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Fusion Genes in Cancer
Wednesday, June 27th
Outline:
Transcription
Fusion genes
Examples of fusion genes in cancer
Summary and conclusions
Chromosomes and Genes
• In humans, every somatic cell has 23 pairs of
chromosomes for a total of 46 chromosomes
in its nucleus (except mature RBC)
• Each chromosome is made up of genes,
and gene expression is a highly regulated
process
Chromatin regulation (epigenetics)
Transcriptional regulation
• Different cell types have different gene
expression patterns, and this results in
cellular phenotype
(skin cell vs. intestinal cell)
Lodish et al. (2000) Molecular Cell Biology
Gene Expression
Beads = Histone proteins
String = DNA
Histone proteins are positively charged
DNA is negatively charged
Acetyl groups neutralize the positively
charged histone proteins
R
Histone Acetyltransferase
(HAT) – adds acetyl group
to histone protein
Histone Deacetylase (HDAC) Removes acetyl group from
histone protein
Nature Reviews Drug Discovery 1, 287-299 (April 2002)
Gene Transcription
Corepressors
Coactivators
DNA
RNA
PROTEIN
Fusions genes in cancer
The Cancer Genome Project website lists at least 326 genes
that have been shown to form gene translocations in cancer
http://www.sanger.ac.uk/genetics/CGP/Census/
Fusion genes
“A fusion gene is a hybrid gene
formed from two previously
separate genes.”
(Wikipedia)
ftp://ftp.sanger.ac.uk/pub4/theses/kong/chapter4.pdf
Fusion genes result in aberrant gene expression
• Gene 2 expression and transcriptional
regulation is now dictated by the Gene 1
promoter and all its regulatory units
N’
C’
• If Gene 1 has a highly active promoter
region, Gene 2 will be overexpressed
Gene 1 Promoter +
Part of Gene 1 coding region
Part of Gene 2 coding region
Promoter
Region
5’
3’
Gene 2
Gene 1
5’
Coding Region
Gene 1
Truncation
Gene 2
Truncation
Coding Region
Gene 1 Promoter
Part of Gene 2 coding region
Gene 1
Gene22
Gene
Coding Region
Promoter Region
Gene 1
Truncation
Gene 2
Truncation
3’
3’
3’
Fusion genes
12 12
12 12
X
Y
X
Genes X and Y
DNA
Is lost
Y
5’
3’
DNA
Is lost
3’
DNA
Is gained
12 12
X
Y
?
5’
Wild Type
3’
TEL Y
Deletion
3’
WT
Promoter
TEL
12 12
WT
21
?
X AML
Y TEL
AML
Promoter
5’
21
Unbalanced
Translocation
5’
5’
DNA is neither
gained nor lost
Balanced
Translocation
AML
TEL
5’
TEL-AML1
(ALL) t(12 ; 21)
Fusion gene detection: cancer diagnostics
• Fusion genes are commonly found in all 4 types of leukemia CML, AML,
CLL, and ALL
CML = chronic myelogenous leukemia
CLL = chronic lymphocytic leukemia
ALL = acute lymphoblastic leukemia
AML = acute myelogenous leukemia
• Leukemia cells can be collected by taking a blood sample from the patient
• Fusion genes are less commonly found in solid tumors, and these tumor
cells can be collected by invasive surgery and biopsy of the tumor
New and more sensitive detection methods are making fusion gene
detection in solid tumors more feasible
• Fusion genes are detected in patient samples using Fluorescent In Situ
Hybridization (FISH) or Polymerase Chain Reaction (PCR)
New methods for future? high throughput sequencing
Fusion genes are detected in patient samples using
Fluorescent In Situ Hybridization (FISH)
Hofer et al. (2009) Cancer Research
Deletion
Wild Type
Insertion
3’
3’
5’
5’
Wild Type
21
TMPRSS2- ERG
ERG
21
3’
21
TMPRSS2- ERG
21
21
TMPRSS2
3’
Deletion
(Intronic deletion)
21
?
5’
?
5’
Unbalanced Translocation
(Rearrangement, Insertion)
Fusion genes are detected in patient samples using
Polymerase Chain Reaction (RT-PCR)
• Extract mRNA from patient tumor sample, use reverse transcriptase to convert
mRNA into cDNA
• Use fusion gene specific primers to amplify cDNA; detect and quantify fusion
gene presence in the patient tumor sample
Fusion gene
Forward primer
Reverse primer
Fusion gene detection: cancer diagnostics
• Fusion genes can serve as prognosis indicators, meaning if the patient
harbors that certain gene fusion in a specific type of cancer the presence
of the fusion can be used as a predictor of cancer aggressiveness
• However, certain fusion genes may indicate poor prognosis, but in some
cases the presence of the fusion gene is actually a good thing for the patient
because certain drugs have been developed that specifically inhibit the fusion
gene
t(8;21) AML1/ETO – Favorable prognosis
t(15;17) PML/RAR – Favorable prognosis
t(9;22) BCR/ABL – Unfavorable prognosis
Hrusak et al. (2002) Leukemia
Examples of fusion genes in cancer
Bcr-Abl
(CML and ALL)
liquid cancer (leukemia)
PML-RARα
(AML)
TMPRSS2-ERG (prostate cancer)
Solid tumors
EML-ALK (lung cancer)
Fusion genes in cancer: Bcr-Abl (CML and ALL)
t(9;22)
• Poster child for fusion genes in cancer due to the development of the
drug Imatinib
“Philadelphia
chromosome”
Prevalence of Bcr-Abl in leukemia patients
CML (Adults) 90%
ALL (Adults) 25-30%
(Children) CML rare in children
(Children) 2-10%
http://www.cancer.gov/cancertopics/pdq/treatment/adultALL/Patient/page1
Fusion genes in cancer: Bcr-Abl (CML and ALL)
BCR – Breakpoint Cluster Region- contains important N-terminal region of
Bcr-Abl fusion protein that is essential for dimerization and Bcr-Abl activation
Abl – nonreceptor tyrosine kinase- The Bcr-Abl fusion protein is a constitutively
Active nonreceptor tryrosine kinase that is overactive in leukemia cell cytoplasm
Fusion protein
N’
C’
http://www.medscape.org/viewarticle/416483_2
Fusion genes in cancer: Bcr-Abl (CML and ALL)
Oncogenic properties of Bcr-Abl lending to the development of leukemia
http://www.medscape.org/viewarticle/416483_2
Fusion genes in cancer: Bcr-Abl (CML and ALL)
Treatment of CML- first line therapy is Bcr-Abl tyrosine kinase inhibitors
First generation – Imatinib (Gleevec)
Small molecule TK inhibitor, binds to and inhibits ATP binding pocket of
Bcr-Abl kinase (can be used in combination with standard chemotherapy)
Problem: drug resistance, mutations in Bcr-Abl that render the kinase no longer
Inhibited by Imatinib.
Solution: develop new inhibitors similar to Imatinib, but better
Second generation – Dasatinib, Nilotinib
Small molecule TK inhibitor, inhibits Bcr-Abl with higher affinity than Imatinib
Fusion genes in cancer: PML-RARα (AML)
t(15;17)
Prevalence of PML-RAR α
Bhatia et al. (2012) Mediterr J Hematol Infect Dis
AML (Adults) 12.5% (Children) 15%
http://flipper.diff.org/app/items/info/482
Fusion genes in cancer: PML-RARα (AML)
• The RARα gene encodes for a transcription factor known as the Retinoic
Acid Receptor. This receptor is important in activating RBC development
and maturation via binding to target genes and inducing their expression.
Thus, RAR is essential for the induction of RBC differentiation and proper
RBC development
• Upon DNA binding, the PML-RARα fusion protein causes RAR to recruit
corepressors to its target genes, inhibiting their transcription activation.
This results in the rapid accumulation of numerous immature RBCs and the
depletion of normal mature RBCs
PML-RAR recruits
corepressors
Corepressors
Ncor
Sin3
RA response element
HDAC
Transcription of
differentiation genes
Fusion genes in cancer: PML-RARα (AML)
Treatment of AML (PML-RARα)
1.) Differentiation inducing agents (e.g. all-trans retinoic acid (ATRA))
• Induce the immature blast cells into terminal differentiation and
replenish the mature red blood cell population in patients
2.) Chemotherapy agents (e.g. cytarabine and anthracycline)
• Kill off the remaining immature AML blasts from patients blood
stream
“The presence of a PML-RARA fusion predicts a favorable response to
differentiation therapy with all-trans retinoic acid (ATRA) and is currently
the most curable subtype of acute myeloid leukemia (AML).”[1-5]
http://www.cancergeneticsitalia.com/dna-fish-probe/pmlrara/
Fusion genes in cancer: TMPRSS2-ERG (prostate cancer)
• The TMPRSS2-ERG fusion gene is present in approximately 50% of prostate
cancer patients
Chromosome 21
Tomlins et al. (2009) European Urology
Fusion genes in cancer: TMPRSS2-ERG (prostate cancer)
• The TMPRSS2-ERG gene fusion results in AR induced overexpression of the
transcription factor ERG in prostate tumor cells
• The prognostic value of TMPRSS2-ERG fusion genes in prostate cancer remains
controversial. There are currently no drugs targeting this fusion gene used in the
clinic to treat prostate cancer. It was discovered at U of M in 2005
ERG
TMPRSS2
ERG
TMPRSS2
ERG
ERG Target Gene
= Androgen Receptor
ERG
ERG
ERG Target Gene
TMPRSS2
ERG
PTEN loss and/or
AKT activation
ERG Target Gene
= Androgen
Invasive carcinoma
St. John et al. (2012) - J Cancer Sci Ther - In Press
Summary and Conclusions
ALL
Some encouraging
proof to not give up
on the cure for cancer
Better detection, new drugs, and better use of classical drugs
Summary and Conclusions
• Selective therapies that target gene fusions in cancer have been successful in
some cases and have increased overall survival rates for many patients
who harbor these fusion genes, especially in leukemia
• The search for new selective therapies will most likely continue to prove
beneficial in many cases, especially when treatment is combined with
classical chemotherapy drugs (e.g. Methotrexate)
• New and more sensitive diagnostic techniques will be invaluable to future
detection methods. This will hopefully yield more information in the detection
of fusion genes in patients samples, especially in solid tumors
“Knowledge is power and knowing is half the battle!” (Mr. Joe, G.I.)
Questions ?