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
R
Gene Expression
Beads = Histone proteins
String = DNA
Histone proteins are positively charged
DNA is negatively charged
Acetyl groups neutralize the positively
charged histone proteins

Nature Reviews Drug Discovery 1, 287-299 (April 2002)
Histone Acetyltransferase
(HAT) adds acetyl group
to histone protein

Histone Deacetylase (HDAC) -
Removes acetyl group from
histone protein
Gene Transcription
DNA

RNA

PROTEIN
Corepressors
Coactivators
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
ftp://ftp.sanger.ac.uk/pub4/theses/kong/chapter4.pdf
A fusion gene is a hybrid gene
formed from two previously
separate genes.
(Wikipedia)
Fusion genes result in aberrant gene expression
N
C
Gene 1 Promoter +
Part of Gene 1 coding region
5
3 Gene 1
Gene 2
Promoter
Region
Coding Region
Gene 1
Truncation
Gene 2
Truncation
Coding Region
Part of Gene 2 coding region
Part of Gene 2 coding region
5
3
Gene 2
Promoter Region
Gene 2 Gene 1
Coding Region
Gene 1 Promoter
Gene 1
Truncation
Gene 2
Truncation
Gene 2 expression and transcriptional
regulation is now dictated by the Gene 1
promoter and all its regulatory units
If Gene 1 has a highly active promoter
region, Gene 2 will be overexpressed
Fusion genes
12 12
? ?
5
3
5
3
Unbalanced
Translocation

12 12
21
5
3
5
3
Balanced
Translocation

21
TEL-AML1
(ALL) t(12 ; 21)
X
X
Y
Genes X and Y
Y
TEL
TEL
AML
DNA
Is lost
DNA
Is gained
12 12
5
3
Wild Type
X
Y
12 12
5
3
Deletion
X
Y
DNA
Is lost
WT
WT
TEL
AML AML
TEL
Promoter
Promoter
DNA is neither
gained nor lost
Y
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)
21 21
5
3
T
M
P
R
S
S
2
-

E
R
G

Deletion
(Intronic deletion)
21 21
? ?
5
3
T
M
P
R
S
S
2
-

E
R
G

5
3
Unbalanced Translocation
(Rearrangement, Insertion)
21 21
5
3
T
M
P
R
S
S
2

E
R
G

Wild Type
Hofer et al. (2009) Cancer Research
Wild Type
Deletion 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
Forward primer
Reverse primer
Fusion gene
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)
PML-RAR (AML)
TMPRSS2-ERG (prostate cancer)
EML-ALK (lung cancer)
liquid cancer (leukemia)
Solid tumors
Poster child for fusion genes in cancer due to the development of the
drug Imatinib
Philadelphia
chromosome
Fusion genes in cancer: Bcr-Abl (CML and ALL)
http://www.cancer.gov/cancertopics/pdq/treatment/adultALL/Patient/page1
t(9;22)
CML (Adults) 90% (Children) CML rare in children
ALL (Adults) 25-30% (Children) 2-10%
Prevalence of Bcr-Abl in leukemia patients
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
N C
Fusion protein
http://www.medscape.org/viewarticle/416483_2
Fusion genes in cancer: Bcr-Abl (CML and ALL)
http://www.medscape.org/viewarticle/416483_2
Oncogenic properties of Bcr-Abl lending to the development of leukemia
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-RAR (AML)
t(15;17)
http://flipper.diff.org/app/items/info/482
AML (Adults) 12.5% (Children) 15% Prevalence of PML-RAR
Bhatia et al. (2012) Mediterr J Hematol Infect Dis
Fusion genes in cancer: PML-RAR (AML)
Upon DNA binding, the PML-RAR 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
The RAR 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
PML-RAR recruits
corepressors
Transcription of
differentiation genes
RA response element
Corepressors
Ncor HDAC
Sin3
Fusion genes in cancer: PML-RAR (AML)
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/
Treatment of AML (PML-RAR)

1.) Differentiation inducing agents (e.g. all-trans retinoic acid (ATRA))




2.) Chemotherapy agents (e.g. cytarabine and anthracycline)
Induce the immature blast cells into terminal differentiation and
replenish the mature red blood cell population in patients
Kill off the remaining immature AML blasts from patients blood
stream
Fusion genes in cancer: TMPRSS2-ERG (prostate cancer)
The TMPRSS2-ERG fusion gene is present in approximately 50% of prostate
cancer patients
Tomlins et al. (2009) European Urology
Chromosome 21
Fusion genes in cancer: TMPRSS2-ERG (prostate cancer)
TMPRSS2 ERG TMPRSS2 TMPRSS2 ERG ERG
ERG
ERG Target Gene ERG Target Gene
ERG
ERG Target Gene
ERG
= Androgen = Androgen Receptor
PTEN loss and/or
AKT activation
Invasive carcinoma
St. John et al. (2012) - J Cancer Sci Ther - In Press
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
Summary and Conclusions
ALL
Better detection, new drugs, and better use of classical drugs
Some encouraging
proof to not give up
on the cure for cancer
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
Questions ?
Knowledge is power and knowing is half the battle! (Mr. Joe, G.I.)