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PubMed1997

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Animals↗

PubMed2005

Biphenylsulfonacetic acid inhibitors of the human papillomavirus type 6 E1 helicase inhibit ATP hydrolysis by an allosteric mechanism involving tyrosine 486.

Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts.

Acetates↗

PubMed2000

Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo.

Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.

3' Untranslated Regions↗

PubMed2005

Mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer.

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.

Codon↗

PubMed2001

Lipopolysaccharide endotoxins.

Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.

ATP-Binding Cassette Transporters↗

PubMed2006

Subtractive hybridization for differential gene expression in mechanically unloaded rat heart.

The objective of this study was to identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. In male Wistar-Kyoto rats, mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by Northern blot analysis, real-time RT-PCR, and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Transplantation caused atrophy (heart-to-body weight ratio: 1.6 +/- 0.1 vs. 2.4 +/- 0.1, P < 0.001). We selected 1,880 clones from the subtractive hybridization procedure (940 forward and 940 reverse runs assessing up- or downregulation). The first screen verified 465 forward and 140 reverse clones, and the second screen verified 67 forward and 30 reverse clones. On sequencing of 24 forward and 23 reverse clones, 9 forward and 14 reverse homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (-49%, P < 0.05) and complex II (-61%, P < 0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level: -42% for complex I (P < 0.01), -57% for complex II (P < 0.05), and -65% for complex IV (P < 0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, with glutamate and succinate as substrates: 85 +/- 27 vs. 224 +/- 32 natoms O.min(-1).mg(-1) with glutamate (P < 0.01) and 59 +/- 18 vs. 154 +/- 30 natoms O.min(-1).mg(-1) with succinate (P < 0.05). Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.

Animals↗

PubMed2004

Functional genomics approach to hypoxia signaling.

Mammalian cells require a constant supply of oxygen to maintain energy balance, and sustained hypoxia can result in cell death. It is therefore not surprising that sophisticated adaptive mechanisms have evolved that enhance cell survival during hypoxia. During the past few years, there have been a growing number of reports on hypoxia-induced transcription of specific genes. In this review, we describe a unique experimental approach that utilizes focused cDNA libraries coupled to microarray analyses to identify hypoxia-responsive signal transduction pathways and genes that confer the hypoxia-tolerant phenotype. We have used the subtractive suppression hybridization (SSH) method to create a cDNA library enriched in hypoxia-regulated genes in oxygen-sensing pheochromocytoma cells and have used this library to create microarrays that allow us to examine hundreds of genes at a time. This library contains over 300 genes and expressed sequence tags upregulated by hypoxia, including tyrosine hydroxylase, vascular endothelial growth factor, and junB. Hypoxic regulation of these and other genes in the library has been confirmed by microarray, Northern blot, and real-time PCR analyses. Coupling focused SSH libraries with microarray analyses allows one to specifically study genes relevant to a phenotype of interest while reducing much of the biological noise associated with these types of studies. When used in conjunction with high-throughput, dye-based assays for cell survival and apoptosis, this approach offers a rapid method for discovering validated therapeutic targets for the treatment of cardiovascular disease, stroke, and tumors.

Animals↗

PubMed2006

ADAMTS1, CRABP1, and NR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis.

BACKGROUND: Gene silencing through CpG island hypermethylation is a major mechanism in cancer development. In the present study, we aimed to identify and validate novel target genes inactivated through promoter hypermethylation in colorectal tumor development. METHODS: With the use of microarrays, the gene expression profiles of colon cancer cell lines before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine were identified and compared. The expression of the responding genes was compared with microarray expression data of primary colorectal carcinomas. Four of these down-regulated genes were subjected to methylation-specific PCR, bisulphite sequencing, and quantitative gene expression analysis using tumors (n=198), normal tissues (n=44), and cell lines (n=30). RESULTS: Twenty-one genes with a CpG island in their promoter responded to treatment in cell lines, and were simultaneously down-regulated in primary colorectal carcinomas. Among 20 colon cancer cell lines, hypermethylation was subsequently identified for three of four analyzed genes, ADAMTS1 (85%), CRABP1 (90%), and NR3C1 (35%). For the latter two genes, hypermethylation was significantly associated with absence or reduced gene expression. The methylation status of ADAMTS1, CRABP1, and NR3C1 was further investigated in 116 colorectal carcinomas and adenomas. Twenty-three of 63 (37%), 7/60 (12%), and 2/63 (3%) adenomas, as well as 37/52 (71%), 25/51 (49%), and 13/51 (25%) carcinomas were hypermethylated for the respective genes. These genes were unmethylated in tumors (n=82) from three other organs, prostate, testis, and kidney. Finally, analysis of normal colorectal mucosa demonstrated that the observed promoter hypermethylation was cancer-specific. CONCLUSION: By using a refined microarray screening approach we present three genes with cancer-specific hypermethylation in colorectal tumors, ADAMTS1, CRABP1, and NR3C1.

ADAM Proteins↗

PubMed2006

The isopeptidase USP2a protects human prostate cancer from apoptosis.

Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death-related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer.

Apoptosis↗

PubMed2004

Curtailing the high rate of late-stage attrition of investigational therapeutics against unprecedented targets in patients with lung and other malignancies.

A greater understanding of the pathogenesis and biology of cancer coupled with major advances in biotechnology has resulted in the identification of rationally designed, target-based (RDTB) anticancer therapeutics, ushering in new therapeutic opportunities and high expectations for the future as well as developmental challenges. Because these agents appear to principally target malignant cells, it is expected that they will produce less toxicity at clinically effective doses than nonspecific cytotoxic agents, but their target requirements are likely to be much more stringent. The innate complexity of the networks that contain elements targeted by these agents also decreases the probability that any single therapeutic manipulation will result in robust clinical activity and success when used alone, particularly in patients with solid malignancies that have multiple relevant signaling aberrations. In contrast, proof of principle and robust antitumor activity may be most efficiently demonstrated in nonrandomized evaluations involving tumors that are principally driven by aberrations of the specific target. The predominant therapeutic manifestation of RDTB agents in preclinical studies is due to decreased tumor growth rates and will likely be similar in the clinic; however, such manifestations are not readily detectable and quantifiable using nonrandomized clinical evaluations. To curtail the increasing rate of late-stage attrition of RDTB agents, which, if maintained, will stymie progress in cancer therapy, the design of initial nonrandomized evaluations, particularly the selection of tumors and patients, must be guided by the principal biological features of the agents. Next, evaluations, some of which must be randomized, can be performed in a wide range of tumor types, depending on the presence and relevance of the target. To validate the concept of RDTB therapeutics and to realize their full potential, radically different development, evaluation, and regulatory paradigms must be adopted.

Antineoplastic Agents↗

PubMed2004

Pharmacogenomic analysis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib.

PURPOSE: To better understand the molecular basis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib, we studied gene expression profiles from a total of 100 patients from a large, multinational Phase III clinical trial (International Randomized Study of IFN-alpha versus STI571). EXPERIMENTAL DESIGN: Gene expression data for >12,000 genes were generated from whole blood samples collected at baseline (before imatinib treatment) using Affymetrix oligonucleotide microarrays. Cytogenetic response was determined based on the percentage of Ph(+) cells from bone marrow following a median of 13 months of treatment. RESULTS: A genomic profile of response was developed using a subset of individuals that exhibited the greatest divergence in cytogenetic response; those with complete response (0% Ph(+) cells; n = 53) and those with minimal or no response (>65% Ph(+) cells; n = 13). A total of 55 genes was identified that were differentially expressed between these two groups. Using a "leave-one-out" strategy, we identified the optimum 31 genes from this list to use as our genomic profile of response. Using this genomic profile, we were able to distinguish between individuals that achieved major cytogenetic response (0-35% Ph(+) cells) and those that did not, with a sensitivity of 93.4% (71 of 76 patients), specificity of 58.3% (14 of 24 patients), positive predictive value of 87.7%, and negative predictive value of 73.7%. CONCLUSIONS: Interestingly, many of the genes identified appear to be strongly related to reported mechanisms of BCR-ABL transformation and warrant additional research as potential drug targets. The validity and clinical implications of these results should be explored in future studies.

Antineoplastic Agents↗

PubMed2004

Synergistic interactions between tamoxifen and trastuzumab (Herceptin).

PURPOSE: HER-2/neu and estrogen receptor (ER) are critical in the biology of breast carcinoma, and both are validated therapeutic targets. Extensive interactions between the signaling pathways of these receptors have been demonstrated. This suggests that targeting both receptors simultaneously may have a dramatic effect on the biology of breast cancer. This hypothesis was tested in cell culture experiments. EXPERIMENTAL DESIGN: ER-positive, HER-2/neu-overexpressing BT-474 human breast carcinoma cells were cultured in the presence of the anti-HER-2/neu therapeutic antibody trastuzumab (Herceptin), the antiestrogen tamoxifen, or both. The effects on cell growth, cell cycle distribution, clonogenicity, survival, and the level and activity of HER-2/neu were examined. RESULTS: The combination of tamoxifen and Herceptin resulted in synergistic growth inhibition and enhancement of cell accumulation in the G(0)-G(1) phase of the cell cycle, with a decrease in cells in S phase. Clonogenicity was inhibited in the presence of each drug and more so by the combination, although prior exposure to drugs did not affect subsequent clonogenicity in drug-free media, and neither drug nor the combination induced apoptosis. Herceptin, but not tamoxifen, inhibited signaling by HER-2/neu. CONCLUSIONS: The combination of tamoxifen and Herceptin is formally demonstrated to result in synergistic growth inhibition and enhancement of G(0)-G(1) cell cycle accumulation. In vitro, the individual drugs or combination produces a cytostatic effect. These results suggest that combined inhibition of ER and HER-2/neu signaling may represent a powerful approach to the treatment of breast cancer.

Antibodies, Monoclonal↗

PubMed2003

Stent-induced expression and activation of the leukocyte integrin Mac-1 is associated with neointimal thickening and restenosis.

BACKGROUND: Increased expression of the beta2 integrin Mac-1 (CD11b/CD18, alphaMbeta2), which is responsible for firm leukocyte adhesion to platelets and fibrinogen at injured vessels, is found in association with neointimal hyperplasia after coronary interventions. The role of Mac-1 in the pathophysiology of restenosis is incompletely defined. To clarify further the role of Mac-1, we determined whether coronary stenting induced activation of Mac-1, which is required for high-affinity receptor-ligand interactions. METHODS AND RESULTS: Expression of CD11b (alpha-subunit of Mac-1) and binding of 8B2 (monoclonal antibody against an activation-dependent neoepitope of Mac-1) on the surface of polymorphonuclear leukocytes were analyzed in 62 patients undergoing coronary stenting using flow cytometric analysis of whole blood obtained from the coronary sinus and femoral vein. Transcardiac CD11b expression increased significantly at 24 hours and maximally at 48 hours after stenting; 8B2 began to increase at 10 minutes and was maximally increased at 48 hours after stenting. These changes were more prominent in patients with subsequent restenosis. Multiple regression analysis showed that the late lumen loss by quantitative coronary angiographic analysis was independently correlated with the CD11b increase (R=0.42, P<0.01) and the 8B2 increase (R=0.55, P<0.001) 48 hours after the procedure. Mac-1 activation, as assessed by 8B2 binding, was the most powerful predictor of late lumen loss. CONCLUSIONS: Coronary stenting produced upregulation and early activation of the leukocyte integrin Mac-1, which is associated with late lumen loss and restenosis. These data support a role for inflammation in neointimal thickening and suggest the validity of targeting leukocyte recruitment for preventing clinical restenosis.

Angioplasty, Balloon, Coronary↗

PubMed2026

Downregulation of Trpv4 and Klf2 in brain microvessels is associated with the progression of neurovascular dysfunction and cognitive impairment in a model of heart failure with preserved ejection fraction.

Vascular cognitive impairment (VCI) shares major risk factors with heart failure with preserved ejection fraction (HFpEF), including obesity, diabetes and hypertension. Yet VCI research often relies on single-stimulus models, whereas patients experience combined risk factors. We therefore assessed cerebrovascular and cognitive phenotypes in an HFpEF model and investigated underlying mechanisms. Male Lean and Obese ZSF1 rats underwent longitudinal assessments of blood pressure, glucose, cardiac function and behavioural performance. Cerebral blood flow and neurovascular coupling were assessed by laser speckle contrast imaging. White matter integrity, blood-brain barrier (BBB) permeability and vascular density were analyzed by (immuno)histochemistry. Cortical microvessels were isolated for transcriptomic profiling, and selected targets were validated using multiplex in-situ hybridization. Obese rats exhibited neurovascular uncoupling and impaired short- and long-term memory and spatial learning, accompanied by brain atrophy and reduced myelin. BBB permeability increased at 22-23 weeks and vascular density at 34-35 weeks in Obese versus Lean rats. Transcriptomic analysis of brain microvessels revealed altered processes related to angiogenesis, vasoreactivity, immune mechanisms and vascular remodelling, with consistent downregulation of Trpv4 and Klf2. Obese ZSF1 rats develop progressive neurovascular dysfunction associated with HFpEF onset and reduced Trpv4 and Klf2 expression in cerebral microvessels, two key vasoprotective genes.

Diastolic dysfunction↗

PubMed2000

Anticonvulsant blood levels: historical review with a pediatric focus.

Epilepsy is heterogeneous and its treatment is often complicated by variable drug responses. Buchtal et al reported a close correlation between serum phenytoin levels, electroencephalographic findings, and clinical status in 1960. They suggested that physicians adjust dosage to attain a "therapeutic level." The concept was enthusiastically received. "Therapeutic serum levels" were proposed for most anticonvulsant drugs, and by 1975, most authorities believed that pharmacokinetic factors explained individual differences in drug response. However, Froscher found that measuring levels did not improve patient outcome. More recently, Schumacher's double-blind study found no correlation between phenytoin levels and seizure control or adverse effects. Pharmacodynamic variables (differences in drug responsiveness) are more important than pharmacokinetic factors for many drugs, especially receptor-active drugs. Pharmacokinetic variables were studied first, and led to a simplistic model. They are less significant than pharmacodynamic factors in the case of warfarin anticoagulation. Anticonvulsant levels can reveal noncompliance and pharmacokinetic differences. They say nothing about pharmacodynamics. Reports of "subtherapeutic levels" imply a need to increase dosage, but this is not supported by outcome data. We still lack evidence that specific drug levels are a valid intermediate target 40 years after Buchtal's paper. Responses to some anticonvulsants could depend primarily on pharmacokinetic factors, while pharmacodynamic factors could be supreme for others.

Anticonvulsants↗

PubMed2006

Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161.

Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs.

Acetylation↗

PubMed2006

Classification, diagnosis and management of myeloproliferative disorders in the JAK2V617F era.

JAK2V617F, a somatic gain-of-function mutation involving the JAK2 tyrosine kinase gene, occurs in nearly all patients with polycythemia vera (PV) but also in a variable proportion of patients with other myeloid disorders; mutational frequency is estimated at approximately 50% in both essential thrombocythemia (ET) and myelofibrosis (MF), up to 20% in certain subcategories of atypical myeloproliferative disorder (atypical MPD), less than 3% in de novo myelodysplastic syndrome (MDS) or acute myeloid leukemia, and 0% in chronic myeloid leukemia (CML). Accordingly, there is now molecular justification for grouping PV, ET, and MF together in a distinct MPD category (i.e., classic, BCR-ABL(-) MPD) that is separate from chronic myeloid leukemia (CML), MDS, and atypical MPD. To date, JAK2V617F has not been described in patients with reactive myeloproliferation, lymphoid disorders, or solid tumor. Therefore, the presence of JAK2V617F strongly suggests an underlying MPD and it is therefore reasonable to consider JAK2V617F-based laboratory tests for the evaluation of polycythemia, primary thrombocytosis, unexplained leukocytosis, bone marrow fibrosis, or abdominal vein thrombosis. Current information on disease-specific prognostic relevance of JAK2V617F is inconclusive and confounded by inter-study differences in the performance of mutation screening assays. Regardless, the discovery of JAK2V617F has reinforced the pathogenetic contribution of JAK-STAT signaling in MPD and identifies JAK2 as a valid drug target.

Disease Management↗

PubMed2006

ARACNE: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context.

BACKGROUND: Elucidating gene regulatory networks is crucial for understanding normal cell physiology and complex pathologic phenotypes. Existing computational methods for the genome-wide "reverse engineering" of such networks have been successful only for lower eukaryotes with simple genomes. Here we present ARACNE, a novel algorithm, using microarray expression profiles, specifically designed to scale up to the complexity of regulatory networks in mammalian cells, yet general enough to address a wider range of network deconvolution problems. This method uses an information theoretic approach to eliminate the majority of indirect interactions inferred by co-expression methods. RESULTS: We prove that ARACNE reconstructs the network exactly (asymptotically) if the effect of loops in the network topology is negligible, and we show that the algorithm works well in practice, even in the presence of numerous loops and complex topologies. We assess ARACNE's ability to reconstruct transcriptional regulatory networks using both a realistic synthetic dataset and a microarray dataset from human B cells. On synthetic datasets ARACNE achieves very low error rates and outperforms established methods, such as Relevance Networks and Bayesian Networks. Application to the deconvolution of genetic networks in human B cells demonstrates ARACNE's ability to infer validated transcriptional targets of the cMYC proto-oncogene. We also study the effects of misestimation of mutual information on network reconstruction, and show that algorithms based on mutual information ranking are more resilient to estimation errors. CONCLUSION: ARACNE shows promise in identifying direct transcriptional interactions in mammalian cellular networks, a problem that has challenged existing reverse engineering algorithms. This approach should enhance our ability to use microarray data to elucidate functional mechanisms that underlie cellular processes and to identify molecular targets of pharmacological compounds in mammalian cellular networks.

Algorithms↗