PHF6 Represses Leukemia Stem Cell Self-Renewal By Dysregulating Cell Cycle Progression

Abstract

Cell fate decision and proliferation of hematopoietic stem cells (HSCs) are tightly regulated to maintain a lifelong stem cell pool size while meeting the continuous demand for mature blood cells. In Acute Myeloid Leukemia (AML), mutations in HSC-regulatory genes are thought to disrupt cell cycle control and differentiation, leading to hyperproliferation of immature myeloid cells. The X-chromosome gene PHF6 (PHD-finger protein 6) shows loss-of-function mutations in 3% of AML patients, indicating its role as a leukemia suppressor. PHF6 is a ubiquitously expressed nucleolar and chromatin binding protein with two ePHD domains. Its knockout in mice is known to cause increased HSC self-renewal, but the functional and molecular consequence of its loss in the context of AML are poorly understood. In this study, we characterize the leukemia suppressing function of PHF6 in AML using an in vivo mouse model. Hematopoietic knockout of Phf6 alone is insufficient to cause hematopoietic malignancy, and we therefore used the retroviral HoxA9 mouse model of AML. We transduced cKO (Vav-Cre; Phf6 flox) or Ctrl (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HoxA9 (cKO+HoxA9 and Ctrl+HoxA9) and performed both in vitro and in vivo studies. cKO+HoxA9 cells showed persistent replating ability in vitro compared to Ctrl+HoxA9, with larger colonies on serial replating indicating increased proliferation. On serial transplantation into irradiated recipient mice, we found higher engraftment ability of cKO+HoxA9 leukemic cells, and reduced survival of secondary recipients (Fig 1A). Limiting dilution transplantation showed that Phf6 cKO increased the frequency of Leukemic Stem Cells (LSCs). Thus, loss of PHF6 increases self-renewal of LSCs, and increased rapidity of AML progression with serial transplantation. To further understand the role of PHF6 in LSC self -renewal, we performed RNA-Seq on an LSC-enriched cell population that we phenotypically characterized with C-Kit+, Ly6C-. Gene Set Enrichment Analysis (GSEA) showed that the cKO LSC transcriptome (compared to Ctrl) positively correlated with leukemic stem cell gene sets, and negatively correlated with gene sets for neutrophil and monocytic differentiation (Fig 1B). On in vitro culture of LSCs, we found that Phf6 cKO cells maintained higher self-renewal rate in vitro, with a shorter G1 phase accompanied by prolonged persistence of an undifferentiated LSC population in culture. In summary, we have found that PHF6 represses LSC self-renewal and inhibits AML progression. Deletion of PHF6 leads to shortening of G1 phase of cell cycle, and maintains undifferentiated leukemic cells. Our future work will involve establishing the molecular mechanism by which PHF6 control self-renewal associated cell cycle progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal