Fusion Genes in Cancer. Wednesday, June 27th
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1 Fusion Genes in Cancer Wednesday, June 27th
2 Outline: Transcription Fusion genes Examples of fusion genes in cancer Summary and conclusions
3 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
4 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, (April 2002)
5 Corepressors Coactivators Gene Transcription DNA RNA PROTEIN
6 Fusions genes in cancer The Cancer Genome Project website lists at least 326 genes that have been shown to form gene translocations in cancer
7 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
8 Fusion genes result in aberrant gene expression N C Gene 2 expression and transcriptional regulation is now dictated by the Gene 1 promoter and all its regulatory units Gene 1 Promoter + Part of Gene 1 coding region 5 Gene 1 Gene 2 3 Promoter Region Coding Region Gene 1 Truncation Gene 2 Truncation If Gene 1 has a highly active promoter region, Gene 2 will be overexpressed Part of Gene 2 coding region Coding Region Gene 1 Promoter Part of Gene 2 coding region 5 Gene 1 Gene 2 3 Promoter Region Gene 1 Truncation Gene 2 Truncation Coding Region
9 Fusion genes DNA Is lost Genes X and Y X X Y Y 5 Wild Type 5 Deletion 3 X Y DNA Is lost DNA Is gained?? 3 TEL 3 Y Promoter 12 WT TEL 12 WT 21 AML Promoter 21 AML TEL 3 X Y AML TEL 5 Unbalanced Translocation 5 5 DNA is neither gained nor lost Balanced Translocation 5 TEL-AML1 (ALL) t(12 ; 21)
10 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
11 Fusion genes are detected in patient samples using Fluorescent In Situ Hybridization (FISH) Wild Type Deletion Hofer et al. (2009) Cancer Research Insertion TMPRSS2 ERG Wild Type TMPRSS2- ERG Deletion (Intronic deletion) TMPRSS2- ERG ?? Unbalanced Translocation (Rearrangement, Insertion)
12 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 Fusion gene Reverse primer
13 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
14 Examples of fusion genes in cancer Bcr-Abl (CML and ALL) PML-RARα (AML) liquid cancer (leukemia) TMPRSS2-ERG EML-ALK (lung cancer) (prostate cancer) Solid tumors
15 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% (Children) CML rare in children ALL (Adults) 25-30% (Children) 2-10%
16 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
17 Fusion genes in cancer: Bcr-Abl (CML and ALL) Oncogenic properties of Bcr-Abl lending to the development of leukemia
18 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
19 Fusion genes in cancer: PML-RARα (AML) t(15;17) Prevalence of PML-RAR α AML (Adults) 12.5% (Children) 15% Bhatia et al. (2012) Mediterr J Hematol Infect Dis
20 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 Sin3 Ncor HDAC Transcription of differentiation genes RA response element
21 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]
22 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
23 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 TMPRSS2 ERG ERG Target Gene ERG ERG Target Gene ERG ERG Target Gene PTEN loss and/or AKT activation = Androgen Receptor = Androgen Invasive carcinoma St. John et al. (2012) - J Cancer Sci Ther - In Press
24 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
25 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
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