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Title: Defining the role of motor neuron and pancreas homeobox 1 (MNX1) in myeloid and lymphoid leukaemia
Authors: Tonin, Stefano
Issue Date: 2015
Publisher: Newcastle University
Abstract: MNX1 is a homeodomain containing transcription factor that has been implicated in the development of a number of human diseases. Loss of function mutations of MNX1 were identified as the primary cause of the rare inherited Currarino syndrome, which includes the development of sacral masses, often teratomas. In one fourth of infant acute myeloid leukaemia (AML) cases the gene is involved in a t(7;12) translocation. In most cases, the translocation leads to the fusion of MNX1 with ETV6 (located on chromosome 12). In other cases, the breakpoint on chromosome 7 is centromeric to the MNX1 locus, but involves a concomitant deletion of the distal portion of chromosome 7q, including MNX1. Thus, a common feature of the chromosome 7 aberration is the inactivation of one copy of MNX1. Furthermore, the MNX1 promoter is frequently hypermethylated, and transcriptionally inactivated, in childhood and adult acute lymphoblastic leukaemia (ALL) (33% of childhood and 39% of adult cases). The present work investigated the functional role of MNX1 in leukaemia cells. AML and ALL cell lines were initially transfected using a Nucleofector device. These experiments provided initial evidence that MNX1 was able to induce apoptosis in leukaemia cell lines, consistent with a tumour-suppressor like role. However, definitive conclusions were hampered by a low transfection efficiency and non-specific toxicity induced by use of the Nucleofector device. To overcome these problems, subsequent experiments to examine the function of MNX1 in more detail were performed using a lentiviral transduction system. This was shown to achieve a much higher transfection efficiency, with no detectable non-specific effects on cell survival. MNX1 was re-expressed in leukemic cell lines either lacking its expression due to promoter hypermethylation or normally expressing this gene. MNX1 expressing cells were rapidly lost from the population in all silenced cell lines, whereas two of the cell lines normally expressing MNX1 showed no or very limited loss. This suggests that re-expression of MNX1 resulted in inhibition of cell growth and/or survival. In AML cell lines which exhibited rapid loss of MNX1 expressing cells, re-expression of MNX1 was associated with induction of consistently high Abstract IX levels of apoptosis (25÷40%). In ALL cell lines, multiple approaches demonstrated that, expression of MNX1 resulted in a strong G1 arrest and a near total loss of cells in S-phase or G2/M. Cell cycle arrest in ALL cell lines was associated with either apoptotic or apoptosis-independent cell death. Therefore, while induction of apoptosis was seen in both ALL and AML lines, cell cycle arrest was restricted to the ALL cell lines, suggesting that the specific biological effects of MNX1 may be lineage dependent. To address the issue of cytogenetic abnormalities involving MNX1 in AML, this study investigated 24 AML cases by fluorescence in situ hybridisation (FISH), using MNX1 and ETV6 flanking probes. Although the cohort resulted negative for the t(7;12) translocation, this analysis confirmed that karyotyping alone is not sufficient to assess the presence of abnormalities involving MNX1, and a FISH screening is required for an accurate detection. Two genes closely related to MNX1, GBX1 and GBX2, which are both homeodomain containing transcription factors and show a high level of sequence similarity to MNX1, were also investigated in this work. This analysis revealed that GBX2 promoter is frequently hypermethylated in ALL patients, both in children (38%) and adults (63%), whereas GBX1 was rarely methylated (10% of cases), suggesting that GBX2 also merits further study for its potential functional role in leukaemia. Overall, these results implicate MNX1 as an important regulator of leukaemia cell growth and survival and identify MNX1-regulated pathways as potential therapeutic targets in ALL and AML.
Description: PhD Thesis
Appears in Collections:Northern Institute for Cancer Research

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