Dr. R. Keith Humphries, Research Supervisor (Medicine)
Dr. Hugh Brock (Zoology)
Dr. Donna Hogge (Medicine)
EXAMINING COMMITTEE
Chair: Dr.
Gerald Weeks (Microbiology & Immunology)
Supervisory
Committee: Dr. R. Keith Humphries, Research Supervisor (Medicine), Dr.
Hugh Brock (Zoology), Dr. Donna Hogge (Medicine)
University
Examiners: Dr. Aly Karsan (Pathology and Laboratory Medicine), Dr.
Kelly McNagny (Medical Genetics)
External Examiner: Dr. Yaacov Ben-David, Sunnybrook &
Women College’s Health Sciences Centre, University of Toronto. Toronto, Ontario
ABSTRACT
Chromosomal rearrangements of the 11p15 locus have been
identified in hematopoietic malignancies, resulting in translocations involving
the N-terminal portion of the nucleoporin gene NUP98. Sixteen different
fusion partner genes have been identified for NUP98 and over half of these are
homeobox transcription factors. By contrast, the NUP98 fusion partner in t(11;20) is
Topoisomerase I (TOP1), a catalytic enzyme recognized for its key role in relaxing
supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1
in murine bone marrow (BM) confers a potent in vitro growth advantage and
a block in differentiation in hematopoietic precursors. In a murine BM
transplantation model,
NUP98-TOP1 expression led to a lethal, transplantable
acute myeloid leukemia (AML). To ascertain if NUP98-TOP1 acts through a novel pathway,
a panel of NUP98-TOP1 mutants was engineered and tested for their
sub-cellular localization and their growth promoting effects. Neither the NUP98- 5’
nor TOP1-3’ portion of the fusion alone, nor a novel VP16-TOP1 fusion had any growth
enhancing effects. Moreover, mutants lacking TOP1 domains known to be
involved in DNA binding were also unable to transform myeloid progenitors. The
TOP1-3’ mutant exhibited ubiquitous GFP expression, while NUP98-5’ and the DNA
binding mutants localized to distinct nuclear bodies. In–vitro mutagenesis was
employed to mutate the TOP1 active-site tyrosine (Y723F), a mutation known to abolish
TOP1 catalytic activity. Similar to NUP98-TOP1, NT-Y723F exhibited a nuclear
localization, had an in vitro growth advantage and induced a lethal, transplantable AML,
suggesting that NUP98-TOP1 induces its leukemogenic effects independent of
TOP1 catalytic, isomerase activity. As observed with expression of other
translocation fusion products, the long latency of disease onset suggests the
acquisition of additional genetic mutations. Two approaches were used to
identify potential NUP98-TOP1 collaborating genes. We chose the strong
candidate gene Meis1, as it has previously been shown to accelerate leukemia
induced by several NUP98-HOX fusions. However, no evidence for collaboration
between Meis1 and NUP98-TOP1 was observed. Our second approach followed the
serendipitous finding of NUP98-TOP1 retroviral integration into the ISCBP locus
in a leukemic mouse. Strikingly, NUP98-TOP1 expression in ICSBP deficient bone
marrow accelerated disease onset. The results of this thesis add to the recognition of NUP98
fusion genes as an important class of leukemic fusion proteins. These studies
further demonstrate the complexity of the molecular pathways involved in leukemogenesis.
PUBLICATIONS
Gurevich
RM, Aplan PD, Humphries RK. (2004) “The NUP98-Topoisomerase
I AML-associated fusion gene has potent leukemogenic
activities independent of an engineered catalytic site mutation.” Blood. Aug 15;104(4):1127-36
SELECTED PRESENTATIONS
Gurevich RM, Aplan PD, Humphries RK. (2004) “Generation of
a pre-leukemic, transplantable cell line from the AML-associated
NUP98-TOP1 fusion gene as a new model to test potential collaborating genes” Blood.
104(11). Poster presentation at the 46th Annual meeting of the American Society
of Hematology. San Diego, CA USA. Dec 2004.
Gurevich RM, Aplan PD, Humphries RK. (2004) “Functional
dissection of the
NUP98-TOP1 fusion gene: overlapping and unique features
with NUP98-HOX fusion genes” Experimental Hematology. 32(7) Supplement. Poster
presentation at the 33nd Annual Meeting of the International Society for Experimental
Hematology. New Orleans, LA. USA. July 2004.
Gurevich RM, Aplan PD, Humphries RK. (2003) “Topoisomerase
Catalytic Activity is Dispensable for the Myeloproliferative Effects of the
AML Associated NUP98- Topoisomerase I (NUP98-TOP1) Fusion” Experimental
Hematology. 31(7) Supplement. Oral presentation at the 32nd Annual Meeting
of the International Society for Experimental Hematology. Paris, France. July 2003
Gurevich RM, Pineault N, Aplan PD, Humphries RK. (2002)
“The NUP98-
Topoisomerase I (NUP98-TOP1) fusion associated with
t(11;20) can induce marked myeloproliferation independent of a mutation designed to
block TOP1 catalytic activity.” Blood. 100(11). Oral presentation at the 44th
Annual meeting of the American Society of Hematology Philadelphia, PA, USA. Dec
2002.
Gurevich RM, Pineault N, Antonchuk J, Aplan PD, Humphries
RK. (2002) “The Nucleoporin NUP98-Topoisomerase I (NUP98-TOP1) fusion gene
induces marked myelomonocytic proliferative abnormalities in murine
hematopoietic cells in vitro and in vivo.” Experimental Hematology. 30(6) Supplement 1. Oral
presentation at the 31st Annual Meeting of the International Society for Experimental
Hematology. Montreal, QC, Canada. July 2002.
AWARDS
Academic
Scholarships:
2002 - 2004 National Sciences and Engineering Research
Council PGS-B
2001 - 2003 Michael Smith Foundation for Health Research
Trainee Award
2001 University of British Columbia Graduate Fellowship
(Declined)
Other
Awards:
2004 American Society of Hematology; Travel Award
2004 Keystone Symposia; Scholarship
2004 Faculty of Medicine, UBC; Travel Award
2004 BC Cancer Research Centre; Travel Award
2003 International Society for Experimental Hematology;
Travel Award
2003 American Society of Hematology; Travel Award
2002 UBC; Department of Medical Genetics Research Half Day
Poster Award
2002 UBC; Albert B and Mary Steiner Summer Research Award
2002 St. Boniface Hospital Research Centre, Institute of
Cardiovascular Sciences, Sr Jaqueline St. Yves Publication Award
GRADUATE STUDIES
Field of
Study: Experimental Hematology
Courses:
MEDG 505 GENOME ANALYSIS Drs. P. Heiter and S. Jones
MEDG 520 ADVANCED HUMAN MOLECULAR GENETICS, Dr. C. Brown
MEDG 521 BIOLOGY & GENETICS OF NEOPLASIA Dr. F. Takei
