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We investigated the role of Hoip, a catalytic subunit of linear ubiquitin chain assembly complex (LUBAC), in adult hematopoiesis and myeloid leukemia by using both conditional deletion of Hoip and small-molecule chemical inhibitors of Hoip. Conditional deletion of Hoip led to significantly longer survival and marked depletion of leukemia burden in murine myeloid leukemia models. Nevertheless, a competitive transplantation assay showed the reduction of donor-derived cells in the bone marrow of recipient mice was relatively mild after conditional deletion of Hoip. Although both Hoip-deficient hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) impaired the maintenance of quiescence, conditional deletion of Hoipinduced apoptosis in LSCs but not HSCs in vivo. Structure-function analysis revealed that LUBAC ligase activity and the interaction of LUBAC subunits were critical for the propagation of leukemia. Hoip regulated oxidative phosphorylation pathway independently of nuclear factor kappa B pathway in leukemia, but not in normal hematopoietic cells. Finally, the administration of thiolutin, which inhibits the catalytic activity of Hoip, improved the survival of recipients in murine myeloid leukemia and suppressed propagation in the patient-derived xenograft model of myeloid leukemia. Collectively, these data indicate that inhibition of LUBAC activity may be a valid therapeutic target for myeloid leukemia.

UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.

The identification of characteristic differences between cancer stem cells and their normal counterparts remains a key challenge for cancer treatment. Here, we investigated the role of immunoglobulin superfamily member 8 (Igsf8, also known as EWI-2, PGRL, and CD316) on normal and malignant hematopoietic stem cells, mainly using the conditional knockout model. Deletion of Igsf8 did not affect steady state hematopoiesis, but it led to a significant improvement of survival in mouse myeloid leukemia models. Deletion of Igsf8 significantly depletes leukemia stem cells (LSCs) through enhanced apoptosis and β-catenin degradation. At a molecular level, we found that activation of β-catenin in LSCs depends on Igsf8, which promotes the association of FZD4 with its co-receptor LRP6 in the presence of Igsf8. Similarly, IGSF8 inhibition blocks the colony-forming ability of LSCs and improves the survival of recipients in xenograft models of myeloid leukemia. Collectively, these data indicate strong genetic evidence identifying Igsf8 as a key regulator of myeloid leukemia and the possibility that targeting IGSF8 may serve as a new therapeutic approach against myeloid leukemia.

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2Δ/Δ) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1+/-Ezh2Δ/Δ) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1+/-Ezh2Δ/Δ mice. Cdkn2a, a major PRC2 target encoding p19Arf, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1+/-Ezh2Δ/Δ erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1+/-Ezh2Δ/Δ mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.

POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

Clonal expansion of non-leukemic cells expressing two novel MLL – ELL variants differing in transforming activity

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2(trap)) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2(trap/trap) mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2(trap/trap) HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2(trap/trap) HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

To analyze the molecular mechanisms of the proliferation and differentiation of hematopoietic cells, we have cloned PTKs from sorted stem cells. We discuss the expression and function of receptor tyrosine kinases, STK/RON, TIE, TEK and HTK which have been cloned from these cells. STK and its ligand, MSP contributed to the motility and phagocytosis of peritoneal macrophages and bone absorption of osteoclasts. Apoptosis was induced in an erythrorid cell line by the binding of MSP (MacrophageStimulating Protein). TIE, TEK and HTK were interestingly expressed in the subpopulations of stem cells and related to the myeloid differentiation. These study will indicate the heterogeneity of stem cells and their diverse differentiation.