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Applications of LA

Explore the applications of LA in gene-drug survival analysis using human cell lines. Discover the gene expression profiles and potential therapeutic targets for diseases like Alzheimer's and Multiple Sclerosis.

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Applications of LA

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  1. Applications of LA Human examplesGene-drug Survival analysis

  2. Schematic illustration of LA

  3. The human examples

  4. Gene expression profile for NCI’s 60 cell lines • For each cell line, the relative mRNA concentrations are measured by cDNA glass array. • Cell lines used in microarray experiment are without drug administration. • Ross D.T. et al. Systematic variation in gene expression patterns in human cancer cell lines. Nat. Genet. 24, 227-235 (2000)

  5. NCI 60 Cell lines OVARIAN (6) IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 SK-OV-3 PROSTATE (2) DU-145 PC-3 LEUKEMIA (6) CCRF-CEM HL-60 K-562 MOLT-4 RPMI-8226 SR MELANOMA (8) LOXIMVI M14 MALME-3M SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 BREAST (8) BT-549 HS578T MCF7 MCF7/ADF-RES MDA-MB-231/ATCC MDA-MB-435 MDA-N T-47D LUNG (9) A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 CNS (6) SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 COLON (7) COLO205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 RENAL (8) 786-0 A498 ACHN CAKI-1 RXF-393 SN12C TK-10 UO-31

  6. How does LA work in cell-lines? Alzheimer’s Disease hallmark gene Amyloid-beta precursor protein (APP)

  7. Alzheimer’s disease The brain tissue shows "neurofibrillary tangles" (twisted fragments of protein within nerve cells that clog up the cell), "neuritic plaques" (abnormal clusters of dead and dying nerve cells, other brain cells, and protein), and "senile plaques" (areas where products of dying nerve cells have accumulated around protein). Although these changes occur to some extent in all brains with age, there are many more of them in the brains of people with AD. The destruction of nerve cells (neurons) leads to a decrease in neurotransmitters (substances secreted by a neuron to send a message to another neuron). The correct balance of neurotransmitters is critical to the brain.

  8. Amyloid beta peptide is the predominant component of senile plagues in brains of MD patients. It is derived from Amyloid-beta precusor protein (APP) by consecutive proteolytic cleavage of Beta-secretaseand gamma-secretase

  9. What is the physiological role of APP? Cao X, Sudhof TC. A transcriptionally active complex of APP with Fe65 and histone acetyltransferase Tip60. Science. 2001 Jul 6;293(5527):115-20.

  10. Abstract of Cao and Sudhof Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase. gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. We now demonstrate that thecytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function ingene expression.

  11. Take X=APP, Y=APBP1 • APBP1 encodes FE65 • Find BACE2 from our short list of LA score leaders. • BACE2 encodes a beta-site APP-cleaving enzyme

  12. Take X=APP, Y=HTATIPHTATIP encodes Tip60 Finds PSEN1 (second place positive LA score leader) Which encodes presenilin 1, a major component of gamma-secretase

  13. Application: finding candidate genes for Multiple sclerosis

  14. Multiple Sclerosis

  15. Multiple sclerosis • 1. MS is a chronic neurological disorder disease, characterized by multicentric inflammation, demyelination and axonal damage, resulting in heterogeneous clinical features, including pareses, sensory symptoms and ataxia. The classical clinical features include disturbances in sensation and mobility. The typical age of onset is between years 20 and 40, making MS one of the most common neurological diseases of young adults. Four genome-wide scans (US, UK, Canada, and Finland) have revealed several putative susceptibility loci, of which the loci on chromosomes 6p, 5p, 17q and 19q have been replicated in multiple study samples. More recently, Professors Aarno Peltonen and Leena Peltonen’s teams have generated a fine map on 17q22-q24 (Saarela et al 2002). They are now interested in the functional aspect of the genes in this region using microarray technology.

  16. Multiple sclerosis study Aarno : How about PRKCA ? KC: it is not in the NCI’s cell-line data, how about PRKCQ? Aarno: No, we are very picky. Daniel: GNF has PRKCA KC: we have not analyzed GNF data yet. Aarno, Daniel, Robert: give it a try

  17. (GNF_ gene expression Atlas; http://expression.gnf.org ). Su et al. • X=MBP(myelin basic protein), Y= PRKCA (a gene in interested chromosome 17q22-24 region), • we get Z=SLC1A3 at the second place. • Returning to the cell-line data, we use SLC1A3 as X to find highest score LA pairs. • MYT1(Myelin transcription factor 1) appears twice in our short list of the best 40 pairs (out of a total of over 73 million possible pairs)!!

  18. A lot more evidences: including connection to HLA (major histocompatibility complex) family of genes in 6p21, the locus which most linkage analysis has pointed to. To summarize: Starting from PRKCA(protein kinase C, alpha), a primary susceptible gene from an MS locus on chromosome 17q22-q24, we use the method of liquid association (LA) to probe for functionally associated genes. A string of evidences from four large-scale gene expression databases suggest a strong connection to SLC1A3(glial high glutamate transporter, member 3). Significant linkage of SLC1A3 to MS is established in a follow-up SNF typing involving high MS-risk population sub-isolate of Finland. Our results open up a new approach of finding susceptible genes for complex diseases.

  19. We have now created a web-page to facilitate MS gene expression study. Genes from chromosome 17q22-24 X=MYT1(myelin transcription factor 1) and Y=FALZ( fetal Alzheimer antigen), we find Z= SLC1A3 (second highest LA score from the negative end).

  20. MYT1 is a zinc finger transcription factor that binds to the promoter regions of proteolipid proteins of the central nervous system and plays a role in the developing nervous system and FALZ has a DNA-binding domain and a zinc finger motif. FALZ is located in chromosome 17q24.3, the region of interest. SLC1A3 is a glial high affinity glutamate transporter, located in chromosome 5p13 which is another region of interested mapped earlier in the Finish study.

  21. Liquid association: A method for exploiting lack of correlation between variables

  22. glutamate-induced excitotoxicity SLC1A3 is highly expressed in various brain regions including cerebellum, frontal cortex, basal ganglia and hippocampus. It encodes a sodium-dependent glutamate/aspartate transporter 1 (GLAST). Glutamate and aspartate are excitatory neurotransmitters that have been implicated in a number of pathologic states of the nervous system. Glutamate concentration in cerebrospinal fluid rises in acute MS patients whilst glutamate antagonist amantadine reduces MS relapse rate. In EAE, the levels of GLAST and GLT-1 (SLC1A2) are found down-regulated in spinal cord at the peak of disease symptoms and no recovery was observed after remission. We consider highly encouraging that several lines of evidence including both genetic association and gene expression association, would be consistent with the glutamate-induced excitotoxicity hypothesis of the mechanisms resulting in demyelination and axonal damage in MS.

  23. International MS whole genome association study(2007). • Affymetrix 500K to screen common genetic variants of 931 family trios. • Using the on-line supplementary information provided, we found two SNPs, rs4869676(chr5:36641766) and rs4869675(chr5: 36636676 ) with TDT p-value 0.0221 and 0.00399 respectively, are in the upstream regulatory region of the SLC1A3 gene. • In fact, within the 1Mb region of rs486975, there are a total 206 SNPs in the Affymetrix 500K chip. No other SNPs have p-value smaller than that of rs486975. • The next most significant SNPs in this region are rs1343692(chr5:35860930), and rs6897932(chr5:35910332; the identified MS susceptibility SNP in the IL7R axon). • The MS marker we identified rs2562582(chr5: 36641117) is , less than 5K apart from rs4869675, but was not in the Affymetrix chip.

  24. A little bit late • IL7R was found long time ago before by LA !!!See the attached the e-mailﾊ sent more than two years ago in 2005 !!! • Begin forwardedmessage:From: Ker Chau Li (local) <kcli@stat.ucla.edu>Date: March 28, 2005 10:17:51 AM PSTTo: Robert Yuan <syuan@stat.ucla.edu>, Aarno Palotie <APalotie@mednet.ucla.edu>, Daniel Chen <pharmacogenomics@yahoo.com>, Denis Bronnikov <denis@ucla.edu>, Palotie Leena <leena.peltonen@ktl.fi>Cc: Ker Chau Li (local) <kcli@stat.ucla.edu> • Subject: IL7R (I thought this e-mail should have been sent out already; but it has not)I take X=SLC1A3, Y=MBP, Z= any gene, using 2002 Atlas data. Two genes are from the short list of genes with highest LA scores.IL7R interleukin 7 receptor and HLA-A IL7R is at 5p13. Interesting coincidence?? other interesting findings include GFAP glial fibrillary acidic protein on 17q21 (Alexander disease)GRM3 (glutamate receptor, metabotropic )CDR1 (cerebellar degeneration-related protein 1)Ighg3 (immunoglobulin heavy constant gamma 3)Iglj3 ( immunoglobulin lambda joining 3)

  25. Liquid association: A method for exploiting lack of correlation between variables

  26. Back to MBP (myelin basic protein) X=MBP=myeline basic protein Y= any gene from a total of 9000 genes Z= any gene from a total of 9000 genes Among the 40 pairs of Y and Z that give the best LA scores, A2M appears many times ( more than 10 ). I use X=A2M, and Y=MBP, Z= any gene The second highest LA-score gene Z is X=MBP, Y= any gene, Z= any gene

  27. A2M and MPDZ A2M(Alpha-2-macroglobulin is a protease inhibitor and cytokine transporter. It inhibits many proteases, including trypsin, thrombin and collagenase. A2M is implicated in Alzheimer disease (AD) due to its ability to mediate the clearance and degradation of A-beta, the major component of beta-amyloid deposits. ) is the only major myelin basic protein-binding protein in human plasma; (Gunnarsson M, Sundstrom P, Stigbrand T, Jensen PE.Native and transformed alpha2-macroglobulin in plasma from patients with multiple sclerosis.Acta Neurol Scand. 2003 Jul;108(1):16-21. ) MPDZ encodes a tight junction protein detected in non-compact region of myelin, and is thought to be required for maintaining the cytoarchitecture of myelinating Schwann cells. (Poliak et al).

  28. Prion (mad cow disease) • Our system can be applied to study clinically important proteins with poorly-understood physiological roles. Here we consider the gene PNRP which encodes the cellular prion protein PrP(c), a glycosyl-phosphatidylinositol-anchored glycoprotein. We shall show how the system embroiders a cellular context to link the prion pathology with the so called conformational diseases - diseases caused by adoption of non-native protein conformations leading to aggregation.

  29. Mad Cow disease - human prion gene Creutzfeldt-Jakob disease is an organic brain syndrome caused by a protein-like particle called a prion. Loss of brain function resembles Alzheimer's disease, but is very rapid in progression. Complete dementia usually occurs by the sixth month, death follows quickly. There is no known cure.

  30. X=PRNP (the gene ecnoding cellular prion protein) Y= any gene, Z= any gene Find Y=SERPINE2(protease nexin 1, known as glial derived neurite-promoting factor 2, highly expressed in nervous system, inhibits uPA) Z=ERBB3(tyrosine kinase receptor for glial-growth factor 2) Prion protein participates in neurite outgrowth and neuronal survival (31). The pathway involves FYN Take X= PRNP Y=FYN (tyrosine kinase), Z= any gene find Z=ERBB3(first place) Recent works show: PrP(sc) is recognized by plasminogen; activity of tPA(tissue plasminogen activator) can be stimulated up to 280-fold by PrP(c ) ; Plasmin accelates the cleavage of PrP(c ) in PrP(c ) -plaminogen complex into Prp (c ) fragments .

  31. Anticancer drug screen

  32. DTP Tumor Cell Lines Screen • Same drug, different tumor, different result • Systematic approach • Initialized in 1980’s • Select a few representative tumors, develop cell-lines (repeatability! fidelity?) • Currently, there are more than 60 cell lines used in the screening. • More than 10,000 compounds tested.

  33. Medicine and the New Genomics • Gene Testing • Gene Therapy • Pharmacogenomics Anticipated Benefits • improved diagnosis of disease • earlier detection of genetic predispositions to disease • rational drug design • gene therapy and control systems for drugs • personalized, custom drugs Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001

  34. Personalized drug developmentsame drug, different responsemeat or poison • 100,000 deaths and 2 million hospitalizations each year in US due to adverse drug response • Pharmacogenomics/pharmacogenetics requires massive computer exploration on heterogeneous databases. • genetic markers, (SNF, haplotypes map) • gene expression profiling (blooming) • drug responsiveness profiling (established) • How to put all three sources of information together? • Pharmacogenomics holds the promise that drugs might one day be tailor-made for individuals and adapted to each person's own genetic makeup. Environment, diet, age, lifestyle, and state of health all can influence a person's response to medicines, but understanding an individual's genetic makeup is thought to be the key to creating personalized drugs with greater efficacy and safety.

  35. III Correlating gene-expression with drug-responsiveness c.line1 c.line2 …….. C.linep gene1gene2 drug1 drug2 x11 x12 …….. x1p x21 x22 …….. x2p … … y11 y12 ………… y1p y21 y22 ……………y2p …..

  36. Experiment procedure Rate of inhibition 48 h at 37°C E.g.: (9-6)/(12-6)*100=50 GI50 is x Drug added at x (M) 48 h at 37°C Drug sensitivity is defined as: -log(GI50) GI50 is the concentration of the drug needed to inhibit the growth of the cells up to 50%

  37. Example of Drug Sensitivity Data Sensitivity profile Cell Line 89 ,M ,-4.00,Non-Small Cell Lung ,NCI-H23 ,1 ,1 ,4.209,3 ,3 ,0.361 89 ,M ,-4.00,Non-Small Cell Lung ,NCI-H522 ,1 ,3 ,6.159,3 ,3 ,1.696 89 ,M ,-4.00,Non-Small Cell Lung ,A549/ATCC ,1 ,4 ,4.186,3 ,3 ,0.322 89 ,M ,-4.00,Non-Small Cell Lung ,EKVX ,1 ,8 ,4.255,3 ,3 ,0.442 89 ,M ,-4.00,Non-Small Cell Lung ,NCI-H226 ,1 ,13 ,4.231,2 ,2 ,0.326 89 ,M ,-4.00,Non-Small Cell Lung ,NCI-H322M ,1 ,17 ,4.000,3 ,3 ,0.000 89 ,M ,-4.00,Non-Small Cell Lung ,NCI-H460 ,1 ,21 ,4.233,3 ,3 ,0.403 89 ,M ,-4.00,Non-Small Cell Lung ,HOP-62 ,1 ,26 ,4.000,3 ,3 ,0.000 89 ,M ,-4.00,Non-Small Cell Lung ,HOP-18 ,1 ,27 ,4.249,3 ,3 ,0.431 GI50

  38. Drug Sensitivity profile • For each chemical compound, the tests are done for all 60 cell lines listed previously. • For each cell line and each compound, there were multiple experiments performed to obtain the average drug concentration.

  39. Drug Sensitivity profile (cont.) • Drugs with similar profiles usually share similar molecular structures and biochemical mechanism of actions. (R. Bai et al. 1991, K. D. Paull et al. 1992, H. N. Jayaram et al., 1992) • Similarity is measured by Pearson’s correlation coefficients. • COMPARE ( gateway to NCI’s anticancer screen database)

  40. Compare Drug sensitivity and Gene expression profiles • Sherf et. al. (2000) compare gene expression profile with the drug sensitivity profile by computing correlation coefficient. • 9706 genes in the expression data set. • 118 chemotherapy agents with known molecular mechanism of drug action • Genes whose profiles correlate well with drug sensitivity profiles are thought to be related to drug functioning. Why?

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