Cystic Fibrosis and the Use of Pharmacogenomics to Determine Surrogate Endpoints for Drug Discovery

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  • Cystic Fibrosis and the Use ofPharmacogenomics to DetermineSurrogate Endpoints for Drug DiscoveryOfer Eidelman, Jian Zhang, Meera Srivastava and Harvey B. PollardDepartment of Anatomy, Physiology and Genetics, and Institute for Molecular Medicine, Uniformed Services UniversitySchool of Medicine, USUHS, Bethesda, Maryland, USA

    ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2231. The Problem of Cystic Fibrosis (CF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2242. The Potential for Application of Pharmacogenomics to CF Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2243. Mechanisms of CF Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

    3.1 Inefficient Trafficking of the Mutant CF Transmembrane Conductance Regulator (CFTR) . . . . . . . . . . . . . . . . . 2253.2 Intrinsic Propensity for Inflammation and Infection in the CF Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2263.3 IL-8 Levels in CF Lung Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

    4. Mechanisms of CPX Therapeutic Effects in CF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2265. Other Potential CF Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

    5.1 CFTR-Interacting Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2275.2 The NFB Signaling Pathway Regulates IL-8 Expression in Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

    6. CF Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2316.1 Pharmacogenomic Data Can be Used to Mine Out Drug- or Gene-Dependent Signaling Pathways . . . . . . . . . . 2316.2 Application of GRASP and GENESAVER Algorithms to Pharmacogenomic Analysis of Cystic Fibrosis . . . . . . . . . . . 232

    6.2.1 The GRASP Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2326.2.2 The GENESAVER Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

    7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

    Abstract Cystic fibrosis (CF) is caused by a mutation in the CFTR gene, encoding a chloride channel. For themost common mutation, F508, the basis of the deficit is the failure of the mutant CFTR channel proteinto traffic properly to the apical plasma membrane of the affected epithelial cell. The trafficking failureresults in loss of the cyclic adenosine monophosphate (cAMP)-activated chloride channel function of theCFTR protein in the plasma membrane. The lung is the principal site affecting patient morbidity andmortality in CF. The main reason is that the CF airway epithelial cells also secrete high levels of theproinflammatory cytokine interleukin (IL)-8, resulting in massive cellular inflammation, infection, tissuedamage and lung destruction. The relationship between the trafficking defect, the loss of chloride channelactivity, and inflammation is not known. However, gene therapy of CF lung epithelial cells with thewild-type CFTR gene can repair the chloride channel defect, as well as suppress the intrinsic hypersecretionof IL-8. Repair of both defective channels and high IL-8 secretion can also be effected by treatment withthe candidate CF drug CPX, which is in clinical trials in CF patients. CPX acts by binding to the mutantCFTR protein, and helps the protein to mature and gain access to the plasma membrane. CPX alsosuppresses the synthesis and secretion of IL-8 from CF epithelial cells, presumably by virtue of its repairof the trafficking defect of mutant CFTR. To guide pharmacogenomic experiments we have thereforehypothesized that the genomic signature of CF epithelial cells treated with CPX should resemble thesignature of the same cells repaired by gene therapy. We have developed two algorithms for identifyinggenes modified by repair of CFTR defects. The GRASP algorithm uses a statistical test to identify the mostprofoundly changing genes. The GENESAVER algorithm allows us to identify those genes whose patternof expression changes in-phase or out-of-phase with IL-8 secretion by CF cells. For the latter algorithm we

    GENOMICS IN DRUG DEVELOPMENT Am J Pharmacogenomics 2001; 1 (3): 223-2381175-2203/01/0003-0223/$22.00/0 Adis International Limited. All rights reserved.

  • modified IL-8 secretion from CF cells by treatment with wild-type CFTR, with CPX, or by exposure tobacteria. The results have supported the hypothesis, and have provided a basis for considering the commonpharmacogenomic expression signature as a surrogate endpoint for CF drug discovery. Significantly, thenature of the hypothesis, as well as the algorithm developed for this study, can be easily applied to phar-macogenomic studies with other goals.

    1. The Problem of Cystic Fibrosis (CF)

    Cystic fibrosis (CF) is the most common inherited autosomalrecessive lethal disease in the United States.[1] Approximately5% of the population carries one mutant cystic fibrosis trans-membrane conductance regulator (CFTR) gene[2-4] and the dis-ease occurs in a frequency of 1 in 2500 live births. Statistically,death occurs in the majority of patients by age 28. Until veryrecently, few afflicted patients survived past early childhood,death often occurring at birth due to intestinal blockage by me-conium ileus. Those who survived later died from nutritionaldeficiencies.[1] The principal reason was that the pancreaticducts became blocked by mucins, thereby preventing pancre-atic enzymes from gaining access to the small intestine. Nutri-tional deficiencies also ensued from blockage of the bile duct,which resulted in inability to recover dietary fats and vitamins.Finally, blockage of the respiratory tract by thick mucous se-cretions led to death from bacterial infections and gross loss ofability to oxygenate the blood.

    Even though CF is a single gene disease, it seems to havepleiotropic physiological consequences. These consequencesinclude effects on a multitude of different cellular functionsincluding the transmembrane transport of Cl- and Na+, Na/Hexchange, mucin secretion, and abnormal inflammatory signal-ing. CF therapy has traditionally been geared towards treatingthe symptoms of the disease. Presently, surgery is often used totreat meconium ileus, and pancreatic enzyme supplements haveprovided an effective mechanism for limiting digestive defects.In addition, physical therapy, DNAse I,[5] ibuprofen,[6] and an-tibiotic therapies have been developed to help treat the obstruc-tive disease in the lung. Whole or partial lung transplants havealso been helpful, although there is a limited transplantationsource. Nonetheless, at the present time the respiratory diffi-culties and ensuing complications of inflammation and lunginfection are directly responsible for the eventual death of over90% of patients with CF at an average age of 28 years.

    The principal basis of CF pathology in the lung seems to bean intrinsically activated proinflammatory process, which isindependent of subsequently acquired infections. The CF air-way shows a great increase in proinflammatory signaling mol-ecules, including interleukin (IL)-8, tumor necrosis factor(TNF)-, and others (see section 3.2 and references 7-10 for

    details). These molecules appear to come initially from the lungepithelial cells, but later come from other cellular constituentsof the inflammatory milieu. The molecular basis of this intrin-sic proinflammatory propensity is not presently known. Thereare also a number of other mutation-dependent defects in lungfunction,[1] including sodium conductance, mucociliary clear-ance and bacterial killing which we will not address here. How-ever, available data make it clear that reintroduction of a wild-type CFTR molecule by gene therapy is sufficient to abrogatethe defective CF intracellular signals, whatever they are (seereferences 11-14 and section 5 for details).

    2. The Potential for Application ofPharmacogenomics to CF Drug Discovery

    In an effort to use the principles of pharmacogenomics todiscover pharmaceuticals that might be useful in treating CF,we have hypothesized that an efficient CF pharmaceuticalshould cause the functional genomics of the CF cell to resemblethat of the wild-type CFTR-repaired CF cell.[15,16] This phar-macogenomic pattern of repair would thus constitute a surro-gate endpoint for drug discovery. It should be emphasized thatsuch an anticipated result has had plenty of predecessors in thegeneral field of pharmacogenomics. Both Bailey et al.[17] andFerrar[18] have predicted that the genetic consequences of ef-fective gene or drug therapy should be assessed at the cellularlevel in terms of specific patterns of global gene expression.Graever et al.[19] have suggested that at the very simplest level,the expectation is that specific patterns of gene expression willbe discerned that could be employed as biologically relevantsurrogate endpoints when searching for more effective drugsor gene targets.

    In its simplest manifestation, this approach should providea composition list for a gene-chip containing the signaturegenes, which, in their ensemble, would be capable of indicatingthe repair of CF defects by a candidate CF drug. One tacticaladvantage to this approach to CF pharmacogenomics is that wealready possess several lead compounds, including the aden-osine A1 receptor antagonists CPX, DAX and certain otherxanthines.[20-22] CPX is currently in clinical trials, and is there-fore available for proof-of-concept experiments. Our initialstudies have identified genes from the TNF receptor (TNFR)/

    224 Eidelman et al.

    Adis International Limited. All rights reserved. Am J Pharmacogenomics 2001; 1 (3)

  • NFB pathway, among others, as important for the proinflamma-tory condition of CF.[16] Yet, not all the delineated genes have anobvious relationship to either CFTR function or to proinflamma-tory processes.

    In this review, we shall first discuss the molecular details un-derlying the function of CFTR in airway epithelial cells, and theactions of CPX on the function of mutant CFTR. We presume thatknowledge of such details may help in interpreting the identifiedgenes and in discovering new ones. In the last part of the review,we shall describe two algorithms, gene ratio analysis paradigm(GRASP) and gene space vector (GENESAVER), which wehave developed in order to mine out the potentially dysfunctionalgenes in a hypothesis-driven manner. We believe both algorithmsmay prove to be of general utility to the pharmacogenomic com-munity.

    3. Mechanisms of CF Pathogenesis

    3.1 Inefficient Trafficking of the Mutant CFTransmembrane Conductance Regulator (CTFR)

    In order to understand how mutant CFTR might cause theintrinsically proinflammatory phenotype of cystic fibrosis, atten-tion should be focused on the exact function of CFTR, and what

    changes occur to CFTR function when the gene is mutated.

    Briefly, the CFTR gene product functions as a cyclic adenosinemonophosphate (cAMP)/protein kinase A (PKA)/adenosine tri-

    phosphate (ATP)-activated chloride channel.[11,23-26] The CFTR

    channel is normally located in the apical membrane of epithelial

    cells.[27] The principal mutation responsible for cystic fibrosis isF508, which is found in approximately 90% of CF chromo-somes.[4,12,28,29] CFTR is a member of the ATP-binding cassette

    (ABC) transporter gene family, and this mutation is located

    within CFTRs first nucleotide binding fold domain (NBF-1).

    This domain has been previously assumed to reside in the cytosol,

    although current functional and crystallographic data suggest a

    partial membrane location.[30,31] The mutation causes only mod-

    est changes in chloride channel activity per se.[27,32,33] Rather, the

    F508 mutation causes the mutant CFTR to be retained in theendoplasmic reticulum and rapidly degraded by a proteosomal

    mechanism. Consequently, mutant CFTR fails to traffic properlyto the golgi and the plasma membrane.[34-37] The biochemical

    evidence for this trafficking arrest is that the mutant CFTR re-

    mains core-glycosylated, and fails to acquire complex N-linked

    oligosaccharides typical of the medial golgi activity.[34,38-41] It

    has therefore been presumed that this trafficking failure is in some

    way responsible for the proinflammatory properties of the CF

    205kDa

    C

    B

    118kDa

    wt F508 0.1% DMSO 0.4 2.0 10 50

    Controls CPX (M)

    Fig. 1. Western blot analysis of CPX-dependent CFTR expression in HEK cells expressing wild-type CFTR (band C; 180kDa) and [F508]-CFTR (band B; 150kDa).Samples of cells, cultured as described by Srivastava et al. 1999,[42] were homogenized in a medium containing a cocktail of protease inhibitors and other reagents,[21]

    and 50g aliquots of protein run on 6% SDS-PAGE gels. The antibody against the CFTR C-terminal 4 residues (monoclonal from Genzyme, Boston, MA, USA) is usedto detect CFTR antigen in the gels, and the complexes are imaged by enhanced chemi-luminescence. kDa = kiloDaltons

    Pharmacogenomics of Cystic Fibrosis 225

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  • airway. As described in section 4, CPX is efficacious in correct-ing the trafficking defect of mutant CFTR.

    3.2 Intrinsic Propensity for Inflammation and Infection inthe CF Lung

    The CF lung has been described as microscopically normal atbirth, with subtle abnormalities in mucus secretion appearingvery early in life.[5] Bacterial infection and objective evidence ofinflammation occur at later times, with a clear temporal evolutionof different principal bacterial pathogens. For example, Staphy-lococcus aureus and Hemophilus influenzae take up residence inthe CF airway early, the mean age of positive culture being 12.4months.[43] By compariso...

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