Mechanisms of Co-Operation of DNMT3A Mutations with JAK2 V617F Through Histone H4 Arginine 3 Provides New Insights in MPN Disease Pathogenesis

Abstract 2823

Myeloproliferative neoplasms (MPN), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary myelofibrosis (PMF), are clonal stem cell disorders characterised by an excess of mature cells of one or more myeloid lineages in the chronic phase, and an associated risk of progression to myelofibrosis and leukemic transformation. Highly specific somatic mutations of JAK2 and MPL are found in the majority of MPN patients, while numerous other somatic mutations have now been identified to be common between MPN and de novo acute myeloid leukemia (AML), including somatic mutations of TET2, ASXL1, CBL, IKZF1, EZH2, IDH1, IDH2 (reviewed in Tefferi, 2010 (Tefferi)), and most recently the DNA methyltransferase, DNMT3A (Abdel-Wahab, et al, Ley, et al, Stegelmann, et al, Walter, et al, Yan et al). We identified somatic heterozygous mutations in DNMT3A affecting amino acids R882 (R882C) and M880 (M880V), detected in peripheral blood mononuclear cells from two JAKV617F-positive PV patients (Rao et al, 2011, accepted for publication).

At the level of chromatin and gene regulation, cooperation of DNMT3A mutations with JAK2V617F can occur through direct interaction of DNMT3A with the chromatin modifying protein PRMT5 which is a direct phosphorylation target of JAK2V617F (Liu F et al, 2011). PRMT5 is an arginine methyltransferase which associates with chromatin and methylates the key arginine (R) residue at the N-terminus of histone H4 (R3), which in turn recruits a DNMT3A complex resulting in methylation of adjacent CpG residues and gene-silencing (Zhao Q et al., 2009). To test the functional affects of DNMT3A mutations M880V and R882C, we have performed site- directed mutagenesis to generate GFP-tagged DNMT3A wild-type and mutant retroviral constructs. These DNMT3A clones will be transduced into cord blood CD34+ve cells and GFP-positive cells selected with flow cytometry. For functional analysis, we will examine DNA methyltransferase activity, changes in histone H4 modifications and CpG methylation.

In a complementary analysis we have identified a change in a JAK2V617F+ve PV patient in the highly conserved HIST1H4C gene resulting in a substitution of Arg³ for Cys in Histone H4. Although there are 15 histone H4 genes encoding identical proteins, gene expression varies significantly in tissues and in cancer cells (W.F Holmes et. al, J.Biol Chem, 2005). We have used Q-PCR, using long primers to differentiate H4 transcripts and measured relative expression levels. We have confirmed that HIST1H4C is the major contributor of the H4 pool making ≥50% of the total H4 RNA in normal CD34+ve cells, Monocytes, lymphocytes and a variety of haemopoietic cell lines, indicating that the change to this critical residue in this gene may be functionally important. An analysis of a matched buccal sample showed that the HIST1H4C R3C variant in the PV patient was germline. Subsequent sequencing of the entire open reading frame of HIST1H4C in 232 normal individuals did not identify any other amino acid substitutions affecting this gene. Given the interaction of this residue with DNMT3A, we are currently investigating functional effects associated with this histone H4 variant. We have generated HEK293 cells with 4-Hydroxy tamoxifen (4-HT) inducible expression of the H4 R3C variant, and following the addition of 4-HT, we are initially assessing changes in histone H4 post-translational modifications, and markers of DNA damage such as gamma H2AX serine 139 phosphorylation.