PRMT1-Mediated Methylation of DUSP4 Determines Megakaryocyte-Erythroid Lineage Choice By Regulating p38 Signaling

Protein arginine methyltransferase 1 (PRMT1) expression level is correlated with short survival rate of acute myeloid leukemia patients.  PRMT1 contributes to leukemogenesis via direct interaction with leukemic fusion proteins such as AML1-ETO and MLL-EEN. Our previous studies have shown that PRMT1 blocks megakaryocyte (MK) terminal differentiation.  Inhibition of PRMT1 enzymatic activity promotes apoptosis and differentiation of acute megakaryocytic leukemia (AMKL) cells. To understand the molecular mechanisms of PRMT1-mediated leukemogenesis, we apply bioorthognal profiling of protein methylation technology to identify PRMT1 substrates.  A dual specificity protein phosphatase, DUSP4 was found a PRMT1 substrate. In literature, high expression of DUPS4 is linked to idiopathic CD4 lymphopenia and silencing of DUSP4 expression causes chemo-resistance in solid tumors and abnormal innate immune response to parasitic infections.  To understand the role of DUSP4 in megakaryocytic differentiation, we ectopically expressed DUSP4 and found that it enhances megakaryocyte development. In liquid culture system, DUSP4 knockdown in human CD34⁺ cells crippled MK differentiation induced by TPO. In TPO+EPO culture system, DUSP4 knockdown in human CD34⁺ cells gave rise to more erythroid cells (CD71⁺) at the expense of MK cells (CD41⁺). Therefore, DUSP4 is essential for MK differentiation. To understand how PRMT1-mediated methylation of DUSP4 blocks MK differentiation, we mapped the DUSP4 methylation site. We found that the DUSP4 methylation mutant has a longer half-life compared to DUSP4 wild type. Further studies confirmed that DUSP4 protein stability is controlled by methylation-induced ubiquitylation.

Although DUSP4 has been shown to dephosphorylate ERK, JNK and p38 kinases in overexpression systems, we found that during megakaryocytic differentiation DUSP4 specifically targets p38. These results are consistent with the findings in literature that p38 is required for erythroid differentiation, while ERK is required for megakaryocytic differentiation. Therefore, DUSP4 is responsible for turning off p38 signaling pathway, which pushes cells to erythroid lineage. Furthermore, we found that DUSP4 is associated with chromatin and dephosphorylates histone H3S10; while the methylation mutant DUSP4 locates in cytoplasm, therefore can still dephosphorylate p38 but not H3S10. Given that H3S10 phosphorylation may play a critical role in endomitosis, we also studied whether overexpression of DUSP4 antagonize aurora kinase activity to promote megakaryocytic differentiation in AMKL cell lines, containing OTT-Mal translocation or trisomy 21/down syndrome. We have tested an array of PRMT1 inhibitors to boost DUSP4 activity for potential curative therapy using AMKL leukemia mouse models. Our data support the notion that targeting PRMT1-DUSP4 pathway is a valid therapeutic approach for AMKL.