 | |
 |
 |
| Home » Funding, Training, & Policies » Training and Career Development » Available Research Training & Career Development Programs » For Postdoctoral Individuals/New Independeent Researchers |
|
Last updated: April 2009 |
K08 Model Application
Note: The model application uses an earlier version of PHS 398. However, current
applications submitted in response to this Funding Opportunity Announcement
(FOA) for Federal assistance must be submitted electronically through Grants.gov
using the SF 424 Research and Related (R&R) forms and the SF 424 (R&R)
Application Guide. The SF 424 (R&R) Application Guide for NIH and Other
PHS Agencies now includes Part 1.7 Supplemental Instructions to the SF 424 (R&R)
for Preparing an Individual Research Career Development Award (CDA) Application
("K" series). Note in particular that the "Candidate" and
"Research Plan" (sections A-D) combined may not exceed 25 pages. Please
note that this new Application Guide is to be used with all Adobe application
packages, including those for the K programs.
This model application contains many of the required elements for a K08 application
but it does not include the face page; budget pages; biographical sketch (including
documentation of licensure to practice medicine in the United States); other
support; letters of reference; statements by sponsor(s), consultant(s), and
collaborator(s); institutional commitment to candidate; literature cited; consortium/contractual
arrangements; consultants; and appendix pages. Note that all K08 applicants
must provide those components in their application. Information of an identifying
and proprietary nature such as the level of effort, the timetable, the name
of the applicant and the names of collaborators, mentors and institutions has
been removed and replaced with the underline symbol "_____."
Potential applicants should realize that the applicant in this model application
has dual doctorates and comes to the K08 program with at least three years of
previous research experience. This applicant has also completed some didactic
course work (graduate courses in biostatistics, cellular and molecular physiology,
etc). Not all potential applicants are expected to have this extensive didactic
and research experience. They should, however, endeavor to describe their experience
and their career development plans with similar levels of detail and clarity.
I. Description and Key Personnel
II. Research Career Award Table of Contents
III. Resources
IV. The Candidate
A. Candidate’s Background
B. Career
Goals and Objectives: Scientific Biography
C. Career
Development Activities during Award Period
1.
Acquire expertise in transcriptional repression
2.
Acquire experience in the analysis of hematopoietic disease
3.
Acquire scientific and career guidance from an advisory committee
4.
Attend Miscellaneous Postdoctoral Sessions
V. Statements
by Sponsor(s), Consultant(s), and Collaborator(s)
VI. Environment
and Institutional Commitment to Candidate
A. Description
of Institutional Environment
B.
Institutional Commitment to Candidate’s Research Career Development
VII. Research Plan
A. Statement
of Hypothesis and Specific Aims
B. Background,
Significance, and Rationale
1. Introduction
2. Significance
C. Preliminary Studies
and Any Results
D. Research Design and Methods
1. Specific Aim 1
2. Specific Aim 2
3. Specific Aim 3
E. Human Subjects
F. Vertebrate Animals
G. Literature Cited
H. Consortium/Contractual
Arrangements
I. Consultants
VIII. Appendix
Principal Investigator/Program Director (Last, first, middle): _____
This proposal describes a 5 year training program for the development of an academic career in Laboratory Medicine. The principal investigator has completed structured residency training in Pathology at the University of _____ and now, will expand upon his scientific skills through an unique integration of interdepartmental resources.
This program will promote the command of transcriptional corepressor biology, as applied to hematopoietic disease. _____ will mentor the principal investigators scientific development. _____ is a recognized leader in the field of transcriptional repression. He is the Chief of Endocrinology and has trained numerous postdoctoral fellows and graduate students. To enhance the training, the program will enlist the expertise of _____, Assistant Professor of Pathology. _____ pioneered the ex vivo bone marrow transduction techniques that will be applied in the analyses of repression. In addition, an advisory committee of highly-regarded medical scientists will provide scientific and career advice.
Research will focus on transcriptional repression in myeloid progenitor cells. Recent work in _____ laboratory demonstrated that aberrant recruitment of corepressors may underlie the disruption of myeloid development in leukemia associated with the AML1-ETO fusion protein. The proposed experiments will entail introduction of AML1-ETOand derivatives into mouse bone marrow and myeloid cell lines. Subsequently, phenotypes will be examined using an assortment of biochemical, molecular, and cellular techniques. The specific aims include: 1) Establishing an in vivo model for the analysis of interactions between AML1-ETO and repression pathways, 2) Determining if corepressor recruitment by AML1 is sufficient to exert the pathogenic effects of AML1-ETO, and 3) Determining the role of ETO domains in the disruption of myeloid development by AML1-ETO. This will be the first detailed functional analysis of the mechanisms of transcriptional repression by AML1-ETO using models that attempt to mimic the pathways of the myeloid progenitor.
The Pathology department of the University of _____ provides an ideal setting
for training physician- scientists by incorporating expertise from diverse
resources into customized programs. Such an environment maximizes the potential
for the principal investigator to establish a scientific niche from which
an academic career can be constructed.
PERFORMANCE SITE(S) (organization, city, state)
Laboratory of _____
Department of _____
Hospital of the University of _____
_____, _____ _____
________________________________________________________________________________________________________________
KEY PERSONNEL. See instructions on Page 11.
Use continuation pages as needed to provide the required information in the
format shown below.
Name Organization
Role on Project
_____ University
of _____
Principal Investigator
_____ University
of _____
Sponsor
_____ University
of _____
Collaborator
Advisory Committee:
_____ University
of _____ Advisory
Committee
_____
University of _____ Advisory
Committee
_____
University of _____
Advisory Committee
_____
University of _____ Advisory
Committee
______________________________________________________________________________________________________________
111PHS 398 (Rev. 4/98)
Page2
BB
LL Candidate (Last, first, middle): ____________________________________________
Page Numbers
1–3. Face Page, Description and Key Personnel, Table of
Contents (Form pages AA, BB, and this substitute page) ............................1-3_________
4. Budget for Entire Proposed Period of Support (Form page EE) ...........................................................................................................
_________
5. Biographical Sketches (Candidate and Sponsor[s]*—Form page FF) (Not
to exceed two pages) ........................................................
_________
6.Other Support (Mentored Career Award Sponsor[s]*—Format pages GG)..........................................................................................
_________
7. Resources (Form page HH) ....................................................................................................................
........................................... _________
a. Statement of Hypothesis and Specific Aims .....................................................................................................................................
_________
b. Background, Significance, and Rationale ...................................(Included
in 25 page limit)...............................................................
_________
c. Preliminary Studies and Any Results ................................................................................
_________
d. Research Design and Methods ........................................................................................
_________
e. Human Subjects* ............................................................................................................_________
f. Vertebrate Animals* ........................................................................................................
_________
g. Literature Cited ..............................................................................................................
_________
h. Consortium/Contractual Arrangements* .........................................................................
_________
i. Consultants* ....................................................................................................................
_________
(Five collated sets. No page numbering necessary.)
Number of publications (not to exceed six): __________
List of key items:
______________________________________________________________________________________________________________
CITIZENSHIP
(____) U.S. Citizen or noncitizen national
(____) Permanent resident of U.S.
(____) If a permanent resident of the U.S., a notarized statement must be
provided by the time of award ______________________________________________________________________________________________________________
Number pages consecutively at the bottom throughout the
application. Do not use suffixes such as 3a, 3b.
Principal Investigator/Program Director (Last, first, middle): _____
RESOURCES
Laboratory: _____ laboratory consists of ~1500 square feet located
on the 6th floor of the Clinical Research Building at the University of _____.
The laboratory contains refrigerators, freezers, a microwave, gel electrophoretic
equipment, microfuges, PCR machines, water baths, a Stratalinker, power supplies,
and most of the small equipment required for the proposed studies. Across the
hall is a tissue culture room with two laminar flow hoods, a phase contrast
microscope, and four CO2 incubators, as well as a walk-in cold room.
Clinical: N/A
Animal: The animal facility is located in the _____ Building adjacent to the
Clinical Research Building. The facility includes an irradiator and two procedure
rooms suitable for bone marrow transplantation experiments. Mouse rooms are
equipped with laminar flow hoods for non-surgical manipulations.
Computer: _____ laboratory is equipped with 3 Power Macintosh computers, zip
drives, a scanner, and printers. Software is available for word processing,
presentations, and sequence analysis. The laboratory has access to the University
of _____ network, the Internet, Medline, and numerous online biomedical journals.
Office: Work will be conducted in the laboratory of _____ where the applicant
has a desk and filing space.
Other: Secretarial assistance is available through the _____ Division. The proximity
of the lab to investigators in the ____ Center (same floor) and _____, Department
of _____ (same building) encourages frequent consultation and interchange of
ideas.
_________________________________________________________________________________________________________________
MAJOR EQUIPMENT: List the most important equipment items already available for this project, noting the location and pertinent capabilities of each.
_____ has two -70 freezers, a gel drier, and Speedvac. _____ share
common space with three ultracentrifuges, two superspeed centrifuges, a phosphoimager,
ice machine, UV/visible spectrophotometer, and beta and gamma counters. The
darkroom contains an X-O-Mat film processor and camera set-up. On the same floor
are additional centrifuges and rotors available during periods of heavy use
and/or repair of Endocrine Section
equipment.
PHS 398 (Rev. 4/98) (Form Page 8) Page HH
a. Candidate’s Background
My career commitment from secondary
school through residency has been the molecular genetics of human disease. I
developed this focus prior to college, in the Secondary Science Training Program
of the _____. I continued to study molecular genetics during college in the
laboratory-oriented Honors Biology Program of the _____. Upon applying to _____
I declared my intent to pursue a career in pathology, specifically for the opportunities
to combine science and medicine. Now, I have completed the structured clinical
pathology rotations of residency, and I am poised to
develop scientific skills in the analysis of transcriptional complexes and the
molecular biology of leukemia. This broadening of skills will help me compete
as an independent investigator, and will ultimately lead to a career in academic
molecular pathology.
My specific scientific concentration
is on the regulation of gene expression. I performed my thesis work in the laboratory
of _____ at _____ studying the DNA elements controlling expression from the
intricate beta-globin gene cluster. We selected a mouse model for our investigations
and performed one of the earliest knockouts combining homologous recombination
with site specific (Cre-lox) recombination technology(24). In addition to advancing
the understanding of globin gene regulation, this work provided a universal
lesson for mouse geneticists by emphasizing the often ignored potential for
a single selectable marker to disrupt expression of multiple genes within a
locus(45). I left the lab with a depth of understanding of locus control and
mouse genetics.
After completing the M.D./Ph.D. program
at _____, I entered residency in clinical pathology at the University of _____.
Clinical rotations focused upon laboratory disciplines including hematopathology,
flow cytometry, and molecular diagnosis. The goal of the program is to launch
the careers of independent scientific investigators while promoting expertise
in a clinical laboratory service. I have completed the structured, core rotations
of residency, and I am initiating the research phase of the program.
b. Career Goals and Objectives: Scientific Biography
Ultimately, my goal is to attain
a tenure-track position in a department of clinical pathology. I intend to devote
approximately _____ of my time to the study of molecular pathways regulating
gene expression in hematopoietic cells. The remaining time will be committed
to the clinical discipline of molecular diagnosis: the diagnostic, prognostic,
and therapy-determining molecular assessment of human disease. This allocation
of time will foster a scientific career while elevating clinical performance.
In March, 1999, I entered the laboratory
of _____, the _____ and an authority on transcriptional repression. The study
of transcription factor complexes, under _____ supervision will serve multiple
objectives in preparing me for an academic career in clinical pathology. My
objectives include:
acquiring a scientific background that will complement my clinical training.
expanding my technical background with new biochemical and cellular skills.
allowing me to update and enhance my skills in the discipline of molecular genetics; a field that has continued to explode in the three years since I completed graduate school.
serving as a transitional period between postdoctoral training and an independent faculty position.
c. Career Development Activities during Award Period
My goal during the next five year period is to develop the skills necessary
to succeed as an independent investigator. My strengths include a command of
the scientific method and technical expertise in molecular genetics. However,
in order to succeed as a scientist in the competitive field of transcription,
I propose to acquire new perspectives and skills complementary to the discipline
of clinical molecular pathology:
1) Acquire expertise in transcriptional repression.
The field of transcription is one of the most rapidly advancing in biology today.
By studying corepressor transcription factors, I will acquire a background in
the fundamental molecular pathways recently shown to be involved in human leukemias.
In order to develop a working knowledge of today’s issues and prime myself
for independent activity, I propose the following:
Research: Perform organized research under the guidance of _____, _____ and a leader in the field of transcriptional repression. Recently, transcriptional biology more commonly associated with nuclear hormone receptors converged with the field of hematopathology. _____ contributed to the recent revelations that linked corepressor complexes with both APL(16) and AML(15). This makes the _____ laboratory the ideal setting for acquiring expertise in the biology of corepressor complexes as they pertain to leukemia. By training in the _____ laboratory, I will be engrossed in the basic science of transcriptional repression. I will acquire new fundamental skills in biochemistry and cell biology including Western blot analysis, immunoprecipitation, and flow cytometry. Additionally, I will benefit from the lab meetings and departmental conferences in which the _____ laboratory participates.
Lectures: Review developments in transcription and stay abreast of emerging topics through participation in a genetics lecture series (1 per week), journal review (1 per week) and transcription seminars (2 per week). My initial goal is to efficiently review the developments that have taken place in genetics during the three year period since I completed graduate school. I have begun this review by attending weekly genetics lectures. Additionally, I intend to follow current developments in the field of transcription through participation in a nuclear hormone receptor journal review and various transcription-related seminars hosted by both clinical departments and the graduate school.
National meetings: Attend the nuclear hormone receptor Keystone
meeting. The field of transcriptional repression is
founded in nuclear hormone receptors. This meeting is likely to provide
insights applicable to repression in
general, including repression in hematopoietic cells.
2) Acquire experience in the analysis of hematopoietic disease.
Ultimately, I intend to study transcription
as it applies to hematopoietic disease. As a graduate student, I
acquired an understanding of embryonic stem cells and homologous recombination
technology. This methodology is not t
consistently or easily used to analyze genes that produce embryonic lethal phenotypes.
I, therefore, intend to
develop skills in alternate approaches to modeling and analyzing hematopoietic
disease.
Research: Develop proficiency in somatic cell gene transfer
and bone marrow transplantation in mice under the
supervision of _____. _____ developed the cell lines(44) and transient transfection
techniques(42, 44) for producing
high titer retroviral cultures. He subsequently used bone marrow retroviral
transduction methodologies to produce
mouse models of chronic myelogenous leukemia(43) and T-cell acute lymphocytic
leukemia(41). _____ has performed
hundreds of bone marrow transplants in mice and continues to perform these
procedures routinely. The _____
laboratory is, therefore, ideal for acquiring skills in mouse models of
hematopoietic disease.
Lectures: Stay abreast of developments in the laboratory analysis
of hematologic disease through participation in
resident’s seminars (1 per week). The clinical pathology residents
hold a weekly conference during which one member
of the program reviews an emerging issue in laboratory medicine. Approximately
half of these sessions focus on
hematological issues. This will provide a regular update on developments
in the analysis of blood diseases.
National meetings: Attend the meeting of the American Society
of Hematology. This conference is likely to be the
most appropriate clinical forum for the presentation and discussion of the
proposed hematopoietic model systems.
3) Acquire scientific and career guidance from an advisory committee.
In order to most effectively promote a career in the field of academic clinical
pathology I have organized an advisory committee composed of physician-scientists
with strengths in the fields of hematology and transcription. Each member of
the committee is an outstanding investigator in his field.
The committee will meet twice yearly. The format of the meeting will include
a formal presentation by the applicant followed by a critical review of the
hypotheses, results, and future directions. Finally, the committee will provide
career guidance. In addition to _____, the committee includes the following
members.
_____ is an Assistant Professor in _____ and a member of the Division of _____. _____ has extensive expertise in ex vivo retroviral transductions of mouse bone marrow(41-44). _____ is already providing valuable advice in the advancement of the mouse model described in specific aim 1.
_____ is an Associate Professor in _____ and the Director of _____. _____ is an authority on mouse models of leukemia and has a background in transcription and molecular hematopathology.
_____ is a Professor of _____ and _____. He has a depth of experience in clinical hematology, leukemia, and bone marrow transplantation.
4) Attend Miscellaneous Postdoctoral Sessions
The University of _____ offers an assortment of postdoctoral courses to strengthen
the training program. These include:
Bioethics: Sessions focus on issues appropriate for both basic and applied science.
Orientation to laboratory animal research: Sessions provide mandated instruction in animal care, as well as, resources in the veterinary school for additional training, as needed.
Safety training: Training includes mandatory radiation, biological, and chemical safety sessions. Training is also offered for the irradiator that will be used in performing bone marrow transplants in mice.
My plan for training in the responsible
conduct of research is as follows. In year 1 of my K08, I will take Course #
_____, Research Ethics and Integrity, 3 units, third term. This course introduces
concepts inherent to the ethical conduct of research with human participants,
issues of scientific integrity, and ethical theories and principles. This course
will fulfill the _____ and the NIH requirements concerning training in the responsible
conduct of research.
In the second year of my K08 I will take Course # _____, Research Ethics, 1
unit, second term. This course presents issues in the responsible conduct of
research, such as research ethics, academic ethics, data management, data ownership,
guidelines of professional conduct, research fraud, academic misconduct, and
conflict of interest, and explains federal and institutional guidelines relating
to research using human and animal models.
Timetable
The department of pathology recognizes
the importance of focused scientific research during the transition from postdoctoral
training to faculty status. Therefore, the initial years of this program will
permit virtually all efforts to be devoted to the career development activities
outlined above. Later years will include fellowship training in molecular diagnosis
and faculty service work. Nevertheless, research will always receive _____ effort.
Year 1: _____
Year 2: _____
Year 3: _____
Year 4-5: _____
a. Description of Institutional Environment
The Department of _____ at the University
of _____ is the ideal setting for training physician scientists. Almost all
residents pursue academic careers. The program advocates free scientific exploration
within any laboratory of the university. This aspect permits the trainee to
customize education using the diverse resources of the entire institution. I
have exploited this strength of the program by entering the laboratory of _____
in the Division of _____. _____ contributed to the recent revelations linking
corepressor complexes (typically associated with nuclear hormone receptors)
to myeloid leukemias(15, 16). The _____ laboratory is, therefore, the ideal
setting for acquiring expertise in the molecular pathogenesis of acute myelogenous
leukemia. Furthermore, the _____ laboratory is optimally situated within the
university to facilitate research. The _____ Building is located among a network
of adjoined buildings. All of the pertinent contacts for this project are readily
accessible. This includes the laboratory of our collaborator, _____ the animal
facility, the histopathology laboratory, and the flow cytometry facility. Additional
relevant scientific resources in the same building include laboratories in the
Department of _____ and the Department _____ The environment is, therefore,
an exceptional one for completing the proposed work.
b. Institutional Commitment to Candidate’s Research Career Development
Aberrant recruitment of corepressor transcription factors (CoRs)
underlies the pathogenic transcriptional activity of the AML1-ETO fusion protein.
AML1-ETO is the product of a common translocation associated with acute myelogenous
leukemia, t(8;21). A zinc finger domain in the C-terminus of ETO interacts with
the nuclear corepressors, NCoR and SMRT. In the context of AML1-ETO, this domain
is essential for repression of several AML1-responsive promoters and for inhibition
of myeloid and monocytic differentiation in cell culture assays. We are interested
in understanding
how the CoRs and uncharacterized ETO domains contribute to the disruption of
myeloid development.
We hypothesize that AML1-ETO will be leukemogenic in mice when
introduced into hematopoietic progenitor cells. Given the embryonic lethality
of AML1-ETO “knock-in” mutations, we will perform ex vivo transduction
of mouse bone marrow with AML1-ETO cDNA. We will transplant the marrow to lethally
irradiated syngeneic recipients and monitor the animals for histopathological
or flow cytometric evidence of leukemia. Successful disruption of the molecular
pathways of the myeloid progenitor cell will provide the optimal system for
functional analyses of
AML1-ETO-corepressor interactions.
We hypothesize that replacing ETO with a corepressor (CoR) will
confer leukemogenic activity upon AML1. To test this, fusion proteins composed
of the DNA-binding domain of AML1 and the repression domains of SMRT and NCoR
will be constructed. Fusion proteins will be analyzed using well-established
tissue culture assays and the mouse model described above. These experiments
will reveal whether CoR recruitment is the sole pathogenic activity of the AML1-ETO
fusion protein.
We hypothesize that ETO domains other than the CoR-binding domain
contribute to the disruption of myeloid differentiation, possibly by participating
in CoR recruitment. In order to test for activity, we will mutate ETO sequences
in the context of AML1-ETO. We will systematically analyze biochemical interactions
of fusion proteins with members of the corepressor complex, expression of AML1
target genes, and inhibition of myeloid differentiation. The properties of AML1-ETO
mutants will identify biologically important regions of the ETO moiety.
Together, the above studies will illuminate the roles of ETO domains and the
CoR complex in the pathogenesis of myeloid leukemia. This information will improve
our understanding of acute myelogenous leukemia and could lead to new diagnostic
markers, prognostic markers, or therapeutic targets in the ETO repression pathway.
Introduction
The t(8;21)(q22;q22) is the second most commonly occurring translocation associated
with acute myelogenous leukemia. It occurs in approximately 20% of adult and
40% of pediatric leukemias of the FrenchAmerican-British M2 subtype(37). The
translocation creates a fusion between the AML1 gene on chromosome 21 and the
ETO gene on chromosome 8. The fusion protein is leukemogenic, although, the
pathways through which AML1-ETO act are just beginning to be understood. This
application focuses on the interactions of AML1-ETO with the transcriptional
repression pathways of
the myeloid progenitor cell.
AML1
AML1 was identified by the cloning of the breakpoint involved in the recurring
t(8;21) translocation of acute myelogenous leukemia (10, 37). AML1 is a well-characterized
member of the core binding factor (CBF) family of transcription factors (also
known as polyoma enhancer binding protein 2 family). It is expressed in hematopoietic
cells and is essential for definitive hematopoietic development (40, 58). The
protein has intrinsic DNA binding activity, conferred by the runt domain (named
for its homology to a Drosophila transcription factor)(34). AML1 synergistically
interacts with an assortment of other
hematopoietic transcription factors including CBFβ, C/EBP, Ets family members,
and myb to activate transcription of hematopoietic target genes (Reviewed (54)).
AML1 also derives transcriptional activating activity from interactions with
the histone acetyl transferase, p300/CBP. Interestingly, the product of the
t(8;21) translocation lacks the C-terminal domain of AML1 (required for p300/CBP
interaction)
(27) but retains the runt domain (sufficient for DNA binding) (Figure 1). Thus,
AML1 domains confer upon AML1-ETO a potential to disrupt expression of AML1
target genes.
AML1
| runt homology (DNA binding) |
activation (p300/CBP binding) |
ETO 
NH1
NH2
NH4
(TAF homology) (dimerization) (CoR binding)
177
752
Figure 1. Maps of the AML1, ETO, and AML1-ETO gene products. The 177 N-terminal
amino acids of AML1 are fused to the 575 C-terminal residues of ETO. Domains
of interest are highlighted including the DNA-binding domain and p300/CBP-interaction
domain of AML1 and the Nervy homology (NH) domains of ETO.
ETO
In contrast to AML1, the activities
of ETO are not clearly understood. ETO is a member of poorly characterized family
of proteins including itself (also called MTG8), ETO-2(9), MTGR1(26), and MTG16
(14). The ETO gene encodes a putative transcription factor, with proline rich
domains, candidate serine and threonine phosphorylation sites, a TAF110-homologous
domain, a dimerization domain, and two zinc-fingers. Four domains are homologous
to those of Drosophila Nervy, a poorly characterized gene expressed in the nervous
system of the fly (Figure 1)(12). The ETO protein product is localized to the
nucleus(11). Expression in hematopoietic cells is debated(11, 36). The only
demonstrated activity has been an ability to transform cells when overexpressed(52,
57). In fact, much of what is known about ETO is derived from the study of the
AML1-ETO fusion protein. Because the function of ETO is unclear, it is difficult
to know how ETO domains contribute to the activity of the AML1-ETO fusion protein.
Inhibition of Myeloid Differentiation by AML1-ETO
The t(8;21) creates a fusion
between the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. The
chimeric gene product of the t(8;21) is comprised of the N-terminal 177 residues
of AML1 and the C-terminal 575 residues of ETO (Figure 1) (36). Consistent with
a role in leukemogenesis, AML1-ETO blocks differentiation of myeloid and monocytic
tissue culture cells (1, 15, 26, 59). Likewise, in mice, AML1-ETO “knock-in”
mutations are embryonic lethal(39, 60) due to the lack of definitive hematopoietic
development. Interestingly, primitive hematopoiesis is normal and cells from
hemizygous yolk sacs and livers can be passaged for several generations longer
than wild-type cells. These findings are suggestive of a putative leukemogenic
activity of the fusion protein. Unfortunately, investigation of this activity
in vivo has been slow. The embryonic-lethal phenotype of the AML1-ETO fusion
in mice prevents transmission through the germline. This hinders detailed functional
analyses of AML1-ETO using reliable transgenic and gene targeting approaches.
Specific Aim 1 will attempt to circumvent this obstacle using an ex vivo bone
marrow transduction approach, under the guidance of an expert in the technique.
Disruption of expression of AML1 target genes by AML1-ETO
Aberrant expression of AML1 target
genes undoubtedly underlies the leukemogenic activity of AML1-ETO. Numerous
target genes involved in myelomonocytic development are regulated by AML1, including
GMCSF, IL-3, and M-CSF receptor, myeloperoxidase, and neutrophil elastase(13,
38, 53, 55, 63, 64) AML1-ETO retains the ability to interact with the promoters
of AML1 target genes through the AML1 runt domain(34). Predictably, the AML1-ETO
fusion protein represses transcription from selected AML1 target genes (13,
32, 35). It has been hypothesized that dominant negative interactions with AML1
contribute to repression(13, 32, 35). Nevertheless, the phenotype of the AML1-ETO
“knock-in” mutation in mice is subtly different from that of the
AML1 “knock-out,” leaving open the possibility that the fusion protein
has unique activities beyond its AML1-inhibitory function(39, 40, 58, 60). Indeed,
the M-CSF receptor, an AML1 target gene, is paradoxically, upregulated by AML1-ETO(46).
Similar, paradoxical, upregulation has been reported for Bcl-2(28), although
it is controversial whether this is consistently observed in leukemic patients(3,
51). Clearly, the proposed mechanisms of AML1-ETO action are incomplete.
Recruitment of Corepressors by AML1-ETO
Recently, our laboratory(15) and
others(33, 56), provided a novel explanation for the transcription-inhibiting
activity of AML1-ETO: the C-terminus of ETO interacts both biochemically and
functionally with corepressor proteins(15, 33, 56) including nuclear receptor
corepressor (NCoR) (23) and silencing mediator for retinoid and thyroid hormone
receptor (SMRT)(5). These molecules are components of multiprotein complexes
that reduce gene expression to levels below baseline. Other members of the complexes
include mammalian Sin3 (mSin3) and histone deacetylases (HDACs) (21). These
molecules confer inhibitory activity on transcription factors such as unliganded
nuclear hormone receptors(4, 6, 23, 49, 61, 62), orphan receptors(8, 50), and
Mad(2, 48). Transcriptional inhibition may occur through chromatin remodeling
or contact with the basal transcriptional machinery, but the mechanism is unproven.
Importantly, the ability of AML1-ETO to inhibit transcription from the neutrophil
protein 3 (NP-3) promoter(33) and to inhibit differentiation of U937 promonocytes(15)
is dependent upon the C-terminal domain of ETO that interacts with CoRs. Therefore,
the AML1-ETO fusion protein may interfere with myeloid differentiation by aberrantly
recruiting corepressor complexes and histone deacetylase activity to the promoters
normally activated by AML1 (Figure 2). Nevertheless, it remains unclear if this
interaction is sufficient to cause leukemia or if additional activities of AML1-ETO
are required. Specific Aim 2 will address this issue.
Figure 2. Model of the role of interactions of fusion protein AML1-ETO with
CoRs (NCoR/SMRT) and the corepressor complex. (a) DNA-bound AML1 interacts with
p300 and potentially with other coactivator molecules. This association results
in transcriptional activation (possibly through histone acetylation and effects
on transcriptional machinery) and normal myeloid development. (b) AML1-ETO recruits
the corepressor complex. This results in transcriptional repression (and possibly
activation of a subset of genes) and a block in differentiation. (15).
Precedent for Aberrant Recruitment of Corepressors in Leukemia
Intriguingly, this link between AML1-ETO and a corepressor complex is the second
example of an interaction between a leukemogenic fusion protein and a transcriptional
corepressor complex. The first example of such an interaction is that causing
acute promyelocytic leukemia (APL). The common APL variant is a retinoic acid-responsive
leukemia commonly associated with a t(15;17) translocation and PML-RARα
fusion protein (Reviewed (17), 25). A rare, retinoic acid-resistant variant
is associated with t(11;17) and the PLZF-RARα fusion protein(7, 17, 25,
30). Both of these fusion proteins are capable of associating with members of
the corepressor complex including NCoR, SMRT, mSin3, and HDAC in the absence
of retinoic acid. (These are the same factors that interact with AML1-ETO, Figure
2b). Amazingly, the responsiveness of disease to retinoic acid therapy is related
to the persistence of the interaction of the fusion protein with the corepressor
complex. (16-18, 20, 22, 31, 47) It, therefore, appears that an aberrant interaction
with corepressors is a recurring mechanism whereby mutant transcription factors
produce leukemia.
Corepressor recruitment may not be sufficient for leukemia
While recruitment of CoRs may be necessary for AML1-ETO (and PML-RARα)
to inhibit myeloid differentiation it is not known if recruitment is sufficient.
It is noteworthy that in both t(8;21) and the t(16;21) variant (between the
AML1 gene and the ETO family member MTG16), almost the entire “ETO”
protein is included in the fusion product(14, 36). No reported translocations
with the AML1 locus truncate the ETO gene, increasing speculation that the entire
protein may contribute to leukemogenesis. In fact, activity has been elicited
from other domains of AML1-ETO in selected
assays. For example, the Nervy-homology 2 domain (Figure 1) permits dimerization
with ETO family members (26) and is necessary for repression of transcription
(32). Furthermore, ETO domains may help explain the counterintuitive activation
of certain AML1 target genes by AML1-ETO(46). Nevertheless, it is not known
if ETO actively contributes to the leukemogenic process in vivo, or if it passively
tethers CoRs to AML1 targeted promoters. Specific Aim 3 will investigate the
roles of conserved ETO domains in the pathogenesis of leukemia.
Selection of model systems for analysis of AML1-ETO
Although the initial characterization of the interaction of AML1-ETO with corepressor
pathways was elegantly described using an assortment of cell lines, additional
information could be extracted from models amenable to myeloid differentiation
assays. Selection of a model system can have a dramatic impact upon the outcome
of transcriptional repression studies. For example, ETO functions as a repressor
when tethered to a heterologous promoter in C33A (cervical carcinoma) cells
but not 293T (renal epithelial) cells(65). Evidently, conclusions drawn from
studies of a given cell line may not provide accurate information about AML1-ETO
activity in vivo. Model systems suited to the evaluation of myeloid differentiation
are available. Several groups have demonstrated that the intact AML1-ETO fusion
protein inhibits myeloid differentiation in progenitor cell culture models(1,
26, 59), although detailed domain mapping has not been attempted. Likewise,
in vivo analyses have suggested that AML1-ETO blocks hematopoietic development,
but follow-up functional analyses have been slowed by the embryonic lethal phenotype
of the AML1-ETO gene fusion(39, 60). Ideally, future studies of AML1-ETO function
will be performed using model systems that recapitulate the repression pathways
of the myeloid progenitor cell.
Significance
Simultaneously, our lab(15) and others(33,
56), made the link between AML1-ETO and corepressor pathways. Nevertheless,
these initial observations have not been expanded with detailed functional analyses
using models that mimic the molecular pathways of the myeloid progenitor cell.
We, therefore, intend to employ a novel mouse model, and a well established
myeloid cell culture model to probe the interactions between AML1-ETO and corepressor
pathways.
First, we will attempt to develop
the mouse model. Because AML1-ETO causes embryonic demise, we will deliver AML1-ETO
somatically, by ex vivo transduction of mouse bone marrow. We hypothesize that
transplanted marrow will cause leukemia in recipient mice. Such a model will
provide, for the first time, an opportunity to evaluate interactions between
AML1-ETO and pathways that are, in fact, active in the hematopoietic progenitor
cell. Because the endpoint of the in vivo studies is the development of a myeloproliferative
disorder, this model will be especially useful for distinguishing pathogenic
activities from biological properties of AML1-ETO unrelated to disease.
Next, we will apply both in vivo
and in vitro models to understand the importance of the interaction of AML1-ETO
with CoRs. While CoR recruitment by AML1-ETO appears necessary, in vitro, for
inhibition of development, it is not clear if it is sufficient to disrupt myeloid
differentiation in the absence of ETO domains. Determining the importance of
CoR recruitment is of fundamental significance to this field because it will
guide investigation either upstream (i.e. to ETO domains) or downstream (i.e.
to CoR-interacting proteins) of the CoR. Finally, we will attempt to understand
if ETO domains, other than the CoR-binding domain contribute to the leukemic
process. These experiments will complement those of specific aim 2. Several
ETO domains have activities in vitro. These domains may act by modifying interactions
with the CoR complex or they may act through independent mechanisms. Clarifying
the role of ETO in the inhibition of myeloid development is likely to provide
insight into both the pathogenic activity of AML1-ETO and mechanisms of repression
in general.
Recruitment of corepressor complexes
may be a common mechanism in leukemogenesis. Examination of the pathways recruited
by AML1-ETO is likely to improve the management of patients with not only t(8;21)
leukemia, but other forms of myeloid leukemia as well. When pathogenic pathways
are dissected using the appropriate model system, every molecular participant
is a potential diagnostic marker, prognostic marker, or therapeutic target in
patients with disease. It is, therefore, vital to learn which molecules are
critical for AML1-ETO to disrupt myeloid gene
expression and development.
The _____ laboratory
recently published data indicating that the C-terminus of AML1-ETO interacts
with corepressors (CoRs) and is necessary for the inhibition of differentiation
of U937 monocytic cells(15) (See appendix). Based upon this data, we propose
to evaluate the mechanisms of transcriptional repression by AML1-ETO using a
mouse model and a myeloid cell culture model.
Efforts have been focused on producing a virus suitable for infection of mouse
myeloid cells. This has entailed:
construction of a retroviral vectors encoding AML1-ETO
expression of the AML1-ETO fusion protein
We have completed these preliminary goals and are ready to begin infections of bone marrow and myeloid cell lines.
Vector Design
Two vectors have been constructed
for the analysis of AML1-ETO in vitro and in vivo. The first, AML1-ETO MigR1
was assembled for in vivo analysis. The mouse stem cell retroviral vector (MSCV)
was selected for its history of prior success in mouse leukemia models(43, 66).
Advantages of the vector include the ability to infect mouse hematopoietic stem
cells, an extended packaging site to maximize retrovirus production, and long-term,
high-level expression from the murine stem cell retroviral LTR(19, 43). The
MigR1 vector, conveniently, expresses green fluorescent protein (GFP) to facilitate
the analysis of infected cells by flow cytometry. A 2.3 kilobase, N-terminal,
FLAG-tagged AML1-ETO cassette was produced by PCR.
Figure 1. Map of AML1-ETO MigR1 retroviral vector for in vivo analyses.
The fidelity of PCR was confirmed by sequencing. The AML1-ETO cassette was inserted
into the Xho1 site of the MSCV MigR1 multiple cloning site. AML1-ETO and GFP
are expressed as a bicistronic message that includes and internal ribosomal
entry site (IRES). A map of the vector is shown (figure1).
The second vector, AML1-ETO MSCV 2.2, was assembled for in vitro analysis. The
2.3 kilobase AML1-ETO cassette, described above, was inserted into the Xho1
site of the MSCV 2.2 multiple cloning site. The selectable PGK-neo gene is located
downstream of AML1-ETO. A map of the vector is shown below.
Flag-AMLI-ETO
Mouse Stem Cell Virus
Figure 2. Map of AML1-ETO MSCV 2.2 retroviral vector for in vitro analyses.
AML1-ETO expression
To insure that the constructs described
above, in fact, express the AML1-ETO fusion protein appropriately, Bosc23 packaging
cells(44) were transfected with both AML1-ETO MigR1 and AML1-ETO MSCV 2.2. 48
hours post-transfection protein extracts were prepared and Western blot analysis
was performed. Identical results have been obtained using anti-FLAG and anti-ETO
(Pharmingen) primary antibodies. Both vectors, AML1-ETO MigR1 and AML1-ETO MSCV2.2
appropriately express the AML1-ETO fusion protein.
Figure 3: MSCV retroviral vectors encode AML1-ETO. Extracts were prepared from
Bosc23 cells transfected with AML1-ETO MigR1 and subjected to Western blot analysis
using an anti-ETO primary antibody recognizing the C-terminus of ETO (Pharmingen).
Extracts were prepared from Bosc23 cells transfected with 1) MigR1 retroviral
vector, 2) AML1-ETO MigR1 retroviral vector, and 3) ETO pCDNA (positive control
expression vector) (extract in lane 3 was diluted 1:100).
Production of high titer retroviral stocks
A critical parameter for the successful development of myeloid leukemia models
using retroviral transduction is the titer of retrovirus used to infect bone
marrow cells(43). The Bosc 23 packaging cell line was developed by Pear et al.
to maximize the production of high titers of retrovirus using transient transfection
techniques(44). 2 x 106 Bosc 23 cells were transfected with 2 µg
of retroviral vector DNA using FuGENE6 (Boerhinger) in the presence of 25µM
chloroquine. 48 hours after transfection, 0.1 ml of BOSC 23 supernatant was
added to NIH3T3 cells as a cocktail of 0.1 ml supernatant, 2.9 ml Dulbeccos
Modified Eagle Medium, 10% fetal calf serum (Gibco), 100 U/ml streptomycin (Gibco),
100 U/ml penicillin (Gibco), plus polybrene (final concentration 4 µg/ml). Additional
media was added 4 hours after infection. Two days after infection, NIH3T3 cells
were harvested and analyzed by flow cytometry using a FACScan flow cytometer
(Becton Dickenson). Titers were calculated as: (percentage of GFP positive cells)
x (number of cells plated) / (volume of virus used for infection). As shown
by the histogram and calculation below, titers of greater than 106
are produced routinely.
Figure 4. Production of High-Titers of Retrovirus. The flow cytometry histogram
of NIH3T3 cells transduced with GFP-expressing retroviral vector. Bosc 23 packaging
cells were transfected with the MigR1 retroviral vector encoding GFP. 48 hours
after transfection, 0.1 ml of supernatant was used to infect NIH3T3 cells. 48
hours later, the infected NIH3T3 cells were analyzed by flow cytometry. 40%
of cells were infected. (40% of cells infected) x (4x105 cells plated)
/ (0.1 ml virus) = titer of 1.6 x 106. Similar results have been
produced with AML1-ETO MigR1.
We propose to examine
the association of AML1-ETO with corepressor (CoR) pathways. We will attempt
to perform these studies using two model systems appropriate for analysis of
myeloid development. In specific aim 1, we will use retroviral transduction
to introduce AML1-ETO into the bone marrow of mice. The proposed ex vivo transduction
/ transplant system was pioneered by our collaborator, _____, who is committed
to providing the training, protocols, and reagents needed to establish the system
in the _____ laboratory. Specific aims 2 and 3 will employ the mouse
model as well as a complementary 32D3 myeloid progenitor cell model. The 32D3
model has been used previously to demonstrate the effects of AML1-ETO(1, 59).
The _____ laboratory has extensive expertise in all proposed molecular analyses
outlined below, including RT-PCR, immunoprecipitations, Western blotting, and
histone deacetylase assays.
We hypothesize that expression of AML1/ETO in murine bone marrow
progenitors will be sufficient to cause a myeloproliferative disorder. Analyses
of AML1-ETO using different cell lines can give contradictory results(65), most
likely due to cell type specific differences between repression pathways. Therefore,
the ideal system for analyzing leukemogenic interactions between CoRs and AML1-ETO
is that which most closely recapitulates the molecular pathways of the myeloblast.
Development of a mouse model will permit the evaluation, for the first time,
of interactions between AML1-ETO and CoRs in vivo. This approach has the potential
to selectively reveal activities of AML1-ETO that are involved in disease. The
approach would distinguish these pathogenic activities from those that are incidentally
detected using in vitro models. An in vivo model, therefore, could be invaluable
in determining the direction of future studies of disease mechanism.
We will attempt to produce leukemia in mice by ex vivo transduction of mouse
bone marrow with vectors expressing AML1-ETO. Transduced bone marrow will be
transplanted to lethally irradiated syngeneic recipient mice. These animals
will be monitored for histopathological or flow cytometric evidence of leukemia.
_____, will supervise development of the model. _____ has performed this procedure
for seven years in several hundred mice and continues to perform the transductions
/ transplants routinely. Therefore, the necessary reagents and expertise are
available to develop
the mouse model for the analysis of AML1-ETO.
AML1-ETO MigR1 vector
A retroviral vector to be used for
in vivo studies was constructed to express AML1-ETO in hematopoietic progenitor
cells and to, subsequently, permit the flow-cytometric recognition of infected
cells using green fluorescence protein (GFP) (see preliminary data). The mouse
stem cell viral vector (MSCV), used in previous leukemia models(43, 66), was
designed to express a bicistronic mRNA encoding N-terminal FLAG-tagged AML1-ETO
and humanized green fluorescent protein (GFP) downstream of an internal ribosomal
entry site (IRES) (43). The vector encodes the N-terminal 177 amino acids of
AML1 fused to the C-terminal 575 amino acids of ETO. Appropriately, a 95 kD
protein is encoded (see preliminary data).
Retrovirus production
A critical parameter for development
of a successful retroviral transduction model is production of high titers (106/ml)
of retrovirus. The Bosc23 packaging cell line was developed by Pear et al. to
achieve this target (44). We can routinely achieve titers in excess of 106,
as measured by GFP expression in infected NIH3T3 cells (see preliminary data).
Bone marrow infection
Bone marrow infections will be performed
as described (41-43). 4x106 bone marrow cells from Balb/c mice will
be harvested from dissected long bones four to five days after treatment with
5-FU (5 mg / mouse). Cells will be transduced by spinoculation(42) on consecutive
days in cocktails of 1 ml retroviral supernatant plus 3 ml DMEM, 10% fetal bovine
serum (Gibco), 5% WEHI-conditioned medium, 6 U/ml recombinant mouse IL-3 (Genzyme),
10,000 U/ml recombinant mouse IL-6 (Genzyme), 5 U/ml recombinant mouse SCF (Amgen),
2 µg/ml polybrene (Sigma), 100 U/ml
streptomycin (Gibco), 100 U/ml penicillin (Gibco), and 2 mM L-glutamine (Gibco).
Transplantation
2.5 x 105 to 5.0 x 105
cells cultured for 48 hours, as described above, will be injected into tail
veins of lethally irradiated syngeneic Balb/c recipient mice.
Monitoring virus expression
To insure that virus is expressed
in the periphery, 100 µl of blood will be collected monthly and analyzed by
flow cytometry for GFP expression. Flow cytometry will be performed in the University
of _____ _____ Center facility using a FACScan flow cytometer (Becton Dickenson).
Monitoring disease onset
Mice will be monitored for disease
by observation daily (decreased activity, tachypnea-denoted by hunched posture,
etc.), blood smear analysis weekly (see below), and flow cytometric analysis
monthly (see below). Mice will be monitored for up to a year based upon the
experience of the _____ laboratory.
Histopathology
Wright stained blood smears of mice
will be analyzed weekly for histopathological evidence of leukemia. Animals
with increased white blood cell counts (>50,000) or circulating blasts will
be sacrificed and examined for gross or histopathological involvement of peripheral
blood, bone marrow, spleen, liver, thymus, or lymph nodes. The applicant has
training in hematopathology and is capable of routine analyses. Additionally,
the expertise of _____, the Director of _____ and member of the advisory committee,
will be sought if unexpectedly complex morphology is observed.
Flow cytometry
Flow cytometry will be used every
other week to monitor animals for signs of disease. We will analyze 100 µl of
blood for abnormal FSC/SSC, Gr1/Mac1, or B220/Thy1.2 profiles. Additionally,
flow cytometry will be performed on peripheral blood, bone marrow, spleen, liver,
thymus, and lymph nodes of mice with leukemia. Analyses will be performed using
GFP to gate virus-infected cells. Second and third fluorochromes will be PE
and PerCP. A comprehensive panel of antibodies will be used to determine the
profile of myeloid and lymphoid markers (including Gr1, Mac1, Ter119, B220,
Thy1.2, CD4, CD8) expressed on leukemic cells. Flow cytometry will be performed
in the University of _____ _____ Center facility using a FacsCalibur dual laser
flow cytometer (Becton Dickenson).
Determination of retrovirus induced disease
In order to demonstrate that the
AML1-ETO retrovirus, in fact, causes disease in the involved animals, proviral
integration will be confirmed by Southern blot analysis and expression of AML1-ETO
protein will be confirmed by Western blot analysis.
Serial transplantation
The standard for declaring a leukemia
model is the ability to serially passage disease from a donor to a recipient.
We will therefore transplant bone marrow cells from diseased mice to sublethally
irradiated syngeneic recipients. Recipient mice will be monitored for disease
as described above.
Alternative approaches
We expect that the outlined retroviral
transduction experiment is the most likely approach to successfully produce
leukemia in mice. Although, AML1-ETO “knock-in” mutations completely
disrupt definitive hematopoiesis, we expect to have better success because we
will be infecting cells somewhat more differentiated than the primitive hematopoietic
stem cells. (These stem cells are less likely to be infected because of their
low proliferation rate). We have opted not to take conventional transgenic approaches
or conditional “knock-in” approaches because we do not know the
exact stage of hematopoietic development during which AML1-ETO must be expressed
to disrupt myeloid development.
We feel that the project has a high
likelihood of success, given the availability of necessary reagents and technical
expertise. Nevertheless, we recognize that producing myeloid leukemia in mice
is subject to biological limitations. For example, mice may develop a myeloproliferative
disorder that does not resemble acute myelogenous leukemia of the French-American-British
M2 subtype. This outcome would not compromise our ability to perform the in
vivo studies of corepressor biology. (However, it could limit the ability to
draw general conclusions pertinent to myeloid development). It is also possible
that disease will be produced with low penetrance. If this occurs, it may be
possible to increase disease frequency using alternative genetic backgrounds
such as p53 -/+ or -/-. If mice develop no evidence of a myeloproliferative
disorder, we can satisfactorily address questions in specific aims 2 and 3 using
well-established cell culture models.
Although numerous models are potentially available for in vitro studies (primary
bone marrow cells, FDCP-mix, MEL, etc.) the differentiation of both U937 cells
and 32D3 cells has previously been shown to be inhibited by AML1-ETO(1, 15,
59). While either cell line would be an acceptable alternative to the mouse
model, we intend to use the 32D3 myeloid progenitor cell line for two reasons.
First, we speculate that 32D3 cells may more closely represent myeloid precursors
than the monocytic U937 cells. Additionally, we will be able to employ the same
ecotropic virus produced
for the in vivo studies. The remainder of this proposal will, therefore, focus
on the 32D3 cell model system.
32D3 cells are a diploid, IL-3 dependent myeloid progenitor cell line that can
be induced to differentiate into neutrophil-like cells when treated with G-CSF(29).
Cells are cultured in Iscoves modified Dulbecco medium (IMDM; GIBCO),10% fetal
bovine serum (Gibco), 100 U/ml streptomycin (Gibco), 100 U/ml penicillin (Gibco)
and 2 ng/ml recombinant murine IL-3 (R+D Systems). Differentiation is performed
by washing cells twice in IMDM and plating cells at a concentration of 2 x 105
cells/ml in IMDM, 10% FBS, 100 U/ml streptomycin, 100 U/ml penicillin and 10
ng/ml recombinant murine G-CSF (R+D Systems). Cells are harvested after 7-12
days for Wright staining or flow cytometry (using a marker such as Gr1). 4-6
clones will be examined for each construct.
We hypothesize that corepressor recruitment
by AML1 may be sufficient to disrupt myeloid development in the absence of ETO
domains. This is based upon the recent observation by the _____ laboratory(15)
and others(33, 56), that AML1-ETO interacts with corepressors (CoRs), NCoR and
SMRT. Addressing the issue of sufficiency is critical to guiding future studies
either upstream or downstream of the CoR. We will attempt to determine if fusing
the CoR repression domains to the AML1 DNA-binding domain can confer pathogenic
activity upon AML1. We intend to examine fusion constructs for the ability to
disrupt myeloid differentiation through transcriptional mechanisms.
Selection of mouse vs. cell culture model
AML1-CoR constructs will be evaluated
using both in vivo and in vitro models. The in vivo mouse model developed in
specific aim 1 is potentially the best assay of activity because it tests specifically
for production of disease. (As discussed, if the mouse model is not produced
successfully, constructs will be tested for the ability to inhibit myeloid differentiation
in 32D3 cells). The model system used, subsequently, in specific aim 2 will
depend on the phenotype of AML1-CoR constructs in the differentiation assay.
Constructs that produce leukemia in mice will be amenable to follow-up studies
in vivo because circulating blasts will be available for analysis. In contrast,
analysis of constructs that do not produce leukemia will best be performed in
32D3 cells because this model can better provide adequate immature myeloid cells
for experimentation. We will perform the expression analyses described below,
using leukemic blasts from mice when possible, but we will not hesitate to employ
the 32D3 system as necessary.
AML1-NCoR and AML1-SMRT Vectors
Fusion genes encoding the first 177
residues of AML1 (the same as those found in AML1-ETO) and the repression domains
of NCoR (residues 1-1445) and SMRT (residues 1-483) will be constructed. (It
is noteworthy that the MSCV retrovirus can effectively package inserted sequences
greater than 7 kb)(43). The fusion genes will be cloned into two retroviral
vectors. First, they will be cloned into MSCV MigR1 containing retroviral sequences,
IRES, and GFP for in vivo analyses. Second, they will be cloned into MSCV 2.2
containing the selectable PGK-neo gene for in vitro
studies(43). Constructs will be evaluated for errors by sequencing and for the
ability to express stable, intact protein by Western blot analysis.
Retrovirus production and cell infection
Retrovirus will be produced using
Bosc23 cells (44) and infections will be performed by spinoculation(42), as
described in specific aim 1.
Inhibition of myeloid differentiation
Vectors expressing AML1-NCoR and
AML1-SMRT will be tested for the ability to inhibit myeloid differentiation.
Mice and 32D3 cells will be analyzed as discussed in specific aim 1 using microscopy
and flow cytometry to monitor myeloid differentiation. The outcome of this experiment
will answer the fundamental question of whether or not CoR recruitment by AML1
is sufficient to disrupt myeloid differentiation.
Disruption of AML1 target gene expression
AML1-ETO is hypothesized to disrupt
myeloid differentiation by perturbing expression of AML1 target genes. It has
been speculated that these specific downstream targets contribute to the leukemic
phenotype. We, therefore, intend to examine expression of selected AML1 target
genes in mouse myeloid cells and 32D3 cells transduced with AML1-CoR fusions.
Infected cells from mice will be collected by sorting for GFP fluorescence whereas
infected 32D3 clones will simply be expanded for analysis. Quantitative RT-PCR
will be performed to detect expression of targets
including IL-3, GM-CSF, and M-CSF receptor. Representative target genes with
the potential to influence disease phenotype have been selected. These includes
both target genes that are increased and decreased by AML1-ETO. Expression profiles
will be compared and contrasted with those of cells transduced with the AML1-ETO
construct. Results should confirm a transcriptional activity of AML1-CoR and
may provide insight into the AML1 target genes participating in disease.
Alternative Approaches
We do not anticipate significant
technical difficulties with specific aim 2. Cloned sequences necessary for construct
design are available in the _____ laboratory and the proposed techniques are
performed routinely.
Interpretation
The _____ laboratory has demonstrated
that the CoR-binding domain is essential for the activity of AML1-ETO (15).
This aim will address the fundamental issue of the sufficiency of CoR recruitment.
If AML1-CoR fusions are sufficient to inhibit myeloid differentiation, then
focus will, appropriately, be directed downstream of the CoR, to CoR-interacting
molecules. Alternatively, if AML1-CoR does not inhibit myeloid development,
additional efforts must be directed toward understanding the unique pathogenic
functions of ETO domains. Completion of this aim is, therefore, critical to
guiding future investigation in the field.
Expression studies are necessary
to confirm that AML1-ETO acts through CoR recruitment. Our working hypothesis
is that AML1-ETO recruits CoRs to exert its transcriptional effects and inhibit
myeloid differentiation. We, therefore, anticipate that AML1-CoR will influence
gene expression to the same degree as AML1-ETO. If the observed profile of gene
expression is not correlated with the differentiation phenotype, the transcriptional
activity of AML1-CoR must differ from the transcriptional activity of AML1-ETO
(e.g. lack dominant negative activity, lack a transforming activity of ETO,
etc.). Dissociation of the differentiation and expression phenotypes would suggest
that the transcriptional effects of CoR recruitment are not identical to the
pathogenic transcriptional effects. Differences in AML1 target gene expression
may, then, provide insight into the genes participating in disease. In summary,
the results of expression studies will be interpreted in the context of the
differentiation analysis.
ETO has four conserved domains named Nervy-homology (NH) for similarity to the Drosophila factor, Nervy(12). The _____ laboratory recently demonstrated that NH4 corresponds to the CoR binding domain and is essential for inhibition of differentiation of U937 tissue culture cells(15). Deletions of other ETO domains, in the context of AML1-ETO, result in compromised interactions with CoRs or reduced transcriptional repression activity (32, 33). Nevertheless, it is unclear if such deletions affect myeloid development. The experiments described in specific aim 3 complement those of specific aim 2. Specific aim 3 has the potential to indicate whether specific domains of ETO have essential CoR-dependent or CoR-independent activities. We will systematically analyze AML1-ETO deletion mutants for the ability to
inhibit myeloid differentiation
disrupt AML1 target gene expression (IL-3, M-CSF receptor, and GM-CSF) and
interact with members of corepressor complexes (NCoR, SMRT, mSin3, and HDAC)
This approach will provide insight into the activities of ETO domains and reveal whether or not they contribute to disease.
AML1-ETO∆NH1, AML1-ETO∆NH2 vectors
We will attempt to produce deletions
in two conserved domains of the AML1-ETO cassette using PCR. The NH1 (TAF110
domain) deletion will lack AML1-ETO residues 277-344. The NH2 (dimerization
domain) deletion will lack AML1-ETO residues 500-520. The FLAG AML1-ETO cDNA
will be released as a Xho1 cassette and cloned into MSCV MigR1 containing retroviral
sequences, IRES, and GFP(43) for in vivo experiments and MSCV 2.2 containing
PGK-neo for in vitro studies. Constructs will be evaluated for errors by sequencing
and for the ability to express stable, intact protein by Western blotting.
Inhibition of myeloid differentiation
Experiments will be performed as described in specific aim 2.
Disruption of AML1 target gene expression
Experiments will be performed as described in specific aim 2.
Interactions with members of the corepressor complex
CoRs function in repression
complexes to produce their transcriptional effects. AML1-ETO interacts not only
with CoRs, but also with other members of the corepressor complex, including
mSin3 and HDAC(33). We will attempt to determine if AML1-ETO mutants are capable
of recruiting corepressor complexes as effectively as intact AML1-ETO. Protein
extracts will be prepared from leukemic blasts or 32D3 cells, as previously
described. Coimmunoprecipitations will be performed using antibodies directed
against the FLAG epitope of the fusion proteins followed by immunoblotting using
antibodies directed against NCoR, SMRT, mSin3, HDAC. Additionally, immunoprecipitates
will be tested for histone deacetylase activity. Undoubtedly, during the course
of these studies, new members of the repression complexes will be published.
(Some have already been identified in the _____ laboratory.) As appropriate,
attempts will be made to evaluate interactions with these proteins, as well
as those listed. The findings of the coimmunoprecipitation experiments will
be interpreted in light of both differentiation and expression studies. These
experiments will provide clues to the ETO domains participating in repression
complex recruitment and to the domains contributing to pathology.
Alternative Approaches
One area of potential difficulty
with the techniques described in specific aim 3 relates to the coimmunoprecipitation
analyses. While the detection of protein-protein interactions is the forte of
the _____ laboratory, it may be difficult to demonstrate such interactions using
the systems described. One potential problem is the level of expression of fusion
constructs. If AML1-ETO mutants are not expressed in cells at a level adequate
for coimmunoprecipitation studies, the model may have to be adjusted. For example,
it may be necessary to design vectors with the potent CMV promoter driving expression
and perform transfections of 32D3 cells by electroporation. An additional area
of potential difficulty is related to the source of starting material. Protein
analysis in myeloid cells can be compromised by proteolysis if the cells show
a degree of differentiation. If such a difficulty arises, we anticipate that
we can circumvent it using the 32D3 cell model.
Interpretation
The systematic approach to analyzing
deletion mutations of AML1-ETO will help clarify the role of AML1-ETO in leukemogenesis.
These experiments will complement those of specific aim 2. In specific aim 2
we simulated constitutive recruitment of CoR in order to look for an effect
of an ETO deficiency. In specific aim 3, we are altering ETO and looking for
deficiencies in either CoR recruitment or other undescribed activities. The
activity of AML1-ETO mutants in myeloid differentiation, gene expression, and
complex recruitment assays will provide clues to the importance of the NH domains,
themselves, and to the role of ETO in transcription.
Differentiation analyses address
the ultimate question of whether or not a given domain contributes to inhibition
of myeloid differentiation. These assays are likely to be the most sensitive
measures of activity because they do not exclude any specific mechanism of inhibition.
Results will be interpreted in conjunction with expression studies and complex
recruitment analyses.
Expression studies will indicate
if AML1-ETO mutants can disrupt AML1 target gene expression. As in specific
aim 2, the activity of AML1-ETO mutants is likely to be correlated with the
differentiation phenotype, if in fact, AML1-ETO exerts its leukemic effects
solely through corepressor recruitment. A discrepancy between the differentiation
and expression phenotypes would suggest that we revealed a unique, non-transcriptional
activity of ETO. This will potentially be significant in unraveling the mechanism
by which AML1-ETO produces leukemia.
Coimmunoprecipitation studies indicate
the degree to which AML1-ETO mutations affect recruitment of the corepressor
complex. Domains outside of NH4 have been implicated in recruitment of the repression
complex(33). These results will be compared to those from differentiation and
expression studies. If AML1-ETO acts through CoR recruitment, then corepressor
complex recruitment should correlate exactly with gene expression and differentiation
studies. Dissociation between activities could signify CoR-independent transcriptional
effects or unique, non-transcriptional activities of ETO. These experiments
will be crucial to examining the intricacies of CoR recruitment by AML1-ETO.
In summary, these studies will provide
insight into the molecular pathogenesis of acute myelogenous leukemia. The project
combines the expertise in transcriptional repression of the _____ laboratory
and the expertise in leukemia modeling of the _____ laboratory. This collaboration
not only advances the proposed science, but also strengthens the proposed training
program. In the process of performing these experiments, I expect to develop
perspectives and skills necessary for my transition to an independent physician-scientist.
The development of an in vivo model for the study of AML1-ETO is the first priority of the proposed work. We are ready to begin transduction/transplantation experiments, although, the time to leukemia development is unpredictable. Experience in _____ laboratory suggests that leukemia should develop within several weeks to a year. We, therefore, anticipate that specific aim 1 will be completed within the first year.
Constructs to be used
in specific aims 2 and 3 will be assembled over years 1 and 2. The analysis
of constructs described in specific aim 2 will be initiated prior to those in
specific aim 3. Constructs will be tested in functional assays as they are completed.
Assays for inhibition of development will be performed first. They will be completed
over years 2-4. Expression studies and coimmunoprecipitation studies will be
performed over years 3-5.
N/A
The University of _____ and _____ has approved a protocol regarding the bone
marrow transplant procedure in mice. The approval letter is attached.
This system was selected because, historically, the murine hematopoietic system
has served as an excellent model for studying cellular development and proliferation.
Tissue culture models may not accurately replicate the hematopoietic stem cell
or provide definitive insight into the molecular pathways involved in the pathogenesis
of leukemia. Furthermore, tissue culture models do not provide insight into
phenotype at the level of the organism.
Bone marrow transplant experiments will entail harvest of marrow, ex vivo transduction,
and transplant to lethally irradiated syngeneic recipients. Balb/C mice (6-12
weeks old and greater than 20g) will receive a lethal dose of irradiation (900
Rads) from a Cs source to be received in two 450 Rad doses given over a 24 hour
period. After the second radiation dose, the mice will receive 1-2 x 106
bone marrow cells from a syngeneic donor via tail vein injection. This quantity
of bone marrow cells results in 100% survival. Because the mice will be immunocomprimised
for approximately 14 days, the recipient mice will be maintained on autoclaved
food and water.
Groups of 14 mice (6 donor mice for 8 recipient mice) will be used in these
studies and experiments will be repeated at least once (12 and 16 mice per group,
respectively.) The bone marrow and spleen of diseased animals will be used in
serial passage to determine the malignant potential of the cells (maximum 16
mice per group, if all recipients develop leukemia.) IACUC has approved the
use of 444 mice for the proposed studies.
Care will be taken to minimize animal distress. Annex II IACUC Guidelines for
Tumor Load Studies will be followed. Recipients will be monitored for physical
evidence of leukemia (cachexia, decreased activity, etc.) or peripheral histopathological
evidence of leukemia (using a drop of tail blood). Mice with evidence of leukemia
or other illness will be euthanized using CO2.