The Acute Promyelocytic Leukemia-Associated Fusion Proteins PML/RAR and PLZF/RAR Interact with PU.1 and Inactivate Its Transcriptional Function

Abstract

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation The block of differentiation is due to a deregulated function of differentiation-relevant transcription factors. We have recently shown that the acute promyelocytic leukemia (APL)-associated fusion proteins (AAFP) PML/RAR and PLZF/RAR, as well as the AML-M2-associated AML-1/ETO fusion protein, respectively related to t(15;17), t(11;17) and t(8;21), block the VitD3-induced monocytic differentiation by sequestering the VitD3-receptor (VDR). VDR is a member of the superfamily of hormon-inducible transcription factors. The aim of the study was to answer the question of whether the AAFP are able to interact directly and thereby to inhibit functionally other transcription factors, such as PU.1, which are indispensable for the normal hematopoiesis. PU.1 is a Ets-related transcription factor which plays an important in role in the stem cell fate decision as well as in the lineage specific monocytic differentiation. Recently it has been shown that the functional inactivation of PU.1 contributes to the leukemogenesis.Here we report that i.) PML/RAR as well as PLZF/RAR strongly interacted in vivo with PU.1 as revealed by mammalian two hybrid assays; ii.) the interaction with PML as well as with PLZF was stronger than that with RAR; iii.) immunofluorescence experiments revealed a diffuse nuclear anti-PU.1 staining pattern with few „dots“ in mock transfected U937 or KG-1 control cells which changed to a „microspeckled“ pattern in PML/RAR- as well as in PLZF/RAR-positive U937 or KG-1 cells; iv.) in the control cells the portion of PU.1 exhibiting localization in „dot“ structures co-localized with PML within the „dots“ of the PML-nuclear bodies (PML-NBs); v.) in PML/RAR- and PLZF/RAR-positive cells PU.1 completely co-localized within the „microspeckles“ of the fusion proteins; vi.) the AAFP inactivated the transactivation of the PU.1 target promotors of IL-18 and of GM-CSFR; vii.) the expression levels of PU.1 were not influenced by the presence of the AAFP; viii.) the overexpression of PU.1 in Sca1+/lin- hematopoietic stem cells (HSC) led in absence of G/GM-CSF to an increase of their „replating efficiency“ and to colonies expressing high levels of the stem cell markers c-Kit and Sca1; ix.) the co-expression of PU.1 in PML/RAR- and PLZF/RAR-positive HSC increased the number of differentiated cells in the CFU of the first plating but did not influence their overall replating efficiency.Taken together these data strongly suggest that the delocalization of PU.1 might withdraw it from its normal functional environment such as the PML-NBs and that this might be an important mechanisms for its functional inactivation by AML-associated fusion proteins. Furthermore it seems that the up-regulation of PU.1 is able to overcome the leukemic differentiation block only at the level of more mature cells but not at the early stem cell level which is in accordance to its dual effect on the hematopoiesis by promoting proliferation/self renewal in normal as well as in leukemic stem cells and by inducing differentiation in more mature hematopoietic precursors.