Insufficiency of Non-Canonical PRC1 Complex Cooperates with an Activating JAK2 Mutation in the Pathogenesis of Myelofibrosis

Introduction: PcG proteins form two main multiprotein complexes, Polycomb repressive complex 1 (PRC1) and PRC2. They repress the transcription of target genes. Polycomb group ring finger protein1 (PCGF1) is a component of PRC1.1, a non-canonical PRC1.1 that monoubiquitylates H2A at lysine 119 in a manner independent of H3K27me3. Several groups including ours showed that the loss of Ezh2, a component of PRC2, promotes the development of JAK2 V617F-induced Myelofibrosis (MF) in mice. However, the role of PRC1.1 in hematologic malignancies is still not fully understood. We found that the deletion of PCGF1 in mice promotes myeloid commitment of hematopoietic stem and progenitor cells (HSPCs), and eventually induces a lethal myeloproliferative neoplasm (MPN)-like disease in mice (Nakajima-Takagi Y, unpublished data). Based on these findings, we investigated the role of PCGF1 in a mouse model of JAK2V617F-induced myelofibrosis.

Methods: We transplanted BM cells from Cre-ERT2, PCGF1flox/flox;Cre-ERT2, JAK2V617F;Cre-ERT2, and JAK2V617F;PCGF1flox/flox;Cre-ERT2 mice into lethally irradiated recipient mice. We deleted PCGF1 by tamoxifen administration 4 weeks after transplantation.

Results: JAK2/PCGF1 KO mice developed lethal MF significantly earlier than the other genotypes (p<0.01). JAK2/PCGF1 KO mice showed progressive anemia and severe thrombocytopenia. Bone marrow analysis of JAK2/PCGF1 KO mice revealed a significant reduction in HSPCs and an increase in the number of granulocyte-macrophage progenitors (GMPs). Erythropoiesis was severely impaired at the later stages of erythroid differentiation. To understand the molecular basis of MF-initiating cells in JAK2/PCGF1 KO mouse, we performed a gene expression analysis of LSKs/GMPs/MEPs isolated from the primary recipients 1 month after TAM injection. Gene set enrichment analysis of RNA-seq data clearly showed de-repression of PRC1 target genes marked with H2AK119ub1 in hematopoietic stem and progenitor cells (HSPCs) from JAK2/PCGF1 KO mice. The gene set of megakaryocyte progenitors was also positively enriched in JAK2/PCGF1 KO HSPCs. ChIP sequencing of H2AK119Ub revealed that the levels of H2AK119Ub at promoter regions were mildly reduced in JAK2/PCGF1 KO LK cells compared with Pcgf1 KO LK cells. Among differentially expressed genes, we found that HoxA cluster genes were de-repressed in JAK2/PCGF1 KO progenitor cells including MEPs following significant reductions in H2AK119Ub levels at the promoter regions. Lin28b-Let-7-Hmga2 pathway genes that are activated in JAK2/Ezh2 KO progenitor cells were not altered in expression in JAK2/PCGF1 KO progenitor cells, suggesting different mechanisms operating in the pathogenesis of JAK2/Ezh2 KO and JAK2/PCGF1 KO MF. A selective AURKA inhibitor has been reported to promote differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF (Wen et al., Nat Med 21:1473, 2015). Following this report, we treated JAK2/PCGF1 KO mice with JAK inhibitors and/or AURKA inhibitors. Both inhibitors improved MF-related phenotypes including impaired erythroid differentiation of JAK2/PCGF1 KO mice.

Conclusions: Our findings suggest that dysregulated PRC1.1 function promotes JAK2V617F-induced MF with mechanisms distinct from MF associated with PRC2 dysfunction.