Cleavage of AML1/MTG8 by asymmetric hammerhead ribozymes

Abstract

The chromosomal translocation t(8;21) is one of the most frequent aberrations associated with acute myeloid leukaemia. It joins the 5 H section of the AML1 gene with the almost complete open reading frame of MTG8 (ETO). The resulting fusion RNA represents a leukaemia-specific target for antisense/ribozyme inhibition. We tested several asymmetric hammerhead ribozymes targeted against the fusion site for their ability to cleave the AML1/MTG8 RNA at low magnesium concentrations. One ribozyme cleaves AML1/ MTG8 RNA with high catalytic efficiency without binding or cleaving the wild-type AML1 transcript. The presence of cellular RNA does not affect the cleavage. Injection of AML1/MTG8 RNA and ribozyme RNA into Xenopus eggs or oocytes causes a specific reduction of AML1/MTG8 protein expression. Asymmetric anti-AML1/MTG8 ribozymes may be valuable modulators of AML1/MTG8 expression in leukaemic cells.Keywords: catalytic RNA; RNA cleavage; acute myeloid leukaemia.The translocation t(8;21) is one of the most frequent chromosomal aberrations in acute myeloid leukaemia (AML) fusing the AML1 gene on chromosome 21 to the MTG8 gene on chromosome 8 [1,2]. This translocation replaces the C-terminal transactivation domain of AML1 by MTG8, the latter lacking only a few N-terminal amino acids. The resulting fusion protein, AML1/MTG8, can still bind to DNA via the runt homology domain of AML1 [3]. AML1/MTG8 leads to the repression of gene expression by recruiting a histone deacetylase complex to the promotor/ enhancer [4±6]. Because AML1 regulates the expression of many genes important for normal haematopoiesis, their repression by AML1/MTG8 may be essential for the malignant transformation of normal progenitors [7]. Both the homozygous disruption of AML1 [8] and the heterozygous insertion of AML1/MTG8 [9,10] by homologous recombination abolish definitive haematopoiesis in the liver of mouse embryos. These results emphasize the central role of AML1 and AML1/MTG8 in haematopoiesis. Moreover, they demonstrate a gain of function for the fusion protein, as the AML1/MTG8 knock-in cells still contain one intact AML1 allele. Therefore, inhibition of AML1/MTG8 expression by an antisense or ribozyme strategy may revert the malignant phenotype.Ribozymes, in particular, have been applied to modulate the expression of leukaemic fusion proteins such as bcr-abl [11,12]. The chromosomal rearrangement creates a fusion RNA, which is specific to the leukaemic cell, and therefore represents a unique target for an antisense molecule or a ribozyme. Recently, Matsushita et al. [13,14] reported that preformed anti-AML1/MTG8 ribozymes delivered with cationic lipids led to a reduced proliferation of a t(8;21) positive cell line, Kasumi-1. Similar results were obtained with anti-AML1/MTG8 oligodeoxynucleotides [15]. However, it remained unclear whether the observed inhibition of cell proliferation was indeed due to a reduction of AML1/ MTG8 or other antisense-or ribozyme-independent effects.Recently, the groups of Sczakiel and Tabler described asymmetric rib...

Histone deacetylase inhibitors induce the degradation of the t(8;21) fusion oncoprotein

“…However, the knockdown was not complete and ribozyme-mediated targeting of the fusion protein, as well as antisense RNA, appeared to be more effective at inducing both cellular differentiation and eventually cell death (Sakakura et al, 1994;Matsushita et al, 1995;Kozu et al, 2000;Szyrach et al, 2001). Thus, DEP may be a potent targeted therapy for t(8;21) AML as it targets multiple pathways, including reducing the levels of RUNX1-ETO.…”

Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

“…Thus, our results suggest that MLT‐induced degradation of AML1‐ETO is independent of activated caspase‐3. Furthermore, knockdown of AML1‐ETO by ribozyme‐mediated targeting of fusion gene results in apoptosis or cell death . Therefore, we speculate that MLT‐induced degradation of AML1‐ETO leads to the apoptosis or cell death.…”

“…17,40 For example, eriocalyxin Furthermore, knockdown of AML1-ETO by ribozyme-mediated targeting of fusion gene results in apoptosis or cell death. 43 Therefore, we speculate that MLT-induced degradation of AML1-ETO leads to the apoptosis or cell death. However, we do not completely exclude the possibility that some unhealthy and dead cells show the lower levels of AML1-ETO protein.MLT has been reported to modulate the ubiquitin-proteasome system, 44 which is the main pathway for the degradation of proteins.…”

Effector Caspases and Leukemia

“…This fusion protein displays dichotomous function since it not only blocks differentiation but also induces growth arrest and apoptotic susceptibility of leukemic cells [62–64]. Similar to PML-RAR α , both apoptosis-independent and -dependent degradation of AML1-ETO have been reported [48, 56, 65–68], but the precise mechanism and biological significance of AML1-ETO degradation remain obscure. Recently, we found that AML1-ETO endows leukemic cells with susceptibility to both extrinsic and intrinsic apoptosis and provided direct evidence showing AML1-ETO as a caspase-3 substrate.…”