Core-binding factor leukemia (CBFL) is a subgroup of acute myeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most common mutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3' untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133(+) stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.
BackgroundCore Binding Factor acute myeloid leukemia (CBF-AML) with t(8;21) RUNX1-MTG8 or inv(16) CBFB-MYH11 fusion proteins often show upregulation of wild type or mutated KIT receptor. However, also non-CBF-AML frequently displays upregulated KIT expression. In the first part of this study we show that KIT expression can be also upregulated by miR-17, a regulator of RUNX1, the gene encoding a CBF subunit. Interestingly, both CBF leukemia fusion proteins and miR-17, which targets RUNX1-3′UTR, negatively affect a common core RUNX1-miRNA mechanism that forces myeloid cells into an undifferentiated, KIT-induced, proliferating state. In the second part of this study we took advantage of the conservation of the core RUNX1-miRNA mechanism in mouse and human, to mechanistically demonstrate in a mouse myeloid cell model that increased KIT-induced proliferation is per se a mechanism sufficient to delay myeloid differentiation.MethodsHuman (U937) or mouse (32D) myeloid clonal lines were used, respectively, to test: 1) the effect of RUNX1-MTG8 and CBFB-MYH11 fusion proteins, or upregulation of miR-17, on KIT-induced proliferation and myeloid differentiation, and 2) the effect of upregulation of KIT-induced proliferation per se on myeloid cell differentiation.ResultsIn the first part of this study we found that stable miR-17 upregulation affects, like the CBF-AML fusion proteins (RUNX1-MTG8 or CBFB-MYH11), a core RUNX1-miRNA mechanism leading to KIT-induced proliferation of differentiation-arrested U937 myeloid cells. In the second part of the study we harnessed the conservation of this core mechanism in human and mouse to demonstrate that the extent of KIT upregulation in 32D mouse myeloid cells with wild type RUNX1 can per se delay G-CSF-induced differentiation. The integrated information gathered from the two myeloid cell models shows that RUNX1 regulates myeloid differentiation not only by direct transcriptional regulation of coding and non-coding myeloid differentiation functions (e.g. miR-223), but also by modulating KIT-induced proliferation via non-coding miRNAs (e.g. miR-221).ConclusionsThe novelty of this study is dual. On the one hand, miRNAs (e.g. miR-17) can mimic the effects of CBF-AML fusion proteins by affecting a core RUNX1-miRNA mechanism of KIT-induced proliferation of undifferentiated myeloid cells. On the other hand, the extent of KIT-induced proliferation itself can modulate myeloid differentiation of cells with wild type RUNX1 function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0283-z) contains supplementary material, which is available to authorized users.
A19 RARB2 is a master tumor suppressor that mediates the growth-inhibitory action of retinoic acid (RA). Homozygosis for epigenetically silent RARB2 alleles, which results into loss of RARB2 tumor suppressor activity, leads to RA-resistance, and apparently precedes the acquisition of morphological transformation of breast epithelial cells (Bistulfi et al., Cancer Research, 2006). RARB2 epigenetic silencing is marked by chromatin repressive changes, including DNA methylation of the RARB2 CpG island (Sirchia et al., Oncogene, 2000). In a mechanistic study, we demonstrated that methylation arises at a specific epicenter in the RARB2 CpG island, which we call the RARB2 methylation epicenter (RME) (Ren et al., MCB, 2005). Here we show that RME methylation is detectable in both benign and ductal carcinoma in situ within the same patient tissue sample. This finding implies sequential epigenetic silencing of RARB2 alleles. Timely identification of the first epigenetic hit in breast tissue of women at risk of breast cancer could prevent progression to RME methylation homozygosity, the consequent loss of RARB2-mediated tumor suppressor function, RA-resistance, and morphological epithelial cell transformation. This work was supported by the Roswell Park Alliance Foundation Award (NS), the Breast Cancer Coalition of Rochester (NS), the Susan Komen Foundation (SR). Citation Information: Cancer Prev Res 2008;1(7 Suppl):A19.
The core binding factor (CBF) transcription factor complex regulates coding and non-coding genes that play a critical role in hematopoiesis. Chromosomal rearrangements involving the two CFB subunits, RUNX1 and CFB beta, are common in acute myeloid leukemia (AML). The fusion proteins resulting from these rearrangements deregulate the transcription of RUNX1-target genes, including microRNAs critical for KIT-mediated proliferation (e.g. miR-221) and myeloid differentiation (e.g. miR-223). We found that overexpression of miR-17, which downregulates RUNX1 level by targeting RUNX1-UTR, recapitulates the biological effects of CBF-AML fusion proteins by affecting the transcription of common coding and non-coding RUNX1-targets. Consistently, increased levels of miR-17 can be detected in AML patient samples displaying increased KIT level, but without evidence of CBF mutations. Based on these finding, we propose that the definition of CBF-AML leukemia can be expanded to accommodate leukemia induced not only by genetic factors but also epigenetic factors, such as microRNAs targeting RUNX1. Acknowledgements: Funding for this study was provided by a Roswell Park Alliance Foundation award, the University of Rochester-RPCI pilot grant, and the Mark Diamond Research Fund. Citation Format: John A. Fischer, Stefano Rossetti, Arani Datta, Alessandro Beghini, Nicoletta Sacchi. Expanding the definition of core binding factor leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 180. doi:10.1158/1538-7445.AM2015-180
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