| Summary: | <p>The t(10;11) chromosomal translocation gives rise to the <italic>CALM-AF10</italic> fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. <italic>CALM-AF10</italic>-driven leukemias are characterized by a perturbed epigenetic and transcriptional state. Specifically, the <italic>HOXA</italic> cluster genes are hypermethylated on Histone H3 lysine 79 (H3K79), which corresponds with their transcriptional upregulation. Conversely, <italic>CALM-AF10</italic> cells display global H3K79 hypomethylation. DOT1L, the H3K79 histone methyltransferase, interacts with the OM-LZ domain of AF10, and the AF10 OM-LZ domain has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. These data have suggested a critical role for the AF10-DOT1L interaction in <italic>CALM-AF10</italic> leukemias. However, the mechanism(s) by which DOT1L-mediated epigenetics are perturbed and the precise role of CALM in leukemogenesis have remained unclear. </p><p>In this dissertation, we examine the contribution of CALM to CALM-AF10-mediated leukemogenesis. We determine that CALM contains a functional nuclear export signal (NES) that mediates steady-state cytoplasmic localization of CALM-AF10. An NES is a highly conserved leucine-rich amino acid sequence that is recognized by the nuclear export receptor, CRM1. Classically, CRM1 binds to NES-containing proteins and mediates their export from the nucleus to the cytoplasm through the nuclear pore complex. Through structure-function analyses, we determine that the CALM-derived NES is necessary and sufficient for CALM-AF10-dependent leukemogenesis. In addition, fusions of NES motifs from heterologous proteins (ABL1, Rev, PKIA, and APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors <italic>in vitro</italic>. From these data, we conclude that a CRM1-dependent NES represents the functional contribution of CALM for CALM-AF10-mediated leukemogenesis. </p><p>In the second part of this dissertation, we examine the mechanism(s) by which the CALM NES imparts transformation potential to AF10. We determine that the CALM NES is essential for CALM-AF10-dependent <italic>Hoxa</italic> gene upregulation and aberrant H3K79 methylation. Using co-immunofluorescence microscopy, we observe increased cytoplasmic localization of DOT1L in the presence of CALM-AF10, suggesting that mislocalization of DOT1L may lead to a global loss of H3K79 methylation. In addition to mediating nuclear export, we find that the CALM-CRM1 interaction is critical for targeting CALM-AF10 to the <italic>Hoxa</italic> locus. Inhibition of CRM1 with Leptomycin B prevents transcription of <italic>Hoxa</italic> genes in <italic>CALM-AF10</italic> leukemia cells. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of CRM1 inhibitors as therapeutic agents for patients with <italic>CALM-AF10</italic> leukemias.</p> === Dissertation
|
|---|
