INTRAMEDULLARY SPINAL CORD LESIONS IN NF1 AND NF2 Sheila Kori.
Integrative Genomic Approaches to Identify Biomarkers and Therapeutic Targets in NF1
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Transcript of Integrative Genomic Approaches to Identify Biomarkers and Therapeutic Targets in NF1
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Integrative genomic approaches to identify biomarkers and therapeutic targets in NF1
Walter J. Jessen, Ph.D.Cincinnati Children’s Hospital
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
“Any living cell carries with it the experiences of a billion years of experimentation by its ancestors.”
Max Delbrück, theoretical physicist
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Principlethe whole is greater than the sum of its parts
Research goal
‣ develop and apply integrative genomic approaches to better organize and evaluate high-throughput genomic data
‣ effectively interpret the results and achieve a greater understanding of the signals and mechanisms regulating disease development and progression
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Neurofibromatosis (NF)
‣ Set of autosomal dominant genetic disorders of the nervous system that cause tumors to form on peripheral nerves
‣ Approximately 50% of those affected have a prior family history of NF
‣ The other 50% are a result of spontaneous genetic mutation
‣ Although most tumors are benign, can cause serious morbidity
Two major forms of NF:
- Type 1 (NF1) von Recklinghausen NF or Peripheral NF‣ Most common hereditary tumor predisposition syndrome‣ Occurs in 1:3500 births‣ Tumors (neurofibromas) form on peripheral nerves
- Type 2 (NF2) Bilateral Acoustic NF‣ Occurs in 1:40,000 births‣ Tumors (schwannomas) form on cranial and spinal nerves
Malignant peripheral nerve sheath tumors (MPNST)‣ Highly aggressive soft tissue sarcomas‣ Localized recurrence, chemo-resistance, frequent metastasis‣ Median age of onset: 26, five year survival: 34%
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
NF1 tumor subtypes
25%
10 – 13%
No effective treatments exist for either neurofibroma or MPNST
Dermal neurofibromas (dNF)‣ Tumors that appear as multiple, firm rubbery
bumps of varying size on the skin‣ Benign, but a significant source of morbidity
Plexiform neurofibromas (pNF)‣ Associated with major nerve trunks‣ Expand within the perineurium to displace
surrounding tissue‣ Capable of becoming malignant
% patients affected
95%
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
NF1 tumor composition is complex
Neurofibroma
Nerve
Tumors contain all the cell types of normal peripheral nerves, including:
Schwann cells are the pathogenic cell type in peripheral nerve tumors
Lines of evidence
1. LOH is observed in NF1-derived Schwann cells but not fibroblasts2. NF1-derived Schwann cells are invasive3. Mice with Schwann cell lineage-specific ablation of Nf1 develop tumors
‣ Schwann cells (ensheath axons)‣ Fibroblasts (give rise to connective tissue)‣ Mast cells (wound healing)‣ Axons (nerve fiber)
Two types of Schwann cells
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Corfas et al., Mechanisms and roles of axon-Schwann cell interactions. J Neurosci. 2004 Oct 20;24(42):9250-60.
A. Myelinated Axon B. Unmyelinated Axons
S Schwann cell nucleus
Ax Axon
Ax
S
Myelin: electrically insulating material (glycolipd and protein) produced by Schwann cells that ensheathes axons; increases speed of electrical impulses
Ax
S
myelin sheath
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
NF1 encodes neurofibromin, a GTPase activating protein
Reduced NF1 expression results in increased Ras activation
Downward J, Cancer: A tumour gene's fatal flaws. Nature. 2009 Nov 5;462(7269):44-5.
RAS activation stimulates downstream signaling pathways
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Very little is known about the pathways that drive NF1 tumor progression
Normal Schwann cell
Dermal neurofibroma
Plexiform neurofibromaNF1-associated
MPNST
NF1 mutation
H-, K-, N-Ras
PDGFRA
EGFR
KIT
cAMP
S6kinase
p53
Rb
CDKN2A (p16)
CDKN2D(p19)
Transformation
Growth factorreceptors
Tumor suppressorgenesSignaling
Benign Malignant
INTEGRATE
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Research objectives: 1.Gain insight into the biological pathways and processes that drive
NF1 tumor formation and transformation2. Identify molecular differences between tumor subtypes
- dermal vs. plexiform - benign vs. malignant
3.Provide candidate genes for diagnostics and treatment strategies
Hypothesis: purified Schwann cells from NF1 tumors will continue to express tumor gene programs in culture
Human cell culture, human tumor, mouse Schwann cell development
Study: Biological pathways that drive NF1 tumor progression
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Profiling gene expression using Affymetrix DNA microarrays
DNA microarray technology:
Enables researchersto simultaneously survey the expressionof a large number of genes.
Microarray or GeneChip:
A tool used to analyzegene expression,consisting of a small glass slide containingsamples of many genes arranged in a regular pattern.
Samples:
Sets of probes which represent gene transcripts
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
What is clustering?
‣ Technique used to group similar genes and samples together
‣ Allows for the identification of potentially meaningful relationships
‣ Genes that have similar patterns of expression are grouped together in clusters
‣ Cluster genes are likely to be co-regulated or part of the same biological process or pathway
‣ Statistics are used to identify over-representation or enrichment of biological processes or pathways in gene clusters
Analyze gene expression data from DNA microarrays by clustering
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Two common types of clustering methods
‣ Hierarchical clustering: subdivides each cluster into smaller clusters, forming a dendrogram (tree-shaped data structure)
Algorithm summary1. Place all points into their own clusters2. While there is more than one cluster, merge the closest pair of clusters
Weakness: doesn’t really produce clusters, user must decide where to split the tree into groups
‣ K-means clustering: subdivides data into a predetermined number of clusters without any implied hierarchical relationship between clusters
Algorithm summary 1. Assign all points to a cluster at random2. Repeat until stable:
a. Compute the centroid for each clusterb. Reassign each point to the nearest centroid
Weakness: must choose k parameter in advance; sensitive to outliers, which can distort centroid positions
➡ Comparative studies have shown that K-means outperforms hierarchical clustering on expression data(Gibbons et al., 2002; Datta and Datta, 2003; Costa et al., 2004)
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Robust Multi-array Average (RMA)
Robust Multi-array Average (RMA): an algorithm for normalizing and summarizing probe-level intensity measurements from DNA microarrays
Boxplot and histogram of signal intensities before RMA pre-processing
The normalization procedure is intended to make the intensity distributions identical across arrays
Boxplot and histogram of signal intensities after RMA pre-processing
Dai et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 2005 Nov 10;33(20):e175.
R: language and environment for statistical computing and graphicsBioconductor: open source software project for genomic data analysis
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Affymetrix probe specificity and annotation issues
Reorganize probes and use updated transcript definitions to increase gene detection confidence and identification
Chip definition file (CDF) and annotation library updates only affect the qualitative attributes of probe sets without any degree of control on the effective matching of probes and genome sequences
Novel system for associating probes with genomic information; custom defined probes meet the following criteria: 1. Probes must have only one perfect match on the genomic sequence2. Because EST sequences are subject to a relatively high error rate, probes must
perfectly match a genomic region that can be aligned with mRNA/EST sequences in the UniGene database
3. Probes must target the same transcript strand 4. Updated probe sets must contain a minimum of 3 probes5. Transcript annotation is based on updated reference sequences (RefSeq)
Dai et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 2005 Nov 10;33(20):e175.
R: language and environment for statistical computing and graphicsBioconductor: open source software project for genomic data analysis
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Analysis of Variance (ANOVA)
Analysis of Variance (ANOVA): a statistical technique for helping to infer whether there are real differences between the means of three or more groups in a population based on sample data
In general, an ANOVA:
‣ measures the overall variation within a group‣ finds the variation between group means‣ combines these to calculate a single test statistic‣ uses this to carry out a hypothesis test
Assumptions with an ANOVA:1. observations are independent2. dependent variable is normally distributed3. homogeneity of variances
➡ The advantage of using ANOVA rather than making multiple comparisons using individual t-tests is that it reduces the probability of a false positive (type-I error)
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Hypothesis testing and error‣ P-value was invented for testing individual hypotheses
‣ Problem with data collected by DNA microarrays, usually involves testing thousands of hypotheses simultaneously
‣ The False Discovery Rate (FDR) is a statistical method used for testing multiple hypotheses that corrects for multiple comparisons
‣ False Discovery Rate (FDR): the expected proportion of false positives (type I errors) among the results declared significant
example: 1,000 genes at an FDR = 0.05- expect a maximum of 50 genes to be false positives (1000 x 0.05)- no such interpretation exists for P-value
‣ At least four factors determine FDR characteristics for a microarray study (Pawitan et al., 2005)
1. proportion of truly differentially expressed genes2. distribution of the true differences3. measurement variability4. sample size➡ Benjamini and Hochberg FDR
Benjamini and Hochberg, Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Statist. Soc. Ser. 1995, B 57 289-300.
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Integrate genomic data from NF1 tumor-derived cell culture samples and tumor samples
Analysis strategy: 1. Identify genes differentially expressed in cultured Schwann cells2. Identify genes similarly deregulated in NF1 cell cultures & human tumors
NHSC
dNFSC
pNFSC
MPNST cell
dNF
pNF
MPNSTPlexiform NF Schwann cells
Normal human Schwann cells
Dermal NF Schwann cells
# samples
10
11
11 MPNST
Plexiform NF
Dermal NF
MPNST cell lines 13
# samples
13
13
6
Human tumorsNormal and NF-derived Schwann cells
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Genes differentially expressed in cultured Schwann cellsPrinciple patterns
Genes upregluated in MPNST cell lines
Genes upregluated in NFSC
Genes upregluated in all
Genes downregulatedin MPNST cell lines andclass 2 NFSC
Genes downregulated in MPNST cell lines
Miller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Two classes of NFSC
Statistical test:ANOVA, FDR ≤ 0.001
Dermal and plexiform neurofibromas mix together
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Genes similarly deregulated in NF1 cell cultures & human tumors
Cell culture Tumors
Functional enrichment
‣Cytoskeletal organization and biogenesis
‣Glycoprotein metabolism
‣Nervous system development
‣Neurogenesis ‣Sphingolipid metabolism
‣Cell adhesion‣Nervous system development
‣Chromosome organization and biogenesis
‣Extracellular matrix organization and biogenesis‣Nervous system development
‣Cell adhesion‣JAK-STAT cascade
‣Skeletal development
‣Cell adhesion‣Morphogenesis‣WNT receptor signaling pathway
dNF and pNFNormal and benign Schwann cells MPNST
Miller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Stage 1 Stage 2 Stage 3 Stage 4
Buchstaller et al. Efficient isolation and gene expression profiling of small numbers of neural crest stem cells and developing Schwann cells. J Neurosci. 2004 Mar 10;24(10):2357-65.
Four stages of Schwann celldevelopment
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Neural crest cell gene signature
Schwann cell precursorgene signature
Immature Schwann cellgene signature
E9 E12 E14 E16 E18 P0E9 E12 E14 E16 E18 P0E9 E12 E14 E16 E18 P0E9 E12 E14 E16 E18 P0
Stage 1 Stage 2 Stage 3
Three activated gene signatures of Schwann cell development
4,75
0 pr
obe
sets
Statistical test:ANOVA, FDR ≤ 0.2
Compare genes to clusters C6 – C11
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Stage 1 Stage 2 Stage 3
Gen
es e
xpre
ssed
in N
F1 c
ell c
ultu
res
and
tum
ors
Miller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Boxes in red (up-regulated) or blue (down-regulated) are statistically significant
Neurofibromas and MPNSTs have gene signatures characteristic of different stages of Schwann cell development
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Results are consistent with recently published data
E8.5
E12.5
E18.5
Stage 1 Stage 2 Stage 3 Stage 4
Developed a mouse model: DhhCre; Nf1 flox/flox
Jianqiang Wu
DhhCre; Nf1 flox/flox mice die by 13 months
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
0 2 4 6 8 10 12 140
25
50
75
100
Nf1 flox/flox; DhhCre (n=28)Nf1 flox/+; DhhCre (n=22)Nf1 flox/flox (n=10)Nf1 flox/+ (n=8)
Months
Perc
ent s
urvi
val
Jianqiang Wu
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Mice have dermal- and plexiform-like neurofibromas N
orm
al m
ouse
Der
mal
neu
rofib
rom
asP
lexi
form
neu
rofib
rom
as
DhhCre; Nf1 flox/floxmice show symptomsof tumor developmentas early as 5½ months of age
Human tumors DhhCre; Nf1 fl/fl mouse model
Wu et al., Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehog-expressing cells. Cancer Cell. 2008 Feb;13(2):105-16.
Jianqiang WuBioinformatics and biology suggest NF1 loss later in
Schwann cell development gives rise to neurofibromas
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
SOX9
Fold change NHSC to dNFSC
Fold change NHSC to pNFSC
Fold change NHSC to MPNST cell lines
Fold changeNHSC to dNF
Fold changeNHSC to pNF
Fold change NHSC to MPNST
9.72 7.76 46.46 27.97 28.08 63.06
‣ Encodes a high-mobility group box-containing transcription factor
‣ Modulates glial specification and differentiation in the peripheral nervous system and spinal cord (Kordes et al., 2005)
‣ Regulates neural crest stem cell survival (Cheung et al., 2005)
Schwann cell cultureSchwann cell culture Human tumor
Perform immunohistochemistry on tumor sections to evaluate protein expression
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Immunohistochemical analysis of SOX9 protein expression
42 NF1 tumor sections (10 independent)
Brown = SOX9+SOX9 is a biomarker for NF1
Anat Stemmer-RachamimovMiller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Neurofibroma Schwann cells MPNST cellsCorresponding phase contrast images
Corresponding phase contrast images
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Test a role for SOX9 in tumor survival
*
‣ Use shRNA to reduce SOX9 expression‣ Infected cells with a lentivirus-expressing shSOX9 or shGFP control‣ Plated 7 days post-selection in puromycin, measured survival (MTS)‣ Plated 3 days post-selection in puromycin, counted cells
p ≤ 0.05
Shyra MillerMiller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
‣ Use shRNA to reduce SOX9 expression‣ Infected cells with a lentivirus-expressing shSOX9 or shGFP control‣ Plated 1–4 days post-selection in puromycin, measured survival (MTS)‣ Plated 3 days post-selection in puromycin, assayed for apoptosis
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
SOX9 is a survival gene for NF1 and a potential therapeutic target
Test MPNST cells for survival
MPNST cells
p ≤ 0.002
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Summary
‣ Gene expression distinguishes benign and malignant NF1 Schwann cell cultures and solid tumors
‣ Gene expression fails to distinguish dermal and plexiform neurofibroma subtypes
‣ NF1 Schwann cell culture and tumor transcription patterns are enriched for genes activated during Schwann cell development
‣ SOX9 is biomarker and survival gene for NF1
‣ Reduction in SOX9 expression kills MPNST cells
Human cell culture, human tumor, mouse cell developmentIdentify enrichment of developmental programs in NF1 tumors
Miller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Model of NF1 tumor formation
Neural Crest Cell
Schwann cellprecursors
Mature Schwann cellsImmature
Schwann cells
Stage 1 Stage 2 Stage 3
Stage 4
Miller*, Jessen* et al., Integrative genomic analyses of neurofibromatosis tumors identify SOX9 as biomarker and survival gene. EMBO Mol Med 2009 July, 1(4);236-248.
Walter Jessen Integrative genomic approaches to peripheral nerve [email protected]
Onging research objectives: 1. Identify core biological processes and pathways for
tumorigenesis and malignancy that are conserved between mouse and human
2.Translate findings from mouse NF1 models to human therapeutics
Hypothesis: there are biological processes and pathways similarly changed in human NF1 tumors and tumors from mouse models of NF1
Mouse tumor, human tumor
Study: Leverage mouse NF1 models for translation to human therapeutics
INTEGRATE
Evaluate three classes of transgenic mouse and human samples
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
MPNST
Control nerve
Neurofibroma
HumanTransgenic mice# samples of
each genotype
5–5–5
4–7–4
3–3–5–3–4 MPNST
Neurofibroma
Normal nerve
# samples
3
26
6
‣ Each data set is referenced to control/normal nerve
‣ Evaluate gene signatures that are shared across tumor subtypes for each species
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Method to identify similarly expressed gene orthologs conserved between mouse and human
Filter for genes similar in neurofibroma
Human Mouse
UP
Human Mouse
DOWNin 80% of samples >1.2 in 80% of samples <0.8
Filter for genessimilar in MPNST UP DOWN
in 80% of samples >1.2 in 80% of samples <0.8
UP DOWNin 80% of samples >1.2 in 80% of samples <0.8
UP DOWNin 80% of samples >1.2 in 80% of samples <0.8
2,212 orthologs similarly expressed
1,016
398 414
758
HumanANOVA (FDR ≤ 0.05)
Nerve vs. NF vs. MPNST
MouseANOVA (FDR ≤ 0.05)
Control nerve vs. NF vs. MPNST
Human Mouse Human Mouse
Identify genesstatistically different
integrate, identify orthologous genes
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Gene orthologs similarly expressed between mouse and human tumors
‣ Axonogenesis
‣ Induction of apoptosis ‣Negative regulation of
MAP kinase activity‣ Regulation of
neurotransmitter levels
‣ Actomyosin structure and organization‣ Negative regulation of
cell cycle progression‣Peripheral nervous
system development
‣ Apoptosis
‣ Negative regulation of MAP kinase activity ‣ Phosphoinositide-
mediated signaling
‣ Regulation of mitosis
‣ Axon ensheathment
‣Axonogenesis‣ Catecholamine
metabolism‣ Peripheral nervous
system development
Functional enrichment
Statistical test:ANOVA, FDR ≤ 0.05
HumanTransgenic mice HumanTransgenic mice Neurofibroma
MPNST
C1 C2 C3 C4
2,21
2 Tr
ansc
ripts
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Gene signatures shared or unique between NF1 tumors and GEM NF1 models
Similar expression patterns Contrasting expression patterns
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Species-specific gene signatures
Human-specific expression patterns Mouse-specific expression patterns
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Perform comparative enrichment analysis on expression signatures(ToppCluster facilitates co-functional enrichment analysis of multiple gene signatures)
http://toppcluster.cchmc.org/17 clusters C11 – C27ToppGene: FDR ≤ 0.05
Generates relationships in high-dimensional space, visualize interaction network using the open source bioinformatics software platform Cytoscape.
ToppGene: uses cluster assignment as a classification parameter and the Gene Set Enrichment Algorithm to identify significant gene set over-representation of several features: gene ontologies, pathways, co-expression, gene-disease, gene-drug, mouse and human phenotypes, microRNAs, cytobands and transcription factor binding site (TFBS).
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Structure based on the force-directed layout paradigmyFiles (Java Graph Layout and Visualization Library) Organic Algorithm
Clusters
Gene
Pathway
Cytoband
Ontology
Gene sets
TFBS
(disease associations)
Protein domain
Drug
Nodes: 2,653Edges: 7,938
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Shared activationApoptosis Cell cycle controlCell proliferationRegulation of MAP kinase activity
Shared repressionCell-cell signalingLipid metabolism MyelinationNervous system development
HS unchanged, MM activatedHS activated, MM repressedCell-cell signalingGlutathione metabolismGABA-B receptor signalingPotassium/calcium transportSmall GTPase mediated signal transduction Synaptic vesicle trafficking
HS unchanged/repressed, MM activatedAngiogenesis, Apoptosis, Immune response,Ras protein signal transductionNote: only ontologies and pathways are listed
Four “Feature Domains”
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Gene orthologs and biological themes shared between mouse and human
‣ Axonogenesis
‣ Induction of apoptosis ‣Negative regulation of
MAP kinase activity‣ Regulation of
neurotransmitter levels
‣ Actomyosin structure and organization‣ Negative regulation of
cell cycle progression‣Peripheral nervous
system development
‣ Apoptosis
‣ Negative regulation of MAP kinase activity ‣ Phosphoinositide-
mediated signaling
‣ Regulation of mitosis
‣ Axon ensheathment
‣Axonogenesis‣ Catecholamine
metabolism‣ Peripheral nervous
system development
Functional enrichment
Statistical test:ANOVA, FDR ≤ 0.05
HumanTransgenic mice HumanTransgenic mice Neurofibroma
MPNST
C1 C2 C3 C4
2,21
2 Tr
ansc
ripts
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
PTPRZ1‣ Encodes a protein tyrosine phosphatase (receptor type Z)
‣ Expression is restricted to the nervous system (Levy et al., 1993)
‣ Plays a critical role in functional recovery from demyelinating lesions (Harroch et al., 2002)
‣ In the top 100 genes discriminating MPNST from 13 other soft tissue sarcomas (Francis et al., 2007)
Transgenic miceTransgenic mice HumanFold change Controls to NF
Fold change Controls to MPNST
Fold changeNerve to NF
Fold change Nerve to MPNST
13.22 7.77 2.56 -2.26
Expression profile suggests PTPRZ1 could be important for tumorigenesis
Use PTPRZ1 to select orthologs that have a similar expression profile and evaluate genetic interactions
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Genetic interaction network analysis
Hypothesis: transcripts having a similar pattern of expression as PTPRZ1 and interacting with genes in the MAP kinase pathway will include critical regulators of survival in NF1
Analysis strategy: 1. Identify the top 100 gene orthologs that
correlate and anti-correlate with PTPRZ12. Add transcripts from clusters C1 and C3
associated with Negative regulation of MAP kinase activity
3. Add ERK genes (MAPK1, MAPK3, MAPK6, MAPK7, MAPK12)
4. Identify genetic interactions, removing those entities that don’t have connections Downward J, Cancer: A tumour gene's fatal
flaws. Nature. 2009 Nov 5;462(7269):44-5.
RAS activation stimulates downstream signaling pathways
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Orange: ERK/MAP kinase genes
Direct interaction
Indirect interaction
Genetic interaction network analysis
Nodes are colored according to the degree of fold change from human nerve to neurofibroma
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
‣ Up-regulated gene targeted by a currently used cancer drug
‣ Directly interacts with a number of genes highly up-regulated in human neurofibroma
- c-Kit- beta-catenin- breast cancer anti-estrogen resistance 1- p21 protein (Cdc42/Rac)- activated kinase 2- arrestin beta 1
Genes associated with cell death, neurological disorders, cell proliferation and survival
All direct interactions up-regulated in neurofibroma
Genetic interaction network analysis
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Genetic interaction network analysis
Pivotal genes in critical signaling pathwaysAktCDKN2A (p16)HIF1ANFkB1VEGFA
All direct interactions up-regulated in neurofibroma
‣ Move one step further down the interaction pathway, number of pivotal genes in critical signaling pathways
‣ Two genes have been targeted therapeutically: KIT and EGFR
‣ Data suggests that the gene is a potential promoter of malignant transformation in NF1
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Dedifferentiated Chondrosarcoma (3)MPNST (4)
Neurofibroma (4)
Gene is over-expressed in a subset of mesenchymal tumors that are aggressive
Myxoid Liposarcoma (6)
Monophasic Synovial Sarcoma (10)
Alveolar Rhabdomyosarcoma (4)
Desmoid Fibromatosis (5)
Embryonal Rhabdomyosarcoma (3)
Henderson et al., A molecular map of mesenchymal tumors. Genome Biol. 2005;6(9):R76. Epub 2005 Aug 26.
‣19 mesenchymal tumor subtypes
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Neurofibroma MPNST
‣DNA repair‣Ensheathment of neurons‣Integrin-mediated
signaling‣Mitotic cell cycle
‣Ras protein signal transduction‣Vesicle-mediated transport
Functional enrichment
Expression signature for the gene in human tumors
854
Tran
scrip
ts
Gene signature
854
7,174
Statistical test:ANOVA, FDR ≤ 0.05
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
Cancer drug is cytotoxic against 5 MPNST cell lines (dose at days 2 and 4 relative to day 0)
% C
ontro
l
[drug] nM
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
‣ Effective method for simultaneous comparison of transcriptional programs between mouse models and human tumors
‣ Human NF1 tumors and mouse NF1 model tumors share activation of genes associated with negative regulation of MAP kinase activity and repression of genes associated with peripheral nervous system development
‣ Genes down-regulated in human NF1 tumors but up-regulated in mouse NF1 models are associated with Ras protein signal transduction and immune response
‣ Use gene interaction network analysis to identify a gene that is a potential promoter of malignant progression in NF1 and a potential therapeutic target
Mouse tumor, human tumorCross-species profiling, genetic network analysis
Summary
Walter Jessen Integrative genomic approaches to identify biomarkers and therapeutic targets in [email protected]
HumanCincinnati, OH – CCHMC Nancy Ratner, Shrya Miller, Atira Hardiman
Boston, MA – MGH/Harvard Anat Stemmer-Rachamimov
Gainseville, FL – University of Florida Margaret Wallace
Barcelona, Spain – LʼHospitalet de Llobregat Concepcion Lazaro, Eduard Serra
Mouse modelsCincinnati, OH – CCHMC Nancy Ratner, Jianqiang Wu, Tilat Rizvi
Paris, France – Fondation Jean Dausset Marco Giovannini, Jan Manent
Bioinformatics/BiostatisticsCincinnati, OH – CCHMC Bruce Aronow, Walter Jessen
Birmingham, AL – University of Alabama Grier Page, Tapan Mehta
FundingDOD: W81XWH-04-1-0273NIH: T32 HL07382-30