Reader - RAP2-28-07

Characterization of a New Fusion Gene in Acute Myeloid Leukemia : 

Characterization of a New Fusion Gene in Acute Myeloid Leukemia Jocelyn Reader Ph.D. Candidate in Human Genetics University of Maryland Baltimore Advisor: Dr. Yi Ning Feb. 28th, 2007

Why a Ph.D. is for Me : 

Why a Ph.D. is for Me Educational and Scientific Research Experiences Professoriate Applied vs. Basic Research Basic research – research driven by scientist’s curiosity or interest in a scientific question Applied research – research designed to solve practical problems rather than acquire knowledge for knowledge’s sake Career Goals and Options

Introduction : 

Introduction Cancer genetics Cytogenetics Studying the structure of chromosome material Cancer Leukemia Acute Myeloid Leukemia http://en.wikipedia.org/wiki/Image:Bcrablmet.jpg

Societal Impact of Cancer : 

Societal Impact of Cancer In 2007 ~1.44 billion new cases of cancer expected ~560,000 deaths due to cancer in the U.S. > 1,500 people a day Leukemia New cases expected : 44,240 Expected number of deaths : 21,790 American Cancer Society; National Cancer Institute

Societal Impact of Cancer : 

Societal Impact of Cancer Cancer is responsible for more estimated years of life lost than any other cause of death Person-years of Life Lost The difference between the actual age of death due to a cancer and the expected age of death

Background: DNA & Chromosomes : 

Background: DNA & Chromosomes http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/chromosome.cfm?key=chromosome http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/gene.cfm?key=gene

Background: The Central Dogma of Molecular Biology : 

Background: The Central Dogma of Molecular Biology Concept of how genes lead to protein formation Protein domains are sequences that give the protein a specific function http://fig.cox.miami.edu/~cmallery/150/gene/c7.17.12.domains.jpg

Background: Etiology of Cancer : 

Background: Etiology of Cancer Campisi J. 2003. Nat Rev Cancer. 3:339-49. Chen Z, Sandberg AA. 2002. Am J Med Genet. 115:130-41. Cancer Uncontrolled growth of abnormal cells Mutations in DNA sequence or gross chromosomal changes Usually occurring in tumor suppressor genes or proto-oncogenes Deletions or translocations

Background: Tumor Suppressor Genes & Proto-oncogenes : 

Background: Tumor Suppressor Genes & Proto-oncogenes Gatekeepers Tumor Suppressors Proto-oncogene Typically involved in cellular growth Can become an oncogene through several mechanisms Campisi et al. 2003

Background: Chromosomal Alterations : 

Background: Chromosomal Alterations Types Deletions Translocations Why study? Role in disease diagnosis, prognosis and treatment Identification of new tumor suppressor genes or proto-oncogenes Outcome Loss of an important gene(s) Create a new gene with a new function

Background: Hematological Malignancies : 

Background: Hematological Malignancies Hematological Malignancies Cancers that affect the blood, bone marrow and lymph node Lymphoma and Leukemia Lymphoma Hodgkin’s disease Non-Hodgkin lymphoma Leukemia

Slide 12: 

Leukemia Speed of cell growth Acute / Chronic Cell lineage Lymphoid / Myeloid Acute lymphoblastic Acute myeloid Chronic lymphoblastic Chronic myeloid Acute Myeloid Leukemia (AML) Arrests cells in early stage of development Most common acute leukemia in adults Background: Hematological Malignancies http://www.bloodlines.stemcells.com/img/Metcalf_Fig3_2.gif

Patient History : 

Patient History 42-year-old male patient with recurrent AML Presented with a possible abnormality on 17p 4 months after last treatment – patient relapsed with same abnormality

Objectives & Experimental Design : 

Objectives & Experimental Design Identify/ verify the chromosomal anomaly in the patient Fluorescence in situ hybridization (FISH) Identify and characterize the genes involved in the chromosomal anomaly Molecular Genetic Techniques RT-PCR Cloning and Sequencing

Molecular Cytogenetics & Genetics Techniques : 

Molecular Cytogenetics & Genetics Techniques Fluorescence in situ hybridization (FISH) http://en.wikipedia.org/wiki/Image:FISH_%28technique%29.gif

PCR & RT-PCR : 

PCR & RT-PCR Reverse transcriptase – polymerase chain reaction (RT-PCR) Start with RNA instead of DNA First reaction use a RNA-dependent DNA polymerase Make cDNA (complementary DNA) PCR products are visualized by agarose gel electrophoresis Polymerase Chain Reaction (PCR) http://en.wikipedia.org/wiki/Image:Pcr.png#file

Cloning of DNA sequences : 

Cloning of DNA sequences http://employees.csbsju.edu/hjakubowski/classes/SrSemMedEthics/Human%20Genome%20Project/plasmid.gif

Identification and Characterization of a New Oncogenic Fusion Gene in Acute Myeloid Leukemia : 

Identification and Characterization of a New Oncogenic Fusion Gene in Acute Myeloid Leukemia

FISH Analysis on 17p : 

FISH Analysis on 17p FISH Analysis TP53 not affected Sub-telomeric probe shows translocation to 11p

Identification of a Candidate Gene for the 11;17 Translocation : 

Identification of a Candidate Gene for the 11;17 Translocation NUP98 11p15.5 Protein that forms part of nuclear pore complex Promiscuous fusion partner gene plays a role in hematological malignancies http://www.infobiogen.fr/services/chromcancer/Genes/NUP98.html

Identification of NUP98s Involvement in 11;17 Translocation : 

Identification of NUP98s Involvement in 11;17 Translocation Created two probes that contained sequence from the 5’ and 3’ end of the NUP98 gene No involvement Fused signal Involvement Split signal

Translocation Breakpoint Mapping on 17p : 

Translocation Breakpoint Mapping on 17p

NUP98 Fusion Gene Partner Candidate Gene : 

NUP98 Fusion Gene Partner Candidate Gene Candidate Gene PHF23 Modified from NCBI Map Viewer http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606&query=542c16&qchr=&strain=All&advsrch=off

RT-PCR Amplification and Sequence Analysis of NUP98-PHF23 Fusion Transcript : 

RT-PCR Amplification and Sequence Analysis of NUP98-PHF23 Fusion Transcript Create DNA primer for NUP98 and PHF23 gene RT-PCR Amplification and visualization Clone PCR product Perform sequence analysis NUP98-PHF23 Gene Fragment Full-length NUP98-PHF23 Fusion Gene

Schematic of Normal and Predicted Proteins : 

Schematic of Normal and Predicted Proteins NUP98-PHF23 fusion protein combination of NUP98 and PHF23 proteins Predicted to have a function different from the normal proteins

PHF23 : 

PHF23 PHD Finger Protein 23 PHD motif Plant Homeodomain Finger Involved in regulating transcription Additional fusion genes leading to AML contain PHD domains NUP98-NSD1 NUP98-NSD3 Jaju RJ, et al. 2001. Blood 98:1264-7. Rosati R, et al. 2002 Blood 99:3857-60.

Summary and Conclusions : 

Summary and Conclusions Summary Types and causes of leukemia Identified a novel fusion gene Conclusion Why study this fusion gene Possible common mechanism of action for these cancer causing genes New therapeutic drug targets Application to other cancers Future Directions Verify oncogenicity of fusion gene

Acknowledgements : 

Acknowledgements Dr. Yi Ning – Advisor Clinical Cytogenetics Laboratory – University of Maryland Baltimore PROMISE Program in Human Genetics Marlene and Stewart Greenebaum Cancer Center