Ezh2 Loss Accelerates JAK2V617F-Driven Primary Myelofibrosis
Authors: Tomioka Takahisa , Sashida Goro , Shide Kotaro , Shimoda Kazuya , Yamaguchi Naoto , Iwama Atsushi 
Published: October 2019

Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes (PRC1 and PRC2). PRC2 contains SUZ12, EED, and EZH2 that catalyze the trimethylation of histone H3 at lysine 27 (H3K27me3) and silence target-genes expression. EZH2 is generally thought to act as an oncogene in lymphoma by silencing tumor suppressor genes through H3K27me3 modifications. However, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as MDS and MPN including primary myelofibrosis (PMF). In a recent study, EZH2 mutations were independently associated with shorter survival in PMF patients, suggesting that EZH2 functions as a tumor suppressor in PMF.

Although JAK2V617F mutant is found in approximately 50% of PMF patients, it remains obscure whether the presence of JAK2V617F mutant predicts survival of PMF patients, and the functional contribution of JAK2V617F to the development of PMF has not been fully delineated. JAK2 has been shown to directly phosphorylate H3Y41 (H3Y41p) and reduce HP1a binding, thereby activating expression of target genes. However, it is unknown how JAK2V617F epigenetically alter expression of target genes in the development of PMF. Given that JAK2V617F mutation is significantly associated with EZH2 mutations in PMF patients, in order to understand how EZH2 mutations contribute to the pathogenesis of JAK2V617F-positive PMF, we generated a novel mouse model of PMF utilizing H2K-JAK2V617F transgenic mice and Ezh2 conditional knockout mice.

We first harvested 5x106 bone marrow cells from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (WT), Cre-ERT;Ezh2flox/flox (Ezh2 cKO), JAK2V617F TG/Cre-ERT;Ezh2wild/wild (JAK2 TG) and JAK2V617F TG/Cre-ERT;Ezh2flox/flox (JAK2 TG/Ezh2 cKO) mice, and then transplanted into lethally irradiated recipient mice. At 4 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 9 months post transplantation. WT and Ezh2 cKO mice did not develop myeloid malignancies. While all 11 JAK2 TG mice died due to PMF-like disease after a long latency as previously reported, 10 out of 10 JAK2 TG/Ezh2 cKO mice immediately developed PMF and died by approximately 50 days post-deletion of Ezh2. JAK2 TG/Ezh2 cKO mice showed a significantly shorter median survival than did JAK2 TG mice (36.5 days versus 245 days, p<0.01). In the peripheral blood, moribund JAK2 TG/Ezh2 cKO mice showed increased mature neutrophils, severe anemia, and thrombocytopenia, compared to WT or JAK2 TG mice at 2 months post transplantation. At the time of sacrifice, JAK2 TG/Ezh2 cKO mice showed a significant hypoplastic bone marrow without an increased myeloblast cells, but also had a marked splenomegaly due to infiltration of myeloid cells compared to JAK2 TG mice. In addition, JAK2 TG/Ezh2 cKO mice showed a severe myelofibrosis in both bone marrow and spleen, indicating that Ezh2 loss obviously promotes JAK2 V617F-driven PMF in vivo.

To understand a molecular mechanism how Ezh2 functions as a tumor suppressor for PMF, we performed gene expression analysis in Lin-Sca1+c-Kit+ (LSK) cells. While Ezh2 cKO LSKs and JAK2 TG LSKs showed up-regulation (>2-fold) of 1044 and 861 genes, respectively, JAK2 TG/Ezh2 cKO LSKs showed up-regulation (>2-fold) of more genes (1306), compared to WT LSKs. As expected, H3Y41p and H3K27me3 target genes were significantly upregulated in JAK2 TG/Ezh2 cKO LSKs, whereas H3K27me3 targets were significantly repressed in JAK2 TG LSKs, consistent with the tumor suppressor role of Ezh2 in PMF. We are now working to understand how dysregulated genes are involved in the progression of JAK2V617F-induced PMF after deletion of Ezh2. In conclusion, we have successfully established the progressive PMF in mice reconstituted with Ezh2 null cells expressing JAK2V617F mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. This model can be utilized for innovating new therapies for PMF.

Disclosures:

No relevant conflicts of interest to declare.