Characterizing the functional role of argonaute protein PIWIL4 in acute myeloid leukemia

PIWIL proteins are stem cell self-renewal and maintenance associated proteins that bind Piwi interacting RNA (piRNA) to mediate epigenetic modifications in lower vertebrates. In solid cancers, PIWIL proteins have been described as putative proto-oncogenes. We could show that a human PIWI like protein-PIWIL4 is overexpressed in majority of AML patients, particularly in patients harboring the MLL-AF9 translocation. The knockdown of PIWIL4 in MLL-AF9 harboring cell lines and primary patient samples leads to a marked depreciation in growth in vitro and in vivo, and a global loss of H3K9me3 marks in cell lines. The loss of growth potential and decrease in global levels of H3K9me3 in cell lines could be rescued via overexpression of wild type PIWIL4, but not by a PIWIL4 mutant lacking the piRNA binding-PAZ domain. No growth inhibition or effect on colony formation was observed upon PIWIL4 depletion in normal human hematopoietic stem progenitor, in vitro. PIWIL4 depleted AML cell lines showed a deregulation of the actin cytoskeleton pathway. Cytoskeletal genes were also deregulated in PIWIL4 high vs PIWIL4 low expressing MLL-AF9 harboring AML patients. Using microarray we could determine that PIWIL4 knockdown induced differential expression of 981 known and predicted piRNAs while through small RNA deep sequencing we could identify piRNA-like-RNA in MLL-AF9 harboring cells. Immunoprecipitation of PIWIL4 identified cytoskeletal proteins, epigenetic enzymes such as MLL1 and NSD1 and heatshock proteins as binding partners. Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML, decreases leukemic growth (while not impacting normal hematopoietic cell growth), shapes epigenetic marks and impacts piRNA expression in this disease