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 ExpressionBeads = Histone proteinsString = DNA

Histone proteins are positively chargedDNA is negatively chargedAcetyl groups neutralize the positively charged histone proteins

Nature Reviews Drug Discovery 1, 287-299 (April 2002)

Histone Acetyltransferase(HAT) – adds acetyl groupto histone protein

Histone Deacetylase (HDAC) -Removes acetyl group fromhistone 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 2Promoter

RegionCoding Region

Gene 1Truncation

Gene 2Truncation

Coding Region

Part of Gene 2 coding region

Part of Gene 2 coding region

5’ 3’Gene 2Promoter Region

Gene 2Gene 1Coding Region

Gene 1 Promoter

Gene 1Truncation

Gene 2Truncation

• 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

1212? ?

5’

3’

5’

3’

Unbalanced Translocation

121221

5’

3’

5’

3’

Balanced Translocation

21

TEL-AML1 (ALL) t(12 ; 21)

X

XY

Genes X and Y

Y

TEL

TELAML

DNA Is lost

DNA Is gained

12 12

5’

3’

Wild Type

X

Y

1212

5’

3’

Deletion

XY

DNA Is lost

WT

WT

TELAMLAML

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 leukemiaCLL = chronic lymphocytic leukemiaALL = acute lymphoblastic leukemiaAML = 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)

2121

5’

3’

TMPR

SS2-

ERG

Deletion (Intronic deletion)

2121

? ?

5’

3’

TMPR

SS2-

ERG

5’

3’

Unbalanced Translocation(Rearrangement, Insertion)

21 21

5’

3’

TMPR

SS2

ERG

Wild Type

Hofer et al. (2009) Cancer Research

Wild TypeDeletion 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 prognosist(15;17) PML/RAR – Favorable prognosist(9;22) BCR/ABL – Unfavorable prognosis

Hrusak et al. (2002) Leukemia

Examples of fusion genes in cancer

Bcr-Abl (CML and ALL)

PML-RARα (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 childrenALL (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 ofBcr-Abl fusion protein that is essential for dimerization and Bcr-Abl activation

Abl – nonreceptor tyrosine kinase- The Bcr-Abl fusion protein is a constitutivelyActive 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 longerInhibited by Imatinib.

Solution: develop new inhibitors similar to Imatinib, but better

Second generation – Dasatinib, NilotinibSmall molecule TK inhibitor, inhibits Bcr-Abl with higher affinity than Imatinib

Fusion genes in cancer: PML-RARα (AML)t(15;17)

http://flipper.diff.org/app/items/info/482

AML (Adults) 12.5% (Children) 15%Prevalence of PML-RAR α

Bhatia et al. (2012) Mediterr J Hematol Infect Dis

Fusion genes in cancer: PML-RARα (AML)

• 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

• 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

PML-RAR recruits corepressors Transcription of

differentiation genes

RA response element

Corepressors

Ncor HDACSin3

Fusion genes in cancer: PML-RARα (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-RARα)

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 ERGERG

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.)