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Abstract

Acute myeloid leukemia (AML) is a form of blood cancer characterized by impaired differentiation and accelerated proliferation due to (epi)genetic dysregulation of hematopoietic stem cells (HSC). The European LeukemiaNet (ELN) classifies AML into three subtypes: favorable, intermediate, and unfavorable. In-house, we found that ~1.4% of AML subtypes (including deletion 7q or translocation (7;12) AML) express motor neuron and transcription factor 1 (MNX1), a homeobox transcription factor (TF) known to be expressed during endoderm differentiation, in motor neurons and in pancreatic cells, but generally not in hematopoietic stem and progenitor cells (HSCs). MNX1-expressing AML typically belongs to the adverse subcategories of AML. MNX1 drives leukemogenesis when overexpressed in human fetal HSCs, and these cells are transplanted into immunosuppressed mice. No therapeutic agent has been identified yet that directly targets MNX1 or reduces MNX1 expression. Therefore, finding a compound that either targets MNX1 or reduces MNX1 expression is essential for treating MNX1- expressing AML cases. GDM-1 is the only AML cell line that expresses MNX1. In these cells, MNX1 is expressed when a hijacked enhancer from the AHI/MYB region (chromosome 6) is juxtaposed with the MNX1 promoter (chromosome 7). In my thesis, I hypothesized that epigenetic modifications could disrupt the enhancer-promoter interaction that drives MNX1 expression. Thus, an epigenetic compound screen was performed, and compounds that affect GDM-1 cell viability were revealed. One compound that reduces MNX1 expression was identified. In particular, decitabine (DAC), a hypomethylating agent, emerged as a promising candidate from this screen. DAC treatment resulted in global hypomethylation and significant downregulation of MNX1 at both the RNA and protein levels. I found that a miRNA-dependent mechanism mediates MNX1 downregulation, while I could rule out two miRNA-independent mechanisms, e.g. DAC mediated changes in TAD structures in which MNX1 is embedded and the silencing via a long-noncoding RNA. miRNA-seq revealed that miR-200a-3p, predicted to bind to the MNX1 3'UTR, is upregulated upon DAC treatment. I validated DAC-mediated hypomethylation at the promoter region of this miRNA by local deep bisulfite sequencing and confirmed the interaction between miR-200a-3p and MNX1 3'UTR by luciferase assay. DAC treatment in patient-derived xenografts (PDX) expressing MNX1 resulted in the same phenotype, indicating the reduction of MNX1 levels through the same mechanism via upregulation of miR-200a-3p. Epigenetic therapies promise a reversible therapeutic strategy for cancer treatment. In summary, this work focused on epigenetic-based therapies for MNX1-expressing AML and filled the gaps in the literature regarding potential therapeutics for MNX1-expressing AML. DAC treatment reduced MNX1 via hypomethylation-mediated activation of miR-200a-3p, which targets the MNX1 3'UTR. Previous work showed the regulation of MNX1 by miR-200a and miR-141-3p in motor neurons and pancreatic insulin-producing cells. This work demonstrated the upregulation of miR-200a-3p in an AML cell line and PDX models and investigated the upregulation of miR-vii 200a-3p upon DAC treatment in the context of AML. These results suggest the potential use of DAC or miR-200a-3p mimics against MNX1-expressing AML cases in a clinical setting.