2.47 ± 0.15, P high CD38 low memory B cells (MFI 3.64 ± 0.42, P An highest calcium influx and a constitutive Erk phosphorylation characterise T and B cells from SLE patients when compared with HC and disease controls. As suspected, STIM1 is over-expressed in SLE, when compared with HC and this expression is similar between T cells (MFI 8.70 ± 0.87) and B cells (MFI 9.00 ± 1.08). Within B cell subsets, STIM1 expression is 3.4 fold highest in transitional SLE B cells (MFI: 16.25 ± 2.18, P Conclusions These results suggest that the differential expression of STIM1 may be an important factor in the process of lymphocyte self-reactivity in SLE, which opens new pathophysiological and therapeutic perspectives.

Targeting B Cell Anergy in Chronic Lymphocytic Leukemia

AbstractAbstract 3863B-Cell Receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones.Previous studies have suggested that in a sizable fraction of CLL patients the B cell clone is functionally anergic and characterized by constitutive phosphorylation of ERK1/2 kinases.We have analyzed all the described anergy-related features in CLL cells and we observed that constitutive ERK1/2 phosphorylation (used as surrogate marker for anergy) together with NF-ATc1 nuclear translocation correlates with the in vitro inability to mobilize intracellular calcium upon anti-IgM stimulation and with reduced expression of surface IgM. The latter features are reminiscent of B cells where chronic Ag-stimulation favours continuous recycling of B cell receptor. Of interest the cells of the pERK(+) subset of patients have higher expression levels of CD22 and CD5, two receptors that are required for the negative regulation of BCR signalling.The activation of the biochemical program that culminates in the induction of B-cell anergy is transient and requires chronic BCR triggering. We showed that Ag removal, obtained by culturing CLL cells in vitro, causes the loss of the anergic signature (reduction of pERK and nuclear NF-ATc1 levels) and, together with the re-expression of surface IgM, restores the ability of CLL cells to fully respond to BCR triggering.These results led us to explore the possibility to interfere with the molecular features of anergy in a therapeutic perspective, using two classes of MAPK inhibitors (targeting MEK1/2 or ERK1/2) and one specific peptide (VIVIT) that blocks NF-AT nuclear translocation.Constitutive ERK1/2 activation was efficiently inhibited after 1 hour treatment with two MEK inhibitors (U0126 and CI1040), and 48 hours treatment with both compounds induced selective apoptosis in the restricted group of pERK(+) patients. In details, pERK(+) samples (n=21) treated with 10μM of U0126 showed a mean survival of 36.58 ± 5.242 (compared to 60.63 ± 6.557 survival of the pERK(-) subset, p=0,01), while pERK(+) samples treated with 10μM of CI1040 (n=17) experienced a mean survival of 36.59 ± 4.145 (compared to 59.17 ± 6.313 survival of the pERK(-) subset; p=0,01). The same results were obtained when we directly inhibited ERK1/2 activity with a new generation compound. By screening 16 samples treated with 10μM of ERK inhibitor, we observed that pERK(+) patients are very sensitive to treatment (29.38 ± 8.064 mean survival) compared to pERK(-) samples (68.12 ± 8.81; p=0,03).Taken together, these data suggest that MEK1/2 can be efficiently targeted in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 represents a good biomarker to predict and monitor treatment response.In parallel, inhibition of NF-ATc1 activation with a cell permeable version of VIVIT peptide, reduced cell viability in the group of NF-AT(+) patients (29.47 ± 7.509 mean survival in the NF-AT(+) group and 53.63 ± 6.044 in the NF-AT(-) subset, p=0,02), As expected, the killing activity also correlated with ERK activation (21.30 ± 4.191 mean survival for the pERK(+) group and 42.95 ± 5.465 mean survival for the pERK(-) subset; p=0,01).The apoptotic effect observed in vitro after the use of ERK1/2 and NF-AT inhibitors is preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation (together with the reduction of surface levels of CD22), and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program is responsible for the survival of leukemic lymphocytes.Combination studies with different inhibitors revealed that the concomitant inhibition of MAPK and NF-AT signalling did not enhance apoptosis, thus confirming that both pathways are directly interconnected. A possible link between ERK and NF-AT is the activation of the SHIP1/CD5 axis whose phosphorylation is lost after inhibitors treatment and re-acquired upon BCR triggering.In conclusion, our results demonstrate that one subset of CLL patients is characterized by the abnormal expansion of B cells with anergic features. In these cells the constitutive phosphorylation of ERK and NF-ATc1 activation can be efficiently targeted for therapeutic purpose, thus opening new clinical perspectives. Disclosures:No relevant conflicts of interest to declare.

Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma

We performed exome sequencing to detect somatic mutations in protein-coding regions in seven melanoma cell lines and donor-matched germline cells. All melanoma samples had high numbers of somatic mutations, which showed the hallmark of UV-induced DNA repair. Such a hallmark was absent in tumor sample-specific mutations in two metastases derived from the same individual. Two melanomas with non-canonical BRAF mutations harbored gain-of-function MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening a larger cohort of individuals with melanoma revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations, which occurred at an overall frequency of 8%. Furthermore, missense and nonsense somatic mutations were frequently found in three candidate melanoma genes, FAT4, LRP1B and DSC1.

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia

c-CBL (CBL) encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by constitutional anomalies that include impaired growth, developmental delay, cryptorchidism, and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.Y371H mutant Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT, and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic, and functional consequences that are reminiscent of disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, and Noonan, Costello, cardiofaciocutaneous, and Legius syndromes.

Abstract 4348: PDGF-Rα expression and activation of the MAPK pathway in disseminated pilocytic astrocytomas

Abstract Background: Pilocytic astrocytomas (PA) are the most common pediatric tumors of the central nervous system. Approximately 5 % of patients with PA present a tumor dissemination. Although the disseminated growth is associated with severe clinical problems, the genetic alterations associated with this biological behaviour have never been investigated. As the histology of the disseminated PAs does not differ significantly to sporadic PAs, we analyzed the mitogen-activated protein kinase (MAPK) pathway for aberrations because genetic alterations of members of this pathway as BRAF and NF-1 have been found to be involved in the pathogenesis of non-disseminated PAs. Methods: Immunohistochemical analysis of the platelet-derived growth factor receptors alpha (PDGF-Rα) and beta (PDGF-Rβ), the epidermal growth factor receptor (EGF-R), and the downstream activated (phosphorylated) extracellular signal-regulated kinase (p-ERK) was performed in a cohort of 18 patients suffering disseminated PAs. In three patients, material of the primary tumor and of relapses could be studied. The BRAF gene was analyzed for mutations by single strand conformation analysis (SSCP) and for gene copy numbers by semiquantitative PCR with fluorescence labelled primers. Mutational hotspots of the K-RAS, H-RAS and N-RAS genes were also analyzed by SSCP. Results: Immunohistochemical analysis showed an increased PDGF-Rα expression in 71.4% of disseminated Pas, but no significant overexpression of EGF-R or PDGF-Rβ. A constitutive ERK phosphorylation was demonstrated in all disseminated PAs investigated. Molecular genetic analysis revealed V600E BRAF mutations in two tumors and an increased BRAF gene copy level in the majority of cases. No RAS mutations were found. Conclusions: This is the first report of molecular genetic analysis of the MAPK pathway in disseminated pilocytic astrocytomas. It underlines the important role of the upstream PDGF-Rα receptor and subsequent MAPK pathway in these gliomas. Its activation represents a potential target for novel strategies in the therapy of disseminated PAs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4348.

The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells

In this study, β2-AR level was found to be up-regulated in MCF-7 cells overexpressing Her2 (MCF-7/Her2). Correlation of β2-AR level with Her2 status was demonstrated in breast cancer tissue samples. Constitutive phosphorylation of ERK, mRNA expression up-regulation of catecholamine-synthesis enzymes, and increased epinephrine release were detected in MCF-7/Her2 cells. β2-AR expression induced by epinephrine and involvement of ERK signaling were validated. The data indicate that Her2 overexpression and excessive phosphorylation of ERK cause epinephrine autocrine release from breast cancer cells, resulting in up-regulation of β2-AR expression. The data also showed that catecholamine prominently stimulated Her2 mRNA expression and promoter activity. The activation and nuclear translocation of STAT3 triggered by isoproterenol were observed. Enhanced binding activities of STAT3 to the Her2 promoter after isoproterenol stimulation were verified. Using STAT3 shRNA and dominant negative STAT3 mutant, the role of STAT3 in isoproterenol-induced Her2 expression was further confirmed. The data support a model where β2-AR and Her2 comprise a positive feedback loop in human breast cancer cells.

CD40 Pathway Activation Status Predicts Response to CD40 Targeted Therapy in Diffuse Large b-Cell Lymphoma.

Abstract 2721Poster Board II-697The role of the CD40 pathway in B-cell cancers is rather enigmatic due to having polar opposite roles in tumor biology. Stimulation of the CD40 receptor can reduce cell viability of NHL cell lines and xenograft tumor models, whereas, in sharp contrast, constitutive CD40 stimulation can actually promote proliferation of NHL cell lines, as well as drive lymphomagenesis and transformation of primary B-cells. Therefore, targeting the CD40 pathway has been challenging due to a knowledge gap in understanding the factors that may dictate a pro- or anti-tumor role. To address this directly, we employed a large panel of NHL cell lines and xenografts models to simulate a patient population and to define the molecular parameters to test in the context of clinical trials. Using baseline gene expression profiling, we found that the in vitro activity of an antibody that stimulates the CD40 pathway, Dacetuzumab (SGN-40), correlated strikingly with a transcriptional profile that was representative of an inactive CD40 pathway. Resistant cells, on the other hand, displayed a transcriptional profile indicative of an activated CD40 pathway. Consistent with these observations, constitutive phosphorylation of ERK, a node downstream of CD40 signaling, was present predominantly in cell lines that were resistant to Dacetuzumab. Fifteen genes, based on their association with in vitro activity and their biology as components of the CD40 network, were selected as a classifier from these pre-clinical data and independently tested in a panel of cell lines and xenografts models. Testing the selected gene classifier by qRT-PCR from baseline fixed tumor tissue of 39 patients treated with Dacetuzumab in phase I and II clinical trials revealed the ability to predict sensitivity in patients, as determined by tumor shrinkage >10%, with an overall accuracy of 80%. Consistent with the observed tumor shrinkage, the classifier positive, predicted to respond to Dacetuzumab, patients also exhibited a prolonged progression free survival (PFS) compared to classifier negative patients, predicted not to respond to Dacetuzumab (HR = 0.23, p = 0.001). Finally, using a novel microarray approach to genome-wide expression profiling on fixed tumor tissue revealed that CD40 pathway activation was indeed an important predictor of response. Overall, these data suggest that sensitivity to Dacetuzumab can be predicted by pre-determining the CD40 pathway activation status of tumors. Disclosures:Dornan:Genentech, Inc.: Employment, Equity Ownership. Burington:Genentech, Inc.: Employment, Equity Ownership. Yue:Genentech, Inc.: Employment, Equity Ownership. Shi:Genentech, Inc.: Employment, Equity Ownership. Advani:Seattle Genetics, Inc.: Research Funding. Yu:Genentech, Inc.: Employment, Equity Ownership. Lau:Genentech, Inc.: Employment, Equity Ownership. Yu:Genentech, Inc.: Employment, Equity Ownership. De Vos:Seattle Genetics, Inc.: Research Funding. Forero-Torres:Seattle Genetics, Inc.: Research Funding. Modrusan:Genentech, Inc.: Employment, Equity Ownership. Koeppen:Genentech, Inc.: Employment, Equity Ownership. Yang:Genentech, Inc.: Employment, Equity Ownership. Du:Genentech, Inc.: Employment, Equity Ownership. Elkins:Genentech, Inc.: Employment, Equity Ownership. Polson:Genentech, Inc.: Employment, Equity Ownership. Offringa:Genentech, Inc.: Employment, Equity Ownership. Whiting:Seattle Genetics, Inc.: Employment, Equity Ownership. Ebens:Genentech, Inc.: Employment, Equity Ownership.

Continual signaling is responsible for constitutive ERK phosphorylation in B-1a cells

B-1a cells constitutively express phosphorylated, activated ERK, but the origin of pERK in B-1 cells has not been determined. To address this issue, we examined specific mediators of intracellular signaling in unmanipulated B-1a cells. We found that constitutive pERK was rapidly lost from B-1a cells following addition of metabolic inhibitors that block src kinase, Syk, PI-3K, and PLC function. We examined Syk and PLC in more detail and found rapid accumulation of phosphorylated forms of these molecules in B-1a cells, but not B-2 cells, when phosphatase activity was inhibited, and this change occurred in the majority of B-1a cells. Further, we showed that inhibition of src kinase activity eliminated “downstream” pSyk and pPLC accumulation in phosphatase-inhibited B-1a cells, indicating a pathway connection. CD86 expression is greater on B-1 than B-2 cells and plays a role in antigen presentation by B-1 cells to T cells. We found that when Syk or PI-3K was inhibited, CD86 expression was diminished in a reversible fashion. All together, these results indicate that continual activation of intracellular signaling leads to constitutive activation of ERK in B-1 cells, with attendant consequences for co-stimulatory molecule expression.

The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.

Multiple Strategies Based on MEK, mTOR and BCL-2 Signal Transduction Inhibition in Acute Lymphoblastic Leukemia (ALL)

In spite of improvements in the last decade, relapses still occurs in the majority of ALL patients, with a long-term survival rate of only 30–40%. Recently, small-molecule inhibitors have been developed for targeting deregulated signal transduction pathways involved in proliferation and apoptosis. Current evidence identifies the Raf/MEK/ERK, the PI3K/AKT/mTOR, and the Bcl-2 pathways as potentially relevant targets for therapeutic intervention. We have previously demonstrated that constitutive ERK phosphorylation is an independent predictor of failure to achieve complete remission in adult ALL (Blood 2007; 109:5473) and thus we evaluated the in vitro activity of MEK-inhibitors. However, neither PD98059 nor PD0325901 affected cell growth, cell cycle distribution, and/or apoptosis in ALL cell lines (IC50 >1 μM for PD0325901). These results were confirmed in primary ALL samples, in which PD98059 significantly (P=0.012) inhibited ERK phosphorylation in 8/12 samples, without inducing cell cycle changes or apoptosis. We next investigated the activity of the mTOR inhibitor Temsirolimus. Temsirolimus displayed a biphasic dose-response in a panel of different ALL cell lines, with a flat curve (35–55% of inhibition) at concentrations ranging between 1 and 5,000 nM and more pronounced growth inhibition at concentrations ≥ 10 μM. The CEM cell line was the most sensitive (IC50: 7 nM), Jurkat showed intermediate sensitivity (IC50: 200 nM), while MOLT-4 were resistant (IC50 > 20,000 nM). Cell growth inhibition was associated with inhibition of cell cycle progression, while apoptosis induction was observed in less than 15% of the cells even at the highest concentration of Temsirolimus (20 μM). We then investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories to A.T.), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, in ALL cell lines and in 10 primary samples. ABT-737 showed a potent dose- and time-dependent growth-inhibitory activity in MOLT-4 (IC50: 198 nM), associated with loss of mitochondrial membrane potential, caspase activation, Bcl-2 cleavage, and ultimately apoptosis induction. Conversely, CEM cells proved resistant (IC50: 12,000 nM). We also found that ABT-737 was highly effective in primary adult and childhood ALL, independent of their chromosomal abnormalities, with a significant decrease in viability (p=0.008) and a remarkable induction of apoptosis (from a mean baseline value of 16.8±8.8% to 43.6±22.8%, p=0.04) at 10 nM ABT-737. A dose-dependent down-regulation of Bcl-2 and Bcl-xL expression was observed in sensitive samples, but not in the only resistant one. In summary, our study shows that ALL cells, do not undergo apoptosis in response to single-pathway inhibition, suggesting the presence of multiple, redundant pathways that preserve leukemic cell survival. The only notable exception was the Bcl-2/Bcl-xL inhibitor ABT-737. Studies are ongoing to identify mechanism-driven combinations of agents that would disrupt multiple signal transduction pathways, resulting in synergistic killing and, ultimately, in novel therapeutic strategies for ALL.

An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

BackgroundVav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.ResultsWe found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.ConclusionOur results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.

Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells.

Overexpressed epidermal growth receptor factor receptors (EGFRs) are thought to contribute to the malignant phenotype of human glioblastomas (GBMs), but the mechanism is not well understood. We found that SKMG-3 cells, a rare GBM cell line that maintains EGFR gene amplification in vitro, produced high levels of EGFR protein. The cells also expressed the related receptors HER2/neu and HER4, but not HER3. Immunoblots and tryptic phosphopeptide maps showed that the SKMG-3 EGFRs were intact and functional and that a subset of these receptors were spontaneously autophosphorylated. EGF treatment stimulated phosphorylation of the EGFRs as well as the downstream effectors Erk, AKT1, stat3 and c-Cbl. Under minimal growth conditions, the unstimulated SKMG-3 cells contained constitutively phosphorylated Erk and AKTI but no detectable stat3 DNA-binding complexes. The EGFR kinase inhibitor PD158780 reduced the constitutive phosphorylation of the receptor and Erk but not that of AKT1. In contrast, inhibition of phosphatidylinositol-3-kinase (PI3K) blocked the constitutive phosphorylation of Erk and AKT-1 but not the EGFR. We conclude that the SKMG-3 cells represent the subset of GBMs with amplified EGFR genes that overexpress intact receptors. The results also suggest that in some GBMs, signals from overexpressed EGFRs contribute to the constitutive phosphorylation of Erk, but these signals may not required for the constitutive activation of PI3K or AKT1.

Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor.

FLT3 is a receptor tyrosine kinase that may play a role in a significant proportion of leukemias. In addition to being aberrantly expressed in acute leukemias, activating mutations of the FLT3 gene have been found in patients with AML, myelodysplastic syndrome (MDS) and more rarely, ALL. Internal tandem duplications (ITDs) of the FLT3 gene have been detected in 17-34% of patients with AML and portend a poor prognosis for these patients. FLT3 receptors containing ITD mutations (FLT3/ITDs) are constitutively activated in the absence of FLT3 ligand (FL) stimulation leading to the activation of downstream signaling proteins, including ERK and STAT 5. FLT3 activity, therefore, is a logical target for therapeutic intervention. AG1296 is a tyrosine kinase inhibitor of the tyrphostin class that shows inhibitory activity for wild-type FLT3, in addition to the PDGF and c-KIT receptors. We examined the inhibitory effects of AG1296 on FLT3/ITDs isolated from AML patients in the IL-3-dependent cell line, Ba/F3, as well as in primary leukemia samples from AML patients. Immunoprecipitation and immunoblotting analyses demonstrated that FLT3/ITDs were constitutively phosphorylated in the absence of FL. The auto-phosphorylation of FLT3/ITDs was inhibited by AG1296 with an IC(50) of approximately 1 microM. FLT3/ITDs were associated with constitutive phosphorylation of ERK, STAT 5A, STAT 5B, CBL, VAV and SHP2 in Ba/F3 cells. The phosphorylation of these downstream signaling molecules was suppressed in a dose-responsive fashion by AG1296. AG1296 inhibited IL-3 independent growth and induced apoptosis in Ba/F3 cells transformed by FLT3/ITDs. AG1296 also inhibited FLT3 auto-phosphorylation, and induced a cytotoxic effect, in primary AML cells. These findings suggest that inhibiting the activity of FLT3 may have a therapeutic value in some leukemias expressing FLT3/ITDs.