Aberrant Signal Transduction Research Articles

Deregulated Intracellular Signaling by Mutated c-CBL in Myeloid Neoplasms

c-CBL encodes a 120-kDa protein involved in intracellular signal transduction in a wide variety of cell types. Recently, frequent mutations of c-CBL have been reported in myeloid neoplasms showing both myelodysplastic and myeloproliferative features, in which most mutations are present in a homozygous state, as a result of allelic conversion in 11q. c-CBL has ubiquitin E3 ligase activity for a wide variety of tyrosine kinases, and thereby, negatively regulates tyrosine kinase signaling. Accordingly, c-CBL seems to have tumor suppressor functions, loss of which promotes tumorigenesis. On the other hand, once mutated, it is converted to an oncogenic protein and commits to myeloid leukemogenesis through a kind of gain of function causing aberrant signal transduction. The inhibition of mutant CBL protein or signaling pathways that it activates would have a role in therapeutics of myeloid neoplasms with CBL mutations.

MLK3 is critical for breast cancer cell migration and promotes a malignant phenotype in mammary epithelial cells

The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3's catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3's contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3-JNK-AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.

Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling

Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax toward attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction.

Palmitoylation stabilizes unliganded rod opsin

S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

Aberrant Signal Transduction and DNA Repair Pathways in Primary Lung Cancer Detected by Protein Profiling and their Association with Clinical Parameters

Background: Because cell signaling and cell metabolic pathways are executed through proteins, characterizing protein signatures in tumor tissues is required to identify key nodes in signaling networks whose alteration is associated with malignancy. This study aimed to determine protein signatures in primary lung cancer tissues. Methods and Findings: Case-matched normal and tumor samples from 101 lung cancer patients, most of them with non-small cell lung cancer, were analyzed with reverse-phase protein array assay for 127 proteins and/or protein phosphorylation sites. Differences in signal intensities between normal and tumor tissues and their association with clinical parameters were analyzed with analysis of variance, some of them were further validated by Western blot and/or immunohistochemical analyses. We found that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction, or lipid metabolism or function as cell surface/matrix proteins. Significant differences were found for 16 and 13 of the 18 molecules, respectively, when squamous cell carcinoma and adenocarcinoma were analyzed separately. The levels of some of those proteins were significantly higher either in tumors of nonsmokers than in those of smokers, or in poorly differentiated tumors than in well or moderately differentiated tumors, or a particular histopathological type of tumors, such as in neuroendocrinal tumors. The levels of 6 molecules could be used to distinguish tumor from the normal with the accuracy of 87%, whereas levels in eight molecules could separate patients into groups with poor or better survival outcomes. Conclusions: Our results demonstrated that molecules involved in DNA repair, signal transductions, lipid metabolism and cell proliferation were drastically aberrant in primary lung cancer tissues. Some of the protein biomarkers could be useful for molecular diagnosis or for predicting clinical outcomes of lung cancer. Aberrant Signal Transduction and DNA Repair Pathways in Primary Lung Cancer Detected by Protein Profiling and their Association with Clinical Parameters Yong He1, Zhen Zhou1, Wayne L. Hofstetter1, Yanbin Zhou1, Wenxian Hu1, Apar Pataer1, Arlene M. Correa1, Yiling Lu2, Jing Wang3, Lixia Diao3, Ignacio I. Wistuba4, Jack A. Roth1, Stephen G. Swisher1, Bingliang Fang1 1Department of Thoracic and Cardiovascular Surgery, 2Department of Systems Biology, 3Department of Bioinformatics and Computation Biology, 4Department of Pathology, The University of Texas M. D. Anderson Cancer Center, USA doi:10.4172/2155-9929.1000075

Single-Cell Biochemical Assays for the Molecular Targets of Disease

Molecularly targeted therapies are at the forefront of clinical science, and are expected to lead to personalization of medical treatments for each patient. Most such therapies are directed at inhibiting specific signal transduction enzymes or pathways, thus creating a critical need for assays capable of measuring the activities of these proteins in disease models and in patient samples. The ability to measure relevant enzyme activity in primary cell samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. Here we report our current efforts to create the analytical and chemical tools needed to directly measure the enzymatic activities of therapeutic targets including protein kinases, lipid modifying enzymes and the proteasome. Fluorescent reagents are under development that report the activity of these various enzymes in model cells lines and primary cells. The basic design incorporates enzyme substrates that are modified to create compounds which can be loaded into cells where they are modified by the enzyme of interest. Work has included modification of peptides to confer membrane permeability and to achieve long intracellular lifetimes. Microelectrophoretic separations combined with low-level fluorescence detection enable the quantitative analysis of these compounds from single mammalian cells. This capability addresses three major issues currently faced in the biochemical analysis of clinical samples: the need for direct measurement of the enzymatic activity of target proteins; sample size requirements that are feasible for clinical implementation; and sample heterogeneity that can mask pertinent aspects related to therapeutic response.

Distinct Signaling Profiles and Drug Responses Identify Subpopulations of Pediatric T-Cell Acute Lymphoblastic Leukemia and Lymphoma Patients.

Abstract 1595▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-621Intensive chemotherapy has dramatically improved cure rates for children with T-cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL). However, short-term and long-term toxicities have also increased while response rates to salvage therapy for relapsed patients remain dismal. Unfortunately, due to limits of technology and the paucity of clinical material available in children that often present with large mediastinal masses, biologic parameters for risk stratification of pediatric T precursor malignancies are limited relative to pre-B ALL. Thus, most T-ALL/LL patients are treated similarly on current protocols. Limited biologic features for risk stratification likely results in overtreatment of some children while precluding patients with high-risk disease from novel therapeutic approaches upfront. Since aberrant signal transduction is a key feature driving malignant cell behavior, we hypothesized that malignant T cells would possess distinct signaling networks relative to normal thymocytes and that differences in signaling profiles could be used to identify distinct subpopulations of T-ALL/LL patients at diagnosis. To test this hypothesis, we developed a phospho-flow cytometry platform to interrogate signaling networks at the single cell level in normal and malignant T precursor cells. Previous studies in mice and humans demonstrated that approximately 95% of thymocytes normally die before exiting the thymus. Since functional T cell receptor (TCR) and IL-7 signals are known to be critical for normal thymocyte survival, we reasoned that malignant T cells might retain dependence on these signaling networks. We thus optimized stimulation conditions with both sub-optimal and saturating concentrations of anti-TCR, anti-CD28 co-stimulation, IL-7, PMA, and pervanadate. Anti-sera to 15 key intracellular protein targets in these signaling networks were validated. To distinguish malignant from normal cells, cell surface staining for 8 epitopes that survive the fixation and permeabilization steps of the assay was established. Fluorescent cell bar coding was utilized for analysis of multiple samples in a single tube, enabling high-throughput screening while minimizing experimental variability and reagent use. Using this novel platform we simultaneously measured the phospho-protein response of 107 signaling nodes in the basal state and in response to stimulation in precursor T cell lines and a panel of banked patient samples. Analysis of pilot data identified the most robust and key signaling nodes for each of the major MAPK, PI3K, and JAK-STAT pathways. Given the limited number of cells in most tissue bank samples, we then focused the platform on the 30 most informative signaling nodes. Simultaneous measurement of pERK, pS6, pSTAT5, pBcl-2, Bcl-Xl, pNFkB, PTEN, intracellular Notch and PI3K levels in the basal state and with stimulation reveals T-ALL/LL patient subpopulations with distinct signaling profiles. Interestingly, a dichotomous pattern of either hypo- or hyper-responsiveness to stimulation is emerging. Measurement of patient signaling networks is also elucidating complex crosstalk between the classically separate MAPK and PI3K signaling pathways. Informed by the patient data, targeted inhibitors are being used to unveil the interdependence of these pathways in response to stimulation and their role in apoptosis. We have also extended this approach to analyze drug responses in patient samples in vitro at the single cell level. Using a Casapse 3 based flow-cytometry assay, we find distinct patterns of sensitivity to dexamethasone, methotrexate, and nelarabine in primary patient samples. The data demonstrates that flow cytometry represents a powerful approach for reliably interrogating the biologic properties of malignant T cells, reveals distinct subpopulations of T-ALL/LL patients that were previously unappreciated, may potentially discover synergistic uses of targeted inhibitors for therapy and perhaps even highlight novel drug targets. This approach warrants further study to determine whether these profiles have prognostic significance and whether in vitro drug responses could be used to predict outcome in the context of clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Jak2 Mutation in Myeloproliferative Neoplasms: Is It Just An Exon Episode?.

Abstract 4982The discovery of the V617F point mutation of JAK2 has revolutionized the understanding of the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs). Further discoveries of other JAK2 exon 12-15 and MPL mutations have extended the scope of the research in this field. However, a very small proportion of patients with polycythemia vera (PV) and 50% of patients with essential thrombocythemia (ET), as well as primary myelofibrosis (PMF), are negative for JAK2 or MPL mutations. This indicates that other forms of the mutations may exist and needs further evaluationRecently, we treated a patient with ET who did not carry JAK2 V617F or JAK2 exon 12 mutations. The genomic DNA of mononuclear cells from heparin-treated bone marrow as well as he gastric tissue from paraffin embedding samples obtained from the patient was extracted using a DNA extraction kit, according to the manufacturer's instruction (Promega, Madison, WI, USA), and stored at -20°C. For mutation analysis of the JAK2 exon 12 and exon 14, genomic DNA was amplified respectively by polymerase chain reaction. And the PCR products either 358bp or 308bp were generated by forward primers within the exon 12 or exon 14 and forward primers within the intron 12 or intron 14(primer sequences and PCR conditions are provided on request). The direct sequencing on ABI-3100-Avant was used for mutation detection.No mutation was found within JAK2 exon 12 or exon 14. However, a C/T mutation was detected in intron 14 at the position 14 downstream of the exon 14 in bone marrow cells. Informatively, this mutation was not found in genomic DNA of the gastric tissue. Further more, this novel mutation was not found in all samples from 59 MPN patients in our institute, among which 23 were diagnosed with PV, 22 ET, 12 PMF and 2 MPN-U. No such mutation was demonstrated in bone marrow mononuclear cells from 10 normal subjects. Preliminary results by flow cytometric analysis showed that hematopoietic cells including granulocytes, erythrocytes and megakaryocytes all showed elevated tyrosine kinase activity.We concluded that mutations in JAK2 mRNA splicing region might also contribute to the aberrant signal transduction in hematopoiesis. And direct sequencing for JAK2 whole genomic DNA might be a suitable method for finding novel JAK2 novel mutation in JAK2 negative patients. Further functional study for evaluation of the effect of nucleotide changes within mRNA splicing sequences in wild type and mutated JAK2 cell lines as well as animal models would provide more convincing evidence of the relationship between JAK2 intron mutation and MPNs.. DisclosuresNo relevant conflicts of interest to declare.

Targeted therapy in T-cell malignancies: dysregulation of the cellular signaling pathways

T-cell malignancies, mainly known as T-cell acute lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin's lymphoma (T-NHL), are aggressive tumors. Although the clinical outcome of the patients has improved dramatically with combination chemotherapy, significant challenges remain, including understanding of the factors that contribute to the malignant behavior of these tumor cells and developing subsequently optimal targeted therapy. Aberrant cell signal transduction is generally involved in tumor progression and drug resistance. This review describes the pathogenetic role of multiple cellular signaling pathways in T-cell malignancies and the potential therapeutic strategies based on the modulation of these key signaling networks.

Phase I Study of the c-raf-1 Antisense Oligonucleotide ISIS 5132 in Combination with Carboplatin and Paclitaxel in Patients with Previously Untreated, Advanced Non-small Cell Lung Cancer

A phase I trial was performed to evaluate the administration of carboplatin/paclitaxel in combination with ISIS-5132, a phosphorothioate antisense oligodeoxynucleotide inhibitor of c-raf-1 kinase expression, in patients with advanced non-small cell lung cancer (NSCLC). Previously untreated patients with stage IIIB/IV NSCLC received ISIS 5132 by continuous intravenous infusion at 2.0 mg/kg/d for 14 days. Starting doses were paclitaxel 175 mg/m(2) and carboplatin targeting an area under the free platinum plasma concentration-time curve (AUC(fp)) of 5 mg . min/ml (dose level 1). The carboplatin dose was then increased to AUC(fp) 6 mg . min/ml (dose level 2) after which the paclitaxel dose was increased to 200 mg/m(2) (dose level 3). The maximum tolerated dose was established by toxicity during the first two 21-day cycles of therapy. The pharmacokinetics of all three agents was determined before and during the ISIS 5132 infusion. Thirteen patients were treated with the carboplatin/paclitaxel/ISIS 5132 combination. Dose-limiting neutropenia occurred in two patients at dose level 3. Grade 3 and 4 nonhematologic toxicities were infrequent and limited to nausea and constipation. The maximum tolerated doses were carboplatin AUC(fp) 6 mg . min/ml, paclitaxel 175 mg/m(2), and ISIS 5132 2.0 mg/kg/d for 14 days. There were no objective responses and the concurrent infusion of ISIS 5132 did not alter the plasma pharmacokinetics of paclitaxel or total platinum. ISIS 5132 can be safely combined with standard doses of carboplatin and paclitaxel. Combining cytotoxic chemotherapeutic agents with inhibitors of aberrant signal transduction mediated by Raf proteins produced no objective responses in the dose and schedule administered in this study.

Dynamic visualization of signal transduction in living cells: From second messengers to kinases

The study of signal transduction, or the highly regulated series of biochemical events which allow a cell to convert a given stimulus into a functional response, has seen a paradigm shift with a recent explosion in the number of genetically encoded FRET-based biosensors capable of detecting spatial and temporal regulation of various signaling events in living cells. The two classes of biosensors discussed, namely kinase activity and second messenger biosensors, utilize two fluorescent proteins (FP) suitable for FRET and convert a signaling event of interest into a conformational change in the biosensor that can be measured as a change in FRET between the two FPs. Individually, these biosensors have been used to elucidate many complex signal transduction mechanisms in various biological systems. However, it has become increasingly clear that it is often more desirable to study multiple signaling events simultaneously, allowing for precise correlation of the temporal profiles of multiple signaling molecules without the complication of cell to cell variability. With the design of spectrally distinct biosensors and new coimaging strategies, simultaneous imaging of multiple signaling events is not only possible, but has aided in mapping the intricate network of cellular signal transduction cascades. Furthermore, as aberrant signal transduction involving second messengers and kinases is implicated in numerous disease states, it is hopeful that these FRET-based biosensors and coimaging strategies can help to unravel the molecular links between altered signal transduction and certain disease states.

Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.

Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells

Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

Novel postremission strategies in adults with acute myeloid leukemia.

Given the high rates of relapse in acute myeloid leukemia (AML), there is tremendous opportunity for the development of new therapeutic strategies in the postremission state. Unfortunately, the currently available modalities for postremission therapy, namely chemotherapy, have proven largely ineffective in changing the natural history of AML. The challenges to overcome therapeutic failure in the minimal residual disease status may relate to an incomplete understanding of the mechanisms and cell populations that are directly related to disease relapse as well as suboptimal ability to identify patients at highest risk for relapse. Being a heterogeneous disease, relapsed AML is unlikely to emanate from one predominant mechanism; instead, there are likely multiple biologic factors at play that allow for clinical relapse to occur. These factors likely include multidrug resistance proteins, aberrant signal transduction pathways, survival of leukemia stem cells, microenvironmental interactions, and immune tolerance. Many novel strategies are in development that target these mechanisms, ranging from chemotherapeutic modalities, to signal transduction inhibitors, to upregulation of antileukemic immune responses. Understanding the underlying mechanisms of leukemic cell survival and resistance has spurred the development of novel therapeutic approaches to overcome these mechanisms in the hope of eradicating minimal residual disease and improving survival in AML.

Activity of the BH3 Mimetic ABT-737 on Polycythemia Vera (PV) Erythroid Precursor Cells

Malignant hemopoietic cells are often characterized by ineffective death pathways, resulting in enhanced resistance to apoptosis and ultimately in the survival/expansion of the abnormal clone. Mechanistic studies have been undertaken to identify the aberrant signal transduction pathway and to develop small-molecule inhibitors targeting deregulated modules. Regulators of apoptotic pathways play a key role in the control of erythroid cell expansion; in particularl Bcl-XL is essential for erythroid cell development and, together with Bcl-2, it protects erythroblast survival from cytotoxic stimuli. Previous studies on erythroid cells derived from polycythemia vera (PV) patients have revealed an increased expression of Bcl-XL associated with cell survival in the absence of erythropoietin. The discovery of the JAK2V617F mutation in the vast majority of PV patients and its association with an increased resistance to apoptosis induced by death receptors prompted us to investigate the correlation between JAK2V617F and the expression of anti-apoptotic Bcl-2 family members and to explore the activity of a Bcl-2 inhibitor on PV erythroid cells. ABT-737 is a synthetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-XL, poorly to Mcl-1, promoting apoptosis as single agent in malignant hemopoietic cells. In this study we analyzed pure populations of primary erythroid precursors obtained from CD34+ cells of healthy donors and PV patients to investigate the expression of Bcl-2, Bcl-XL and Mcl-1. We found that the expression of Bcl-XL and Bcl-2 was increased in PV patients compared to controls, while Mcl-1 levels did not significantly differ. Then we analyzed Bcl-2 and Bcl-XL expression in erythroblasts derived from PV patients clustered on the basis of a different JAK2V617F allele burden. We found that on average the expression of both Bcl-2 and Bcl-XL was comparable to controls in PV erythroblasts with low/null mutation rates while both proteins were significantly (P< 0.001 and P<0.05, respectively) overexpressed in those with a high percentage of mutated JAK2. Subsequently, we investigated whether ABT-737 was effective in inducing apoptosis in normal and PV erythroblasts. A dose-response induction of apoptosis was elicited by ABT- 737 in PV erythroblasts with elevated JAK267F allele burden with higher effects than in normal cells starting from 500nM (P<0.05). The preferential effects of ABT-737 on PV erythroblasts were further documented by differences in ABT-737-induced mitochondrial depolarization and caspase cleavage. Treatment with ABT-737 did not induce significant alterations in cell cycle distribution on both PV and normal erythroid cells. The activity of ABT-737 was then studied in semisolid cultures. BFU-E colony formation was inhibited in PV-derived CD34+ cells but not in normal controls. To investigate the mechanisms underlying ABT-737 inhibition of erythroblast expansion we analyzed GATA-1 levels that resulted significantly (P<0.05) decreased in PV erytroblasts but not in normal cells, indicating a differential extent of caspase-mediated degradation of GATA-1. Taken together these results indicate that ABT-737 promotes apoptosis of mutated JAK2 PV erythroid cells with a preferential activity compared to normal erythroid cells, supporting a potential therapeutic role of this molecule in patients with PV.

Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement

Aberrant GM-CSF signal transduction pathway in juvenile myelomonocytic leukemia assayed by flow cytometric intracellular STAT5 phosphorylation measurement

Aberrant signal transduction pathways in myeloproliferative neoplasms

The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.

A Novel Functional Screen in Human Cells Identifies MOCA as a Negative Regulator of Wnt Signaling

Aberrant Wnt signal transduction is involved in many human diseases such as cancer and neurodegenerative disorders. The key effector protein of the canonical Wnt pathway is beta-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate gene transcription that leads to expression of Wnt target genes. In this study we provide results obtained from a novel functional screen of a human brain cDNA library used to identify 63 genes that are putative negative Wnt regulators. These genes were divided into eight functional groups that include known canonical and noncanonical Wnt pathway components and genes that had not yet been assigned to the Wnt pathway. One of the groups, the presenilin-binding proteins, contains the modifier of cell adhesion (MOCA) gene. We show that MOCA is a novel inhibitor of Wnt/beta-catenin signaling. MOCA forms a complex with beta-catenin and inhibits transcription of known Wnt target genes. Epistasis experiments indicate that MOCA acts to reduce the levels of nuclear beta-catenin, increase the levels of membrane-bound beta-catenin, and enhances cell-cell adhesion. Therefore, our data indicate that MOCA is a novel Wnt negative regulator and demonstrate that this screening approach can be a rapid means for isolation of new Wnt regulators.

Deregulation of Signaling Pathways in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), aberrant signal transduction enhances the survival and proliferation of hematopoietic progenitor cells. Activation of signal transduction in AML may occur through a variety of genetic alterations affecting different signaling molecules, such as the FLT3 and KIT receptor tyrosine kinases (RTKs) and members of the RAS family of guanine nucleotide-binding proteins. These mutant signaling proteins are attractive therapeutic targets; however, developing targeted therapies for each genotypic variant and determining the relationships between different genotypes and critical functional dependencies of the leukemic cells remain major challenges. As the large number of mutant signaling proteins that have been identified in AML are likely to reflect activation of a more limited number of downstream effector pathways, such as the RAF/MEK/ERK and PI3K/AKT cascades, targeting these unifying pathways may represent a more broadly applicable therapeutic strategy. Furthermore, integrative genomic studies combining DNA sequencing, DNA copy number analysis, transcriptional profiling, and functional genetic approaches hold great promise for identifying additional signaling abnormalities in AML that are relevant to leukemogenesis and can be exploited therapeutically. Eventually, it may become possible to use pathogenesis-oriented combinations of signal transduction inhibitors to improve the cure rate in AML patients.

Update on targeted therapies for small cell carcinoma of the lung

Small cell lung cancer (SCLC) is an aggressive malignancy with a poor prognosis. It can be very sensitive to radiation and cytotoxic chemotherapy and is potentially curable if all sites of disease can be encompassed in a radiation field. For the vast majority of patients relapse is the norm, with only limited gains from further lines of chemotherapy. In spite of the recognition of a number of molecular targets based on preclinical studies, targeted therapy for small cell lung cancer has yet to demonstrate any significant clinical benefit. This review summarizes the various targeted pharmacological approaches employed with a view to extending survival in extensive stage SCLC or improving on remission induction in limited stage disease. Pharmacological agents have targeted aberrant signal transduction pathways, proteins involved in cell cycle progression, apoptosis and angiogenesis. Additionally, immunomodulatory approaches have been used to stimulate an immune response against tumor associated antigens. Though the clinical data thus far is discouraging, drugs are still undergoing evaluation and more targets continue to be identified. There is considerable scope for further research and in particular combining newer therapies with existing treatment modalities to improve outcomes.