Dr. de Jong’s research focuses on the genes that regulate normal hematopoietic stem cell (HSC)
function in the bone marrow and examines whether those genes may contribute to the development
of leukemia when their expression is abnormal. She is using the zebrafish animal model to uncover
new genetic regulators of HSCs using an unbiased screening approach. Her screen utilizes a
competitive hematopoietic transplant assay using mutagenized donors to find mutants with inhibited
or enhanced engraftment. The genes discovered in this screen will be important for normal HSC
function and will likely also be important for leukemia development.

When she arrived at the University of Chicago, Dr. de Jong created a new transgenic donor
zebrafish line to overcome some of the disadvantages of the previous line she had been using.
However, this new CG1-Tg fish has not been robust. Dr. de Jong acquired a new clonal fish line,
CG2, from a collaborator at Northwestern University, Sergei Revskoy, MD, PhD, which has allowed
her to create a line to perform her initial marrow transplant experiments that will begin soon.
Phospholipase C gamma1 (PLC 1) and histone deacetylase 1 (HDAC1) are candidate genes of
interest to Dr. de Jong, and these two heterozygous mutant fish lines are currently being bred to the
CG2 clonal line in order to test adult carriers of these mutations in the competitive hematopoietic
transplantation assay described. If these genes are important for HSC function, she expects the
mutant marrows will have a decreased repopulating ability and recipients will have decreased
engraftment. This result would open the door to a whole new area of research in her lab, as she
examines the mechanism of these genes in leukemia to determine whether they could be therapeutic
targets.

Over the next 4-6 months Dr. de Jong aims to perform the first hematopoietic transplants with
clonal CG2 fish, using CG2-Tg (ubi:GFP) donors; perfect the protocol for mutagenizing the clonal
CG2 fish that will be used as donors for the screen; and continue mating the PLC 1 and HDAC1
mutants into the CG2 lineage, so that the transplant experiments will be ready to proceed.

In the past year, we have continued to make progress on the work proposed in this grant. My research focuses on the genes that regulate normal hematopoietic stem cell (HSC) function in the bone marrow, and examines whether those genes may contribute to the development of leukemia when their expression is abnormal. I am taking advantage of the zebrafish animal model as a unique discovery mechanism to uncover new genetic regulators of HSCs using an unbiased screening approach. Our screen utilizes a competitive hematopoietic transplant assay using mutagenized donors to find mutants with inhibited or enhanced engraftment. The genes discovered in this screen will be important for normal HSC function and will likely also be important to understand how leukemia develops. 

This year we have focused on establishing and characterizing a fluorescent transgenic donor zebrafish that will be used for the HSC screen. This is a unique clonal zebrafish line called CG2 that we made to express green fluorescent protein (GFP) under the control of the ubiquitin promoter. We now have a stable transgenic line of these CG2-Tg(ubi:GFP), and we have also made a similar transgenic line expressing DsRed, a red fluorescent protein. The CG2-Tg(ubi:GFP) fish will be mutagenized to perform the screen, while the CG2-Tg(ubi:DsRed) fish will be the wild type competitive donors. Unfortunately the process of generating transgenic animals was hindered in the clonal fish line, so that the time required to complete the process has been prolonged compared with wild type counterparts. Now that we have confirmed these fluorescent transgenic donor animals are finally ready, we are able to begin the mutagenesis process in the coming months, followed by the first competitive transplantation experiments using mutant marrow donors to begin the screen.  

In addition to the unbiased screen, we have also been studying two candidate genes in the competitive transplant assay described; phospholipase C gamma1 (PLC[Symbol]1) and histone deacetylase 1 (HDAC1). These mutant lines have been meticulously bred into the CG2 background and individual animals typed to select the immunologically matched mutants to be used as transplant donors. This immunotyping strategy is critical for our experiments, and has required significant effort, as very little is known about the zebrafish tissue histocompatibility genes, which play a central role in immune matching of any tissue for transplantation. Our work characterizing the zebrafish histocompatibility genes was presented at three different national and international meetings in 2012, and a manuscript is currently being prepared to be submitted for publication in January 2013. While these histocompatibility experiments were not described in the original grant proposal, this work has been essential to understanding the tissue matching for competitive transplant assays using our two candidate mutants. Moving into the future, this work will be critically important for our ongoing transplantation experiments, and for many other zebrafish labs studying transplantation, as well as tumor biology, immunology or infectious diseases.  

Finally, we have started using the HDAC1 heterozygous mutant fish to make T cell leukemias for transplantation. We chose this model in part to facilitate our experiments characterizing the histocompatibility genes, but also because we are interested in understanding how HDAC1 might contribute to leukemia. Since HDAC inhibitors are currently being tested in a number of clinical trials, we postulated that those zebrafish missing one copy of the HDAC1 gene would have less aggressive leukemias. However, our preliminary data indicate the opposite result! We observe that fish with only one copy of HDAC1 actually develop leukemias that are more aggressive and grow faster than leukemias in wild type sibling fish. We are currently working to verify the results from these first experiments, as this unexpected result opens a new avenue of research for my lab. I have written a letter of intent for a grant from the Collaborative Pediatric Cancer Research Program, which was accepted, and we will submit the final grant application in mid January 2013. 

I would like to take this opportunity to sincerely thank the members of the Cancer Research Foundation for your generous support of my work. As you know, federal research funding is extremely competitive, and it is difficult for newly established junior faculty to secure NIH funding without a track record of prior independent publications and preliminary data. I expect that our work on the zebrafish histocompatibility genes will be published in 2013, my first publication as an independent investigator. With our HSC screen sufficiently underway at that point, I believe I will be able to write a competitive R01 grant for the NIH with an excellent chance of being funded. I’m grateful for the support of the CRF, which was invaluable in bringing me to this point in my career. I’m excited that our work has opened new doors to understanding hematopoietic stem cells and leukemia, and I look forward to more discoveries down the road.