Analyzing dysregulation of intracellular signaling pathways in HTLV-1 transformed cells using Massively Parallel Signature Sequencing (MPSS)

1. HTLV-1 induced Adult T-cell Leukemia (ATL):
“The Phantom Menace”
Harshawardhan Bal, Ph.D.
Department of Cancer Immunology and AIDS
Dana-Farber Cancer Institute

2. Adult T-cell Leukemia
ATL identified as a distinct clinical entity in 1977
ATL is a fatal malignancy of mature CD4+ T-cells
Clonal expansion of CD4+CD25+ T-cells
Impaired cell-mediated immunity, opportunistic infections, lymphoid organ infiltration

3. Human T-cell Lymphotropic Virus Type I
Causative agent of ATL identified in 1980
The first retrovirus shown to be associated with human disease
Cellular receptor is the major glucose transporter GLUT1

4. Organization of the HTLV-1 Genome
ORFs involved in viral infectivity, maintenance of high viral loads, host cell activation
p12, p27, p13, and p30
tax, rex
Regulatory genes: transcriptional/post-transcriptional regulators of viral gene expression
HTLV-1 transactivator Tax, the major viral oncoprotein

5. Pleiotropic Effects of HTLV-1 Tax
Tax inhibits apoptosis in infected T-cells
Tax causes genomic instability
Tax affects host DNA repair mechanisms
Tax disregulates the cell cycle machinery
Tax causes neoplastic transformation

6. ATL Clinical Course (“The Dark Side”)
ATL cells lose HTLV-1 gene expression
Selective methylation of 5’ LTR and silencing of viral gene Txp
Nonsense or missense mutation of the tax gene in fresh ATL cells
No cell-free infectious HTLV-I particles shed
ATL develops after a latency period of > 30 years
Chronic CTL response indicates HTLV-1 genes are expressed persistently
Aggressive clinical course, MST of 3-6 months
No treatment currently exists for ATL
Infects 15-25 million people worldwide including Southeastern United States

7. Massively Parallel Signature Sequencing (MPSS)
Global transcription profiling platform
Comprehensive and quantitative measurement, sequences > 1 M beads
Provides 17-20 mer “signatures” of sampled transcripts
Transcript abundance as “transcripts per million (TPM)”
Very high dynamic range - single mRNA copy per cell
3’ UTR signatures identify homologous members of genes
Not limited to a pre-determined set of genes
Powerful discovery research tool

8. Massively Parallel Signature Sequencing (MPSS)

9. Massively Parallel Signature Sequencing (MPSS)
Sequence

10. MPSS Data Format
Signature p-value Control TPM/SD Exp TPM/SD Class Annotation
Class 1: Transcripts with poly (A) signal + poly (A) tail
Class 2: Transcripts with poly (A) signal
Class 3: Transcripts with poly (A) tail

11. Activated CD4+ T-cells vs Acutely HTLV-1 Transformed Cells
PHA/IL-2 activated CD4+ T-cells
Immortalized (IL-2 dependent)
MPSS
comparison
Transformed (IL-2 independent)
Abundantly
express Tax
Acutely transformed (B1 and C5 cells)
Activated CD4+ T-cells vs B1 Activated CD4+ T-cells vs C5

12. Mining MPSS Data
Signature p-value Control TPM/SD Exp TPM/SD Class Annotation

13. Mining MPSS Data
Which signatures/transcripts are the most reliable?
Which signatures/transcripts are differentially expressed?
What are the molecular functions of these differentially expressed signatures?
Which signatures/transcripts interact with each other?
What are the common pathways that they participate in?
Which pathway members are simultaneously up- or downregulated?
How do these transcripts contribute to leukemogenesis?

14. Pipeline for Analysis of MPSS Data
Signatures with UniGene Ids (Activated vs Transformed)
Filter signatures by p-value (< 0.001) AND Class (1-3)
Filter signatures by TPM
Upregulated signatures Downregulated signatures
Classification by molecular function/biological process
Apoptosis Cell cycle Oncogenesis
Anti-apoptosis DNA repair

15. Querying MPSS Data for Differential Expression

16. Viewing MPSS Data Graphically
[ Upregulated ]
[ Downregulated ]
(Exp TPM, Control TPM)

17. Analysis of Deregulation (Clustering with Spotfire)
1.37E4 0
c472tpm b1tpm c5tpm
Control HTLV-B1 HTLV-C5
0 500 1000 TPM

18. Data Integration, Text Mining and Analysis
Perl
PubMed
Data integration
MPSS
UniGene
………
………
……… LocusLink
………
OMIM
GOTM
Protein-protein interactions
Known disease correlations
Relation to apoptosis, cell cycle, DNA damage, …
Previously reported interactions with HTLV-1 genes

19. Detailed Gene Summaries
Extract gene aliases (HUGO names), RefSeq summaries and associated GO terms

20. PubMed Literature Search
Extract previously reported findings (if any) from PubMed using keywords

21. Text Mining Abstracts for Interactors & Related Genes
Genes reported in PubMed literature that Genes that interact with XRCC5 and
are known to interact with XRCC5 are simultaneously detected by MPSS

22. Upregulation of Anti-apoptotic Genes

23. Upregulation of NF-kB Subunits/Inducing Kinases

24. Upregulation of BCL2 Family/NF-kB: Role of Akt1
NF-kB stimulates the expression of Akt1
Akt1 regulates T-cell survival via activation of NF-kB
Akt1 regulates the expression of anti-apoptotic BIRC3 and BCL2
Akt1 phosphorylates BAD and releases pro-apoptotic bcl-xL
Akt1 induces Txp of c-FLIP which inhibits TNFR-induced apoptosis
Hypothesis: Akt1 is constitutively activated in ATL

25. Downregulation of TNFR Superfamily

26. Deregulation of TNFR Superfamily: Role of GITR

27. Deregulation of TNFR Superfamily: Role of GITR
A T helper cell activation marker
Highly expressed on CD3+CD4+CD25+ regulatory T cells
T regs profoundly suppress host immune responses and antitumor immunity
GITR involved in the negative regulation of T-cell activation and T-cell apoptosis
Hypothesis: Inhibition of GITR-mediated signaling may abolish T-cell survival in ATL

28. Deregulation of TNFR Superfamily: Role of CD27/CD27L
CD27/CD70 interactions are important for maturation/activation of B- & T-cells
CD70-expressing glioma cells induce release of soluble CD27 from PBMCs
CD70 mediates immune escape of human malignant gliomas
Inverted profile observed in lymphoproliferative diseases such as CLL and NHL
Hypothesis: Overexpression of CD70 may be linked to enhanced T-cell survival in ATL

29. Deregulation of TNF Superfamily Associated Genes
FADD CASP8
TNFα TNFR1 TRADD RIPK1
CRADD CASP2

30. Survival Pathways in Acutely HTLV-1 Transformed Cells
HTLV-1 Tax/?
(PI3K)
p p p
bcl-xL +p +p
BAD Akt? IKK NF-κB IκB
NIK/COT
+ +
+ +
+ +
bcl-xL BCL2 BIRC3
NF-κB BCL2
Neoplastic c-FLIP
transformation bcl-xL
CASP8
Cytochrome c
Genetic
alterations
Drug
resistance FADD
CRADD RIPK1
TRADD
TNFR1/TNFR2/DR3 Apoptotic signals
TNFα

31. MPSS Analysis of ATL: Insights & Conclusions
Survival of HTLV-1 transformed T-cells due to activation of NF-kB and PI3K/Akt pathways
and downregulation of TNF-mediated apoptotic pathways
Akt1 and NF-kB may act in concert to form a perpetual potentiating loop and enhance
T-cell survival
Downstream effects of survival pathways likely mediated by anti-apoptotic BCL2 (BCL2,
bcl-xL) and IAP family members (BIRC3)
Putative Tax-independent mechanisms of NF-kB activation explained via Akt1
Akt1 and BCL2/bcl-xL may explain drug resistance and genetic alterations in ATL
Elevated GITR may suppress T-cell immunity and antitumor responses
High CD70/low CD27 may help HTLV-1 evade immune response

32. Acknowledgements
The Marasco Lab @ DFCI (2003-2004)
Aki Murakami Jihua Cheng Aimee Tallarico Leslie Matthews
Quan Zhu Keiko Azuma Jianhua Sui Wayne Marasco
Wei Wang & Tom Vasicek, Lynx Therapeutics
(and George Lucas, Industrial Light & Magic)

33. Contact
Harshawardhan Bal, M.Pharm., PhD
harsh.bal@gmail.com
Sponsored by
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