PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown.

Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34⁺ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34⁺ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells.

To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP⁺ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia.

To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro.

In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

No relevant conflicts of interest to declare.