Non-coding rnas in the pathogenesis of core-binding factor acute myeloid leukemia
Fischer, John Adams
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Core-binding factor acute myeloid leukemia (CBF-AML) is a subgroup of acute myeloid leukemia (AML), classically defined by the presence of non-random chromosomal rearrangements involving the core-binding factor (CBF), a master hematopoietic transcription factor complex composed of two subunits, the CBFα subunit (also known as RUNX1) and the CBFβ subunit. The two most common of these rearrangements are the t(8;21) and the inv(16), which result in the production of two chimeric CBF fusion proteins, RUNX1-MTG8 and CBFβ-MYH11, respectively. These fusion proteins directly contribute to the leukemogenic process by deregulating the transcription of RUNX1-target genes involved in myeloid differentiation and proliferation. Emerging evidence shows that, in addition to coding genes, CBF regulates the transcription of non-coding RNAs (e.g. microRNAs), and that non-coding RNAs can regulate CBF level/function. The overall objective of this thesis was to test whether non-coding regulatory RNAs regulated by CBF, as well as non-coding regulatory RNAs regulating CBF itself, play a mechanistic role in the pathogenesis of CBF-AML. By using mouse and human myeloid cell models, we found that ectopic expression of RUNX1-MTG8 or CBFβ-MYH11 induced block of myeloid differentiation and increased proliferation, concomitant with the downregulation of coding genes and microRNA genes critical for these two processes. Among others, the two fusion proteins induced downregulation of miRNA-223, a well-known RUNX1-target and regulator of myeloid differentiation, as well as miR-221/222, which target KIT, a major pro-proliferative receptor tyrosine kinase. We demonstrated that miR-221/222 is a direct RUNX1 target, and that its downregulation by CBF-AML fusion proteins directly contributes to KIT upregulation and increased proliferation. Consistently, increased KIT expression in CBF-AML patient samples correlated with decreased levels of both miR-221 and miR-222. In addition, the two CBF-AML fusion proteins led to the downregulation of the macrophage colony-stimulating factor 1 receptor (CSF1R), and consequent block of monocytic differentiation. Interestingly, these two effects could be recapitulated by ectopic expression of natural anti-sense CSF1R RNA, a long non-coding RNA that plays a role in CSF1R epigenetic silencing during B-cell lymphopoiesis. We further tested whether increased KIT-mediated proliferation could per se contribute to the block of myeloid differentiation by stably expressing KIT at different levels in mouse myeloid 32D cells. Apparently, increasing KIT level was sufficient to enhance proliferation and delay differentiation in response to the granulocyte colony stimulating factor (G-CSF). This effect was not only proportional to the level of KIT, but also due to time- and dose-dependent response to the G-CSF. Remarkably, the KIT-induced delay of differentiation could be counteracted by either increasing the concentration of G-CSF or by inducing KIT inhibition with imatinib, a potent receptor tyrosine kinase inhibitor. Finally, since KIT is also frequently upregulated in AML without CBF cytogenetic rearrangements, we investigated whether other factors interfering with RUNX1 function/expression could recapitulate some of the effects of the CBF-AML fusion proteins. We demonstrate here that one of these factors is overexpression of miR-17, which is known to reduce RUNX1 protein expression by targeting the 3'UTR of RUNX1. We found evidence that miR-17 and KIT can be concomitantly upregulated in non-CBF-AML patient samples. Moreover, ectopic expression of miR-17 in human myeloid cells was sufficient to repress microRNAs deregulated by the CBF-AML fusion proteins, including miR-223 and miR-221, as well as induce block of myeloid differentiation and increased proliferation. Overall, our results show that interference with RUNX1 regulation of coding and non-coding genes critical for myeloid differentiation and proliferation, can be caused both by CBF-fusion proteins and upregulation of microRNAs (e.g. miR-17) targeting RUNX1, suggesting that the definition of CBF-AML could be extended to include any factors interfering with RUNX1 dosage and or function. In light of these findings, microRNAs targeting RUNX1 could be considered for potential therapeutic application, alone or in combination with KIT inhibitors and myeloid differentiation inducing cytokines.