Peter D. Aplan, M.D.
37 Convent Drive Room 6002
Bethesda, MD 20892
A large number of hematologic malignancies are associated with non-random, recurrent chromosomal translocations. These translocations typically either activate latent proto-oncogenes or create novel fusion genes. Our approach to studying leukemia in general is as follows:
- Identify and clone the genes located at translocation breakpoints.
- Test the oncogenic potential of these genes in animal models.
- If the animals develop leukemia, or other hematologic malignancy, study the process of malignant transformation over time (i.e., can occult, pre-malignant lesions be identified in clinically healthy animals).
- Identify collaborating events (i.e., genes and pathways) that, when abrogated, can collaborate with the gene of interest.
- Use the animal model for pre-clinical testing of cancer treatment modalities.
1. We developed a transgenic mouse model of T-cell leukemia by overexpressing SCL and LMO1 in the thymus. We identified mutations of the Notch1 gene in 70% of these mice, demonstrating that Notch1 activation was a progression event that collaborated with SCL/LMO1. The Notch1 mutations are important in maintaining the malignant phenotype of these cells, as T-cell lines established from SCL/LMO1 mice with Notch1 mutations are sensitive to gamma-secretase inhibitors. We used cre-lox technology to generate mice in which SCL can be activated conditionally. These mice show impaired thymocyte differentiation, with a dramatic increase in immature CD4-/CD8- thymocytes.
2. We have cloned several chromosomal translocations that generate NUP98 fusion proteins. We generated transgenic mice that express NUP98HOXD13 (hereafter NHD13) or NUP98TOP1 in the hematopoietic compartment. The NHD13 mice develop a highly penetrant myelodysplastic syndrome (MDS) that resembles the human disease in terms of peripheral blood cytopenias, dysplasia, apoptosis, and progression to frank leukemia. We used retroviral tagging experiments to identify genes that collaborate with NHD13 in the process of leukemic transformation, and identified a number of expected (Meis1) collaborating genes, as well as several novel and unexpected collaborators (MN1, Erg, Mir29a). We have shown for the first time that MDS is transplantable, by transplanting bone marrow from NHD13 mice with MDS. We generated "knock-in" ES cells that express NHD13 under control of endogenous NUP98 regulatory elements, and have derived immortal, IL-3 dependent hematopoietic precursors from these ES cells.
3. Evaluation of CALM-AF10 as an oncogenic fusion. The CALM-AF10 fusion is caused by a t(10;11) chromosomal translocation seen in a wide spectrum of acute leukemia, but most commonly in T-ALL. We have generated transgenic mice that express the CALM-AF10 fusion in the hematopoietic compartment, 50-70% of these mice develop acute leukemia, primarily AML, but often with lymphoid features.. These mice also show increased expression of HOXA cluster genes and impaired thymocyte differentiation, suggesting that the cause of leukemia is, at least in part, impaired blood cell differentiation caused by overexpression of HOXA cluster genes.
Gross chromosomal rearrangements (GCR), including chromosomal translocations, deletions, inversions, and amplifications are present in the majority of cancer cells. These GCRs can lead to production of oncogenic fusion genes, amplification of proto-oncogenes, and deletion of tumor suppressor genes. However, the molecular mechanisms which lead to these GCR are largely unknown. Therefore, our specific goals are:
- To establish one or more model systems in which we can reproducibly generate GCR.
- To determine if certain genomic regions or sequence motifs are likely to be involved in these GCRs.
- To investigate what gene(s) might be important in preventing these GCRs.
An unsolved, fundamental question concerning chromosomal translocations can be phrased as follows: "Do recurrent, non-random translocations occur between 'recombinogenic' regions of the genome that are extraordinarily susceptible to breakage/religation events, or are the regions involved not particularly recombinogenic, but simply regions that lead to the production of oncogenic fusion proteins which confer a growth advantage to the cell?" To help address this question, we developed an in vitro system in which we can produce chromosomal aberrations, through the use of the Isce-I restriction endonuclease, that do not provide a growth advantage to the cell. Using this system, we have learned that gross chromosomal rearrangements are a rare result of improper DNA repair of a double strand break, with a frequency roughly 1% that of small interstitial deletions.
We are using this system to determine if gross chromosomal rearrangements will be more common if additional breaks are introduced, via genotoxic chemotherapy such as etoposide or bleomycin and/or VDJ recombination. Our preliminary results suggest that the use of etoposide and I-SceI does not lead to increased numbers of GCRs. However, the use of RAG1/RAG2 on a substrate that contains a VDJ recombination signal sequence has led to larger deletions, on the scale of 100s of kb. In addition, we are using this system to test whether purine/pyrimidine repeat regions are recombinogenic.
Goldberg L, Gough SM, Lee F, Dang C, Walker RL, Zhu YJ, Bilke S, Pineda M, Onozawa M, Jo Chung Y, Meltzer PS, Aplan PD. Somatic mutations in murine models of leukemia and lymphoma: Disease specificity and clinical relevance. Genes Chromosomes Cancer. 2017;56(6):472-483.
Gough SM, Lee F, Yang F, Walker RL, Zhu YJ, Pineda M, Onozawa M, Chung YJ, Bilke S, Wagner EK, Denu JM, Ning Y, Xu B, Wang GG, Meltzer PS, Aplan PD. NUP98-PHF23 is a chromatin-modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD histone reader function. Cancer Discov. 2014;4(5):564-77.
Onozawa M, Zhang Z, Kim YJ, Goldberg L, Varga T, Bergsagel PL, Kuehl WM, Aplan PD. Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome. Proc Natl Acad Sci U S A. 2014;111(21):7729-34.
Gough SM, Slape CI, Aplan PD. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood. 2011;118(24):6247-57.
Chung YJ, Choi CW, Slape C, Fry T, Aplan PD. Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell. Proc Natl Acad Sci U S A. 2008;105(37):14088-93.
Related Scientific Focus Areas
Genetics and Genomics
This page was last updated on June 15th, 2017