Scientific Report 2007

Molecular and Experimental Medicine

Division of Oncovirology

Molecular Mechanisms of Cancer Development and Protein Modification by a Ubiquitin-Like Modifier

D.-E. Zhang, O.A. Malakhova, L.F. Peterson, M. Yan, A. Boyapati, J.-K. Luo, W. Zou, J.R. Biggs, J.-H. Kim, E.-Y. Ahn, J. Wang, A.J. Okumura, F. Okumura, B. Abdulla, X. Yin, M.-C. Lo, Y. Chen, S. Matsuura, W.-J. Shia, C. Burkat, X. Cong, S. Mauen

Transcription Factor AML1 and its Fusion Protein AML1-ETO in Blood Cell Differentiation and Cancer Development

Acute myeloid leukemia is a major hematopoietic malignant neoplasm characterized by the proliferation of a malignant clone of myeloid progenitor cells. One of the most common targets of chromosomal translocations implicated in this neoplasm is the gene AML1 (RUNX1). The gene was isolated through a study of t(8;21) chromosomal translocation; the results revealed that the runt homology domain of AML1 is fused to a gene termed ETO (MTG8) to form a fusion protein called AML1-ETO. Subsequent studies indicated that the protein AML1 is crucial for normal hematopoiesis. We previously discovered that AML1 synergistically activates the expression of a critical myeloid gene, the gene for the M-SCF receptor, with 2 other important transcription factors, C/EBP and PU.1.

To study the effect of AML1-ETO on hematopoiesis, we produced various mouse models, including homologous recombination, transgenic, and retrovirus-mediated gene expression and bone marrow transplantation, in which wild-type AML1 was replaced by AML1-ETO. Currently, we are identifying the molecular pathways regulated by AML1 in blood stem cells, cofactors involved in the synergy among various transcription factors and AML1-ETO–associated development of leukemia, and critical target genes of AML1 and AML1-ETO in hematopoiesis.

A Novel Ubiquitin-Specific Enzyme, UBP43

In studying genes differentially expressed in AML1-ETO mice, we isolated the gene for a novel enzyme, UBP43 (USP18), which belongs to a family of ubiquitin-specific proteases. Like phosphorylation and dephosphorylation, ubiquitylation and deubiquitylation are mechanisms for protein modification. Recently, we showed that UBP43 is the only currently known enzyme that removes a ubiquitin-like modifier, ISG15, from ISG15 conjugates. In mice that lacked the gene for UBP43, UBP43-deficient bone marrow cells were hypersensitive to treatment with type I interferon and died via apoptosis in the presence of interferon. Most important, in UBP43-deficient cells, interferon induced a prolonged Stat1 tyrosine phosphorylation, DNA binding, and interferon-mediated gene activation. UBP43-deficient mice are resistant to certain viral and bacterial infections and to the development of leukemia. Currently, we are analyzing molecular pathways affected by UBP43.

Role of ISG15 Conjugation in Immune Responses

The gene for ISG15 was originally cloned as a gene highly upregulated by interferon and encodes a small ubiquitin-like protein. Unlike ubiquitin and other ubiquitin-like modifiers, ISG15 is not present in lower eukaryotes, such as yeast, indicating that it may be associated with specialized functions in higher eukaryotic cells. Upon viral infection, bacterial infection, or other stress stimulation, ISG15 protein can be detected in cells both in free and in conjugated form (ISGylation). Using high-throughput Western blot analysis and mass spectrometry, we have identified ISGylated proteins that are involved in various cellular functions. We also have identified an ISG15-conjugating enzyme and several ISG15 ligases. Regulation of protein ISGylation may provide valuable treatments to control cell function and survival. We are using techniques such as gene depletion, protein interaction, biochemical purification, and gene regulation to study the biological function of this interesting protein modification.

Publications

Biggs, J.R., Peterson, L.F., Zhang, Y., Kraft, A.S., Zhang, D.-E. AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex. Mol. Cell. Biol. 26:7420, 2006.

Boyapati, A., Yan, M., Peterson, L.F., Biggs, J.R., Le Beau, M.M., Zhang, D.-E. A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy. Blood 109:3963, 2007.

Dao, C.T., Luo, J.K., Zhang, D.-E. Retinoic acid-induced protein ISGylation is dependent on interferon signal transduction. Blood Cells Mol. Dis. 36:406, 2006.

Malakhova, O.A., Kim, K.I., Luo, J.K., Zou, W., Kumar, K.G.S., Fuchs, S.Y., Shuai, K., Zhang, D.-E. UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity. EMBO J. 25:2358, 2006.

Okumura, A.J., Peterson, L.F., Lo, M.-C., Zhang, D.-E. Expression of AML/Runx and ETO/MTG family members during hematopoietic differentiation of embryonic stem cells. Exp. Hematol. 35:678, 2007.

Okumura, F., Zou, W., Zhang, D.-E. ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP. Genes Dev. 21:255, 2007.

Peterson, L.F., Boyapati, A., Ahn, E.-Y., Biggs, J.R., Okumura, A.J., Lo, M.-C., Yan, M., Zhang, D.-E. Acute myeloid leukemia with the 8q22;21q22 translocation: secondary mutational events and alternative t(8;21) transcripts. Blood 110:799, 2007.

Peterson, L.F., Yan, M., Zhang, D.-E. The p21^waf1 pathway is involved in blocking leukemogenesis by the t(8;21) fusion protein AML1-ETO. Blood 109:4392, 2007.

Rempel, L.A., Austin, K.J., Ritchie, K.J., Yan, M., Shen, M., Zhang, D.-E., Henkes, L.E., Hansen, T.R. Ubp43 gene expression is required for normal Isg15 expression and fetal development. Reprod. Biol. Endocrinol. 5:13, 2007.

Yan, M., Kanbe, E., Peterson, L.F., Boyapati, A., Miao, Y., Wang, Y., Chen, I.M., Chen, Z., Rowley, J.D., Willman, C.L., Zhang, D.-E. A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis. Nat. Med. 12:945, 2006.

Yan, M., Luo, J.K., Ritchie, K.J., Sakai, I., Takeuchi, K., Ren, R., Zhang, D.-E. Ubp43 regulates BCR-ABL leukemogenesis via the type I interferon receptor signaling. Blood 110:305, 2007.

Zhou, G.B., Kang, H., Wang, L., Gao, L., Liu, P., Xie, J., Zhang, F.X., Weng, X.Q., Shen, Z.X., Chen, J., Gu, L.J., Yan, M., Zhang, D.-E., Chen, S.J., Wang, Z.Y., Chen, Z. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vivo. Blood 109:3441, 2007.

Zou, W., Kim, J.-H., Handidu, A., Li, X., Kim, K.I., Yan, M., Li, J., Zhang, D.-E. Microarray analysis reveals that type I interferon strongly increases the expression of immune-response related genes in Ubp43 (Usp18) deficient macrophages. Biochem. Biophys. Res. Commun. 356:193, 2007.

Zou, W., Wang, J., Zhang, D.-E. Negative regulation of ISG15 E3 ligase EFP through its autoISGylation. Biochem. Biophys. Res. Commun. 354:321, 2007.