Powerpoint

Characterization of a New Fusion Gene in Acute Myeloid Leukemia

Jocelyn ReaderPh.D. Candidate in Human

GeneticsUniversity of Maryland Baltimore

Advisor: Dr. Yi NingFeb. 28th, 2007

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

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

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 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

http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/chromosome.cfm?key=chromosomehttp://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/gene.cfm?key=gene

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

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

Gatekeepers Tumor

Suppressors Proto-oncogene

Typically involved in cellular growth

Can become an oncogene through several mechanisms

Campisi et al. 2003

Background: Chromosomal Alterations

Deletion

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

Hematological Malignancies Cancers that affect the blood, bone marrow

and lymph node Lymphoma and Leukemia

Lymphoma Hodgkin’s disease Non-Hodgkin lymphoma

Leukemia

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

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

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

Fluorescence in situ hybridization (FISH)

http://en.wikipedia.org/wiki/Image:FISH_%28technique%29.gif

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

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

FISH Analysis on 17p

FISH Analysis TP53 not

affected Sub-telomeric

probe shows translocation to 11p TP53 (Orange)

17p13.2 (Green)

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

Created two probes that contained sequence from the 5’ and 3’ end of the NUP98 gene

No involvement Fused signal

Involvement Split signal

Nup98cen (Green)Nup98tel (Orange)

RP1-4G17

RP11-542C16

Translocation Breakpoint Mapping on 17p

RP11-186B7 (Green)RP1-4G17 (Orange)

RP11-542C16 (Green)

NUP98 Fusion Gene Partner Candidate Gene

Candidate Gene

PHF23

Component Map

RP11-542C16

RP1-4G17

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

Create DNA primer for NUP98 and PHF23 gene

RT-PCR Amplification and visualization

Clone PCR product Perform sequence

analysis

1. NUP98-PHF23 Gene Fragment

2. Full-length NUP98-PHF23 Fusion Gene

NUP98-PHF23

5’-CCGATGTCAGACCCTAAGAAGAAGGAAGAGGCCCCCGACAGTGCTACCTTGCTTGAGAAG-3’

P M S D P K K K E E A P D S A T L L E K

Schematic of Normal and Predicted Proteins

COOHNH2FG FGGLEBS

NUP98

920 aaNUP98-PHF23

FG GLEBS FGNH2 COOHPHD

797 aa

403 aa

NH2 COOH

PHF23

PHD

Normal and Fusion ProteinsRNP binding domain

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

normal proteins

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

NUP98/NSD3

Jaju RJ, et al. 2001. Blood 98:1264-7. Rosati R, et al. 2002 Blood 99:3857-60.

NH2 COOHPHD

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

Dr. Yi Ning – Advisor Clinical Cytogenetics Laboratory –

University of Maryland Baltimore PROMISE Program in Human Genetics Marlene and Stewart Greenebaum

Cancer Center