Lecture 3 (M. Ochs)

From Expression Measurements to Cell Signaling Inference
Michael Ochs The Sidney Kimmel CCC Johns Hopkins University
Focusing on Cell Signaling

Role in Cancer
Makes it a worthy target Potential for improved treatment
Established Biology
Large body of knowledge built from decades of mechanistic studies But need to remain open to updates that affect models
Oncology Biostatistics and Bioinformatics
Focus on Expression
Microarrays Mature, Global
We understand artifacts and normalization We have validated predictions
Methylation, SNPs, CNVs

Array technologies maturing, useful Sequencing mixed (SNPs best)
Mutations
Overall immature for global measurements
Cancer Revisiting Hallmarks

EGFR ERBB2 RAS RAS p53 pRb p53
RAS VEGF
FAK NEDD9 p53 hTERT
Hanahan and Weinberg, Cell, 100, 57, 2000
Emerging Hallmarks
Cytokines and Receptors
p53 MDM2 ATM BRCA1 BRCA2

Cytokines and Receptors
Hanahan and Weinberg, Cell, 44, 646, 2011
Measuring Signaling
Signaling Protein Levels
very hard in vivo for signaling proteins efforts using 2D gel have been successful, but expensive and not scalable not a global measure, only what you seek
Transcript Levels
easily measured from biopsies/tumors but
Expression of Signaling Proteins

General Relation of mRNA to Protein
correlation coefficient of 0.36 (yeast) human prostate cancer (60% concordant meaning only same direction of change)
Signaling
driven by post-translational modifications signaling proteins have low expression
Transcriptional Signatures for Signaling M

F
H

Signaling Protein
M A
B

F C
D

H E
Transcribed
Gene
A

B

C

D

E

Use Expression as Downstream Marker!!
Transcription as Biomarker for Cell Signaling

Isolate Signature in Presence of Overlapping Regulation Determine Significance of Signature for Activity of Transcriptional Regulators Link Transcriptional Regulators to Signaling Processes
Cell Cycle Data Revisited

******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ******************** ********************
Time series of microarray measurements

(Spellman et al, Mol Biol Cell, 9, 3273, 1999) (Cho et al, Mol Cell, 2, 65, 1998)
6000 yeast genes
Validation Gold Standard Coregulation
Cherepinsky et al, PNAS, 100, 9668, 2003"

ROC Curve for Clusters Revisited
Random Line!
Matrix Factorization
To address multiple regulation
Analytic Solution
Principal Component Analysis
Computational Solutions
Independent Component Analysis Nonnegative Matrix Factorization Bayesian Matrix Factorization
Principal Component Analysis

Identifies Directions in Data in Terms of Decreasing Variance Each Principal Component (PC) is Orthogonal to all Previous PCs
Npc dim(D)
PCA of Cell Cycle Data

1 2 3
M phase 4 5
G1 phase 6
S-G2 phase
PCA
Captures Some Cell Cycle Behavior
Peaks in correct places Shape and overall slope incorrect
Fails to Correctly Link Genes

Poor recovery of gold standard links
Issue of Orthogonality
Biology does not match condition of orthogonality of gene expression
Goal of Analysis
condition M
condition 1

gene 1
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gene N
* * * * * * * * * *

pattern 1

pattern k

D: Data
vs
Mock

gene 1
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gene N
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* * * * * * * * * *
pattern 1
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pattern k

P: Patterns of
Behavior

condition M

condition 1

A: Distribution of
Genes in Patterns

Patterns Nonorthogonal in General

condition M
condition 1

* * * * * * * * * *
pattern 1
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* * * * * * * * * *
pattern k

Link Conditions Time Series

t
Normal
P: Patterns of
Behavior
typically
1/n 0 normalized
to sum to 1

show when process is on show change in activity
Patients
Tumor
group patients with similar expression behaviors
Distributions
A: Distribution of
Distributes Behavior of Genes in Patterns

pattern 1
pattern k
gene 1
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gene N
* * * *

Genes Among Patterns

How much of a genes expression is due to a given pattern
Isolates genes together despite multiple regulation Useful for Gene Set Analysis
Issues
Nonorthogonality
No known analytic solution to best patterns (nonorthogonal basis vectors) Infinite number of potential bases (rotation of any basis set is new basis set)
Bias over Variance

Solutions require introducing bias Typically sparseness and positivity
Original NMF
Nonnegative Matrix Factorization
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X!
Lee and Seung Nature, 401, 788, 1999
NMF
Populate matrices randomly (i.e., from a distribution)
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Minimize a Cost Function

Dij Div(D || M ) = Dij log Dij + Aik Pkj ij k Aik Pkj k
Pick next element

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By Moving to Local Minimum in Cost

P P
A
k
Dk / ( Akh Ph )
A
k
A A
P
k
D k / ( A h Phk )
P
k
NMF Procedures
Choose a Dimensionality Populate the Matrices Step through Matrix Elements, Updating each in turn Stop when Reach Best Fit Repeat from Other Random Starting Points
Bioinformatics
Fox Chase Cancer Center
NMF Issues
Guaranteed Local Minimum = Almost Guaranteed Global Non-Minimum Updating Sequence through Matrix Encourages Trapping Need to Determine Dimensionality No Ability to Include Uncertainty Information (all data equal variance) No Sampling of Distribution
Bioinformatics
NMF of Yeast Cell Cycle Pick Best Fit from 200 Runs
Wang et al, BMC Bioinfo, 28, 7, 2006
NMF Issues
Poor Sampling
Local maxima lead to poor sampling of actual distribution Lack of information on uncertainty of parameter estimates
Poor Recovery in Complex Cases

Demonstrate later for human tumor data
Markov Chain Monte Carlo Bayesian Decomposition

MCMC Allows Sampling
Provides more thorough exploration of model space Provides estimates of uncertainties for each parameter
MCMC Expensive
Gibbs sampling computationally complex Potential in newer stochastic methods
Markov Chain Monte Carlo

Find A and P Simultaneously
)
We can only estimate relative probabilities of possible solutions

Markov Chain Monte Carlo is

used to explore the possible solutions
(Gibbs sampler with simulated annealing)

Ochs et al, J Magn Res, 137, 161, 1999 Moloshok et al, Bioinformatics, 18, 566, 2002
Fertig et al, Bioinformatics, 2010
Sampling of Posterior Distribution
Likelihood Posterior
Prior
p M |D =
p D|M p M
) ( ) p (D )
Evidence (Marginal of Data)
Bayes Equation (independently by Laplace)
The Prior
Atomic Domain 1D Infinitely Divisible
Atoms are created ex vacuo with a prior uniform in space, exponential in amplitude,
* * * * * * * * **** * * * * **** * * * * * * * * **** **** **** **** ****
Positive Additive Distribution
Atomic Domain is divided into multiple sections mapping to matrix; one domain for each matrix
Genes in Patterns
In this case, A and P are positive
A"
Prior Qualities
Atoms
Point Masses Described Completely by Location and Amplitude
Distribution
Bias toward Few Large Atoms Bias toward Minimizing Structure Include Correlations by Kernel Function
*Sibisi and Skilling, J Royal Stat Soc B, 59, 217, 1997
Specific Priors
For 0 = 4800
Number of Atoms P(N) = (1-a)aN with a = 0/(0+1) 0 is expectation on N (hyperparameter) Flux of Atoms P(z) = q-1e-z/q with q expected flux (hyperparameter)
3500 For q = 200
CDF for N
150
CDF for z
MCMC
Move
Birth Death
Step
Number of Steps is ~ 1/2[N + Ga(N)]
First for A Then for P
Birth Death
Exchange
Birthing Atoms
probability
Position from U(0,232) Flux from positive Normal Distribution based on gradient and curvature of LogLikelihood As average flux increases, distribution shifts to low amplitude
With low average atom flux

Flux
probability
With high average atom flux
Flux
Effect of logLikelihood
decreasing gradient
The distributions for choosing atomic flux adjusts to both changes in average flux of present atoms and changes in the logLikelihood around a chosen point Oncology Biostatistics
and Bioinformatics
decreasing curvature
Accepting Changes
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Test change in likelihood using standard MCMC criteria Note: Only using relative likelihood changes, ignoring normalization (evidence)
A"
Destroying Atoms
Pick Atom at Random Destroy if Effect on LogLikelihood is within allowable MCMC criteria
Exchanging Flux/Moving Atoms

Atomic Domain 1D Infinitely Divisible Movement involves trying to move atom between neighbors
Pick Atom at Random Find its Neighbor
Look at Joint LogLikelihood and choose fluxes based on random sample from joint LogLikelihood
Bioinformatics
BD Patterns of Cell Cycle Data

0.3!
0.25!
G1
0.2! 0.15!
M G1
M/G1! G1A! S/G2! M! G1B! Oscillator!
S/G2
0.1!
0.05!
oscillator
0! M/G1! G1! G1! S! S! G2! M! M! M/G1! M/G1! G1! G1! S! G2! M! M! M/G1!
Moloshok et al, Bioinformatics, 18, 566, 2002
Bayesian Decomposition
Sensitivity
ROC analysis equivalent to NMF results Matrix factorization identifies known coregulation in yeast data
1 - Specificity
Bayesian Decomposition
Finds P Matrix
Determines patterns in the data using bias of positivity and minimal structure For time series, can see make sense
Simultaneously Finds A Matrix

Links genes together despite multiple regulation Column of A becomes SIGNATURE

GIST Experiment Targeted Therapy

GIST-T1 cells in triplicate Agilent arrays Gene level estimates Keep genes with known TF regulation
Matrix Factorization of GIST Data

Time 48 hr
Time = 0

gene 1
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gene N
* * * * * * * * * *

pattern 1

pattern k

D: Data

gene 1
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gene N
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pattern 1
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pattern k

P: Patterns of
Behavior

Time = 48 hr

Time = 0

A: Distribution of
Genes in Patterns

Patterns in the Data

Mathematically - Basis Vectors
Decreases with IM Increases with IM
Transient with IM
Increasing time with IM

Estimating Transcription Factor (TF) Activity

Build a Gene Set of All Targets
Genes known to be regulated by a TF High quality annotations important
Build a Statistical Test for TF

Use sum of individual gene tests Test for significance
Inference on TF Activity
A: Distribution of
For each gene Genes in Patterns

gene 1
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gene 1363
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and from MCMC 500,000 samples from posterior distribution Zip = ip / ip
pattern 1

pattern 5

Calculate Z-score Calculate Z-score for TF
1 Ztp = Z rp R r Gt
Significance of Ztp
Need distribution of Ztp for
Different Rs Different Patterns
Permutation Test
For each pattern generate Ztp for random collection of R genes

Linking to Pathway Activity For now Visually

Convert TF p-value to TF Activity
Rescale p < 0.5 for over-representation to a 0 - 1 scale Rescale p < 0.5 for under-representation to a -1 - 0 scale
Visualize as Blue (Low Activity) - Yellow (High Activity) with Pathway Maps Look for Coordinated Changes
Network Activity TF Target Genes Not Shown
Decreases with IM Transient with IM Increases with IM

Biological Validation DNA Damage to p53 Activation
Biological Validation STAT3 and ELK1 Activity

STAT3 and ELK1 Activity
Advantages of MCMC Sampling

coGAPS: sampling NMF: best 50 of 500
Decreases with IM
Transient with IM
Increases with IM
Significant High Active TF Significant Low Inactive TF
Summary of Matrix Factorization

Matrix Factorization Recovers Coregulation better than Clustering MCMC Sampling Recovers Signatures of Biology better than NMF Costs
Bias of NMF > Bias of Clustering Complexity of MCMC > Complexity of NMF
Tumor Responses Method Works in Clinical Data
Cell Line Increases with IM
Tumor Pattern A
RTOG 0132 Phase II Trial
Initial Clinical Response

Group A vs Group B
>20% Shrinkage
Logistic Regression
RTOG 0132 Survival

Good Initial Tumor Response May Correlate with Cancer Stem Cell Activation 3 Year PFS Does NOT Correlate with Initial Response
DESIDE
Differential Expr for Signaling Determination
Inference on Activity of Downstream Signaling Effectors Global Unbiased Measure Multiple Signaling Proteins Potentially Give Same Transcriptional Signature Some Important Signaling Molecules Change Translation Only (mTOR)
The Role of Methylation

Transcription Factors
Function by turning on genes by binding to DNA in promoters Promoters must be accessible
Methylation
Of promoters silences genes Of histones can also silence genes
Transcription - Epigenetics
TF
TFBS
BTM
CGCGATACGCG
~1500 5000 bases
Copy Number Effects

Loss of Gene
Obviously this will also affect the expression of gene LOH can also affect amount of transcript
Measurements
Because of tumor heterogeneity (some normal tissue, infiltrates), exact measurement is difficult
Simplest Correction
Gene Set for Transcription Factor
Group of genes regulated by TF If gene CANNOT be regulated, remove from the Gene Set
Filtered Gene Set Analysis

Each set is adjusted for methylation and CNV Requires cut-off and patient-specific measurement
Filtered Gene Set Test

Patient 1
Patient M

TF Targets Gene A Gene B Gene C Gene D Gene E
TF Targets 1 Gene A Gene B Gene D Gene E
gene 1
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Gene C * * * * * * * * * *
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Gene B * * * * * * * * * *
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Gene E * * * * * * * * * *
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gene N
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Methylation

TF Targets TF Targets Gene A TF Targets Gene A TF B A Gene Targets Gene TF B A Gene Targets Gene Gene TF Targets M C Gene BTargets Gene Gene TF BAA C Gene Gene Gene B A DGene Gene Gene C Gene Gene B D Gene C Gene Gene C E Gene D Gene Gene C E Gene D Gene Gene C E Gene Gene EDD Gene Gene E D Gene Gene E
New Statistic on Genes Needed

Gene Set Analysis
Depends on ranking of genes between two groups But have ELIMINATED ONE GROUP, each patient enters individually
Generate a t-like statistic

Provides a signed statistics suitable for mixed
A Simple Modified t-Statistic

= x is x iN t is iN
xis expression of gene i in patient s xiN mean expression of gene i in normal samples
iN standard deviation of gene i in normal samples
HNSCC Data
ARRA Funded Biomarker Study
69 Samples: 44 HNSCC, 25 UPPP
Transcripts Promoter Methylation

16330 X 69 12033 X 69
Copy Number Variation
14599 X 69
Joe Califano, Patrick Hennessey
Exploring Impact of FGSA Filtered Gene Set Analysis

Vary Cutoffs
Methylation and copy number to see impact on estimation of p-value Compare with GSA
Biological Validation
See predicted changes and try to validate Problem: tumor samples are difficult to work with for validation
Gene Sets
TRANSFAC Professional 2010.4
1325 TFs filtered to keep only those with 5 unique targets 230 TFs for Analysis
FGSA
TFs can have fewer targets for any individual patient due to filtering
Most Varying Significant TFs

Color Key and Histogram
Count
pTF2512 Methylation Beta as 0.NN CNV as N.N
0 50
150
0.2
Value
0.4
0.6
HSF2A HSF1L GABPbeta NFYA STAT3 STAT1alpha E2F1 p50:RelAp65 cFos:cJun NFkappaB1p50 LRF STAT5 AP2 E2F4 NFkappaB1 PPARalpha:RXRalpha RXRalpha:PPARgamma RXRalpha:PPARalpha STAT3:STAT3 STAT1:STAT1 sp4 AhR:arnt usf1:USF2 PPARalpha ATF1 NF1 MyoD
pTF100
pTF1515
pTF1512
pTF1508
pTF1500
pTF4515
pTF4512
pTF4508
pTF4500
pTF3515
pTF3512
pTF3508
pTF3500
pTF2515
pTF2512
pTF2508
pTF2500
Highlights
Some TFs Show Changes
Methylation and CNV affect p-value estimates of activities for some TFs
Some TFs Show No Significant Change Methylation Dominates over CNV

Total CNV range captured in each methylation range by clustering
Methylation Expression
FADS2 correlation: 0.335633632853448

Normal HPV HPV+
10
Log2 Expression
0.0
0.2
0.4
0.6
0.8
1.0
Methylation Beta
NR1D1 correlation: 0.00821586442546115

8.5
Normal HPV HPV+
Log2 Expression
6.5
7.0
7.5
8.0
0.0
0.2
0.4
0.6
0.8
1.0
Methylation Beta
FABP1 correlation: 0.0441809836195627

Normal HPV HPV+
Log2 Expression
4.0
4.5
5.0
3.0
3.5
0.0
0.2
0.4
0.6
0.8
1.0
Methylation Beta
Summary
Abstraction
Reduce complexity of signaling networks Link networks to effectors (TFs)
Bias
NMF/MCMC necessary but introduces Bias TF activity defined by known target genes
Experimental Validation
Biological complexity means models limited
Acknowledgements
Research Signaling and TFs: Ghislain Bidaut (CRC-Marseille) Andrew Kossenkov (Wistar) Computational Modeling: Elana Fertig, Luda Danilova Collaborators HNSCC: Joseph Califano GIST: Andy Godwin (KUMC) Genetics: Giovanni Parmigiani (DFCI, Harvard) Olga Favorova (RSMU)
New MCMC Techniques: Network Modeling: Elana Fertig, Alexander Favorov Donald Geman (JHU) Laurent Younes (JHU) Genetics of Complex Diseases: Alexander Favorov

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Lecture 3 (M. Ochs)

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From Expression Measurements to Cell Signaling Inference

Michael Ochs The Sidney Kimmel CCC Johns Hopkins University

Focusing on Cell Signaling

Methylation, SNPs, CNVs

Cancer Revisiting Hallmarks

FAK NEDD9 p53 hTERT

Oncology Biostatistics and Bioinformatics

Hanahan and Weinberg, Cell, 100, 57, 2000

p53 MDM2 ATM BRCA1 BRCA2

Cytokines and Receptors

Hanahan and Weinberg, Cell, 44, 646, 2011

Expression of Signaling Proteins

Transcriptional Signatures for Signaling M

Use Expression as Downstream Marker!!

Transcription as Biomarker for Cell Signaling

Oncology Biostatistics and Bioinformatics

Cell Cycle Data Revisited

Time series of microarray measurements

6000 yeast genes

Validation Gold Standard Coregulation

Cherepinsky et al, PNAS, 100, 9668, 2003"

ROC Curve for Clusters Revisited

Oncology Biostatistics and Bioinformatics

Principal Component Analysis

Oncology Biostatistics and Bioinformatics

PCA of Cell Cycle Data

Oncology Biostatistics and Bioinformatics

Fails to Correctly Link Genes

Oncology Biostatistics and Bioinformatics

Patterns Nonorthogonal in General

Link Conditions Time Series

show when process is on show change in activity

group patients with similar expression behaviors

Genes Among Patterns

Bias over Variance

Oncology Biostatistics and Bioinformatics

Lee and Seung Nature, 401, 788, 1999

Minimize a Cost Function

Pick next element

By Moving to Local Minimum in Cost

Oncology Biostatistics and Bioinformatics

Fox Chase Cancer Center

Fox Chase Cancer Center

Oncology Biostatistics and Bioinformatics

Wang et al, BMC Bioinfo, 28, 7, 2006

Poor Recovery in Complex Cases

Oncology Biostatistics and Bioinformatics

Markov Chain Monte Carlo Bayesian Decomposition

Markov Chain Monte Carlo

Markov Chain Monte Carlo is

Fertig et al, Bioinformatics, 2010

Sampling of Posterior Distribution

Evidence (Marginal of Data)

Oncology Biostatistics and Bioinformatics

Bayes Equation (independently by Laplace)

* * * * * * * * **** * * * * **** * * * * * * * * **** **** **** **** ****

Positive Additive Distribution

*Sibisi and Skilling, J Royal Stat Soc B, 59, 217, 1997

3500 For q = 200

First for A Then for P

With low average atom flux

Oncology Biostatistics and Bioinformatics

With high average atom flux

* * * * * * * * **** * * * * **** * * * * * * * * **** **** **** **** ****

Oncology Biostatistics and Bioinformatics

Exchanging Flux/Moving Atoms

Pick Atom at Random Find its Neighbor

* * * * * * * * **** * * * * **** * * * * * * * *

* * * * * * * * **** * * * * **** * * * * * * * *