Characterization of a New Fusion Gene in Acute Myeloid Leukemia

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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

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Why a Ph.D. is for Me

• Educational and Scientific Research Experiences
• Professoriate
• Applied vs. Basic Research
• Basic research research driven by scientists
  curiosity or interest in a scientific question
• Applied research research designed to solve
  practical problems rather than acquire knowledge
  for knowledges sake
• Career Goals and Options

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Introduction

• Cancer genetics
• Cytogenetics
• Studying the structure of chromosome material
• Cancer
• Leukemia
• Acute Myeloid Leukemia

http//en.wikipedia.org/wiki/ImageBcrablmet.jpg
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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
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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

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Background DNA Chromosomes
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ary/Illustration/chromosome.cfm?keychromosome htt
p//www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary
/Illustration/gene.cfm?keygene
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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
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Background Etiology of Cancer

• 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

Campisi J. 2003. Nat Rev Cancer. 3339-49.
Chen Z, Sandberg AA. 2002. Am J Med Genet.
115130-41.
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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
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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

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Background Hematological Malignancies

• Hematological Malignancies
• Cancers that affect the blood, bone marrow and
  lymph node
• Lymphoma and Leukemia
• Lymphoma
• Hodgkins disease
• Non-Hodgkin lymphoma
• Leukemia

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Background Hematological Malignancies

• 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

http//www.bloodlines.stemcells.com/img/Metcalf_Fi
g3_2.gif
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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

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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

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Molecular Cytogenetics Genetics Techniques

• Fluorescence in situ hybridization (FISH)

http//en.wikipedia.org/wiki/ImageFISH_28techniq
ue29.gif
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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/ImagePcr.pngfile
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Cloning of DNA sequences
http//employees.csbsju.edu/hjakubowski/classes/Sr
SemMedEthics/Human20Genome20Project/plasmid.gif
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Identification and Characterization of a New
Oncogenic Fusion Gene in Acute Myeloid Leukemia
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FISH Analysis on 17p

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

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Identification of a Candidate Gene for the 1117
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/Gene
s/NUP98.html
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Identification of NUP98s Involvement in 1117
Translocation

• Created two probes that contained sequence from
  the 5 and 3 end of the NUP98 gene
• No involvement
• Fused signal
• Involvement
• Split signal

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Translocation Breakpoint Mapping on 17p
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NUP98 Fusion Gene Partner Candidate Gene

• Candidate Gene
• PHF23

Modified from NCBI Map Viewer http//www.ncbi.nlm.
nih.gov/mapview/map_search.cgi?taxid9606query54
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RT-PCR Amplification and Sequence Analysis of
NUP98-PHF23 Fusion Transcript

• NUP98-PHF23 Gene Fragment
• Full-length NUP98-PHF23 Fusion Gene

• Create DNA primer for NUP98 and PHF23 gene
• RT-PCR Amplification and visualization
• Clone PCR product
• Perform sequence analysis

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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

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PHF23

• Additional fusion genes leading to AML contain
  PHD domains
• NUP98-NSD1
• NUP98-NSD3

• PHD Finger Protein 23
• PHD motif
• Plant Homeodomain Finger
• Involved in regulating transcription

Jaju RJ, et al. 2001. Blood 981264-7. Rosati R,
et al. 2002 Blood 993857-60.
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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

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Acknowledgements

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