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MOZ–TIF2-mediated destruction of CBP/p300 is blocked by calpain inhibitor 2

Leukemia volume 24, pages 1359–1361 (2010)Cite this article

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The oncogenic fusion protein MOZ–TIF2 is one of several fusion products resulting from reciprocal chromosomal rearrangements in the MOZ/MYST3/KAT6A gene in acute myeloid leukemia.1 Previous studies have shown that MOZ–TIF2 transforms haematopoietic progenitors2, 3 and induces acute myeloid leukemia in a murine bone marrow transplant model.2, 4 MOZ–TIF2 induces widespread changes in histone acetylation,3, 5 subverting the development of haematopoietic progenitors towards leukaemia. One mechanism critical to the leukaemogenic action of MOZ–TIF2 is its association with the lysine acetyltransferases CBP and p300.3 This interaction requires a C-terminal activation domain (AD1) present in TIF2.6, 7 The AD1 sequence forms an amphipathic α-helix that is conserved in proteins such as Ets2, E1A, p160s and IRFs,6 and which docks with the SID (SRC1 interaction domain) of CBP/p300 to form a stable four-helix bundle.7 Mutations in the AD1 α-helix render MOZ–TIF2 incapable of transforming haematopoietic cells and unable to induce leukaemia in a mouse model,2, 3 indicating that recruitment of CBP/p300 is essential for cell transformation. A consequence of this interaction is the strong depletion of intracellular levels of CBP and p300 via a proteasome-independent mechanism, accompanied by a loss of transcription at p53 and nuclear receptor target genes, including the RARβ2 gene.3, 5 However, the mechanism by which MOZ–TIF2 promotes depletion of cellular CBP/p300 is unknown.

CBP and other proteins associated with leukaemias are often found to localize to PML or nuclear bodies. Cells expressing MOZ–TIF2 display a loss of CBP staining in nuclear bodies.3 We therefore assessed whether CPI-2 could restore the accumulation of CBP in nuclear bodies. The adherent cell line HEK293 was chosen for this experiment as it gives high quality images of CBP-stained PML bodies, and also we have previously shown that the effects of MOZ–TIF2 on gene expression and CBP/p300 levels are maintained in this background.3 HEK293 cells were transfected with YFP–MOZ–TIF2 expression vector. After 24 h to allow for expression, cells were treated with CPI-2 (2 μM) or vehicle for 8 h. The cells were then stained by immunocytochemistry using α-CBP antibodies. As shown in Figure 1c, in contrast to non-transfected cells, cells expressing YFP–MOZ–TIF2 and treated with vehicle showed a reduced overall level of CBP staining and in many cases a complete loss of CBP from PML bodies. However, cells pre-treated with CPI-2 showed close to normal levels of CBP, and many cells showed CBP to be present in nuclear bodies, irrespective of the presence of YFP–MOZ–TIF2 (Figure 1c). To quantify this effect, we repeated the HEK293 transfections using either YFP or YFP–MOZ–TIF2 vectors. After 8 h of treatment with vehicle or CPI-2 (2 μM), YFP+ cells were collected by cell sorting, and scored for the presence of CBP in nuclear bodies (n=85 minimum). As shown in Figure 1d, >95% of control cells (for example, non-transfected or expressing YFP only) scored positive for the detection of endogenous CBP in nuclear bodies, whereas only 16% of YFP–MOZ–TIF2 cells displayed any CBP staining in nuclear speckles. However, pre-treatment of YFP–MOZ–TIF2-expressing cells with CPI-2, but not vehicle induced a strong increase in the numbers of cells scoring positive for the detection of CBP in nuclear bodies (61%; Figure 1d). In contrast, CPI-2 treatment had no significant effect on CBP staining to nuclear bodies in controls, that is, in the absence of MOZ–TIF2 (Figure 1c and data not shown).

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Acknowledgements

This work was supported by the Association of International Cancer Research (KBK), Leukaemia & Lymphoma Research (HMC), Cancer Research UK and the Wellcome Trust. Our thanks are due to Prof. Saverio Minucci (Institute of Oncology, Milan), Dr. Paolo Salomoni (UCL Cancer Institute) and our colleagues in the Gene Regulation Group for advice, reagents, technical assistance and useful discussions. We acknowledge receipt of a travel bursary to KBK from the European Association for Cancer Research.

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  1. Gene Regulation Group, School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, UK

    K B Kindle, H M Collins & D M Heery

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  1. K B Kindle
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Correspondence to D M Heery.

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Kindle, K., Collins, H. & Heery, D. MOZ–TIF2-mediated destruction of CBP/p300 is blocked by calpain inhibitor 2. Leukemia 24, 1359–1361 (2010). https://doi.org/10.1038/leu.2010.92

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