Noncanonical EZH2 drives retinoic acid resistance of variant acute promyelocytic leukemias.

Cancer cell heterogeneity is a major driver of therapy resistance. To characterize resistant cells and their vulnerabilities, we studied the PLZF-RARA variant of acute promyelocytic leukemia, resistant to retinoic acid (RA), using single-cell multiomics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells. We identified a subset of cells resistant to RA with proliferation, DNA replication, and repair signatures that depend on a fine-tuned E2F transcriptional network targeting the epigenetic regulator enhancer of zeste homolog 2 (EZH2). Epigenomic and functional analyses validated the driver role of EZH2 in RA resistance. Targeting pan-EZH2 activities (canonical/noncanonical) was necessary to eliminate leukemia relapse-initiating cells, which underlies a dependency of resistant cells on an EZH2 noncanonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach. Beyond RA resistance and acute promyelocytic leukemia context, our study also demonstrates the power of single-cell multiomics to identify, characterize, and clear therapy-resistant cells.

The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms.

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3- and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3- T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3- and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Efficacy of the novel tubulin polymerization inhibitor PTC-028 for myelodysplastic syndrome.

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.

Bmi1 counteracts hematopoietic stem cell aging by repressing target genes and enforcing the stem cell gene signature.

Polycomb-group proteins are critical regulators of stem cells. We previously demonstrated that Bmi1, a component of polycomb repressive complex 1, defines the regenerative capacity of hematopoietic stem cells (HSCs). Here, we attempted to ameliorate the age-related decline in HSC function by modulating Bmi1 expression. The forced expression of Bmi1 did not attenuate myeloid-biased differentiation of aged HSCs. However, single cell transplantation assays revealed that the sustained expression of Bmi1 augmented the multi-lineage repopulating capacity of aged HSCs. Chromatin immunoprecipitation-sequencing of Bmi1 combined with an RNA sequence analysis showed that the majority of Bmi1 direct target genes are developmental regulator genes marked with a bivalent histone domain. The sustained expression of Bmi1 strictly maintained the transcriptional repression of their target genes and enforced expression of HSC signature genes in aged HSCs. Therefore, the manipulation of Bmi1 expression is a potential approach against impairments in HSC function with aging.

Bcor insufficiency promotes initiation and progression of myelodysplastic syndrome.

BCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (Bcor ΔE4/y ) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (Bcor ΔE9-10/y ), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. Bcor ΔE9-10/y mice developed lethal T-ALL in a similar manner to Bcor ΔE4/y mice, whereas Bcor ΔE9-10/y hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2 Tet2 Δ/Δ Bcor ΔE9-10/y mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2 Δ/Δ Bcor ΔE9-10/y MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in Bcor ΔE9-10/y progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

Akt inhibition synergizes with polycomb repressive complex 2 inhibition in the treatment of multiple myeloma.

Polycomb repressive complex 2 (PRC2) components, EZH2 and its homolog EZH1, and PI3K/Akt signaling pathway are focal points as therapeutic targets for multiple myeloma. However, the exact crosstalk between their downstream targets remains unclear. We herein elucidated some epigenetic interactions following Akt inhibition and demonstrated the efficacy of the combined inhibition of Akt and PRC2. We found that TAS-117, a potent and selective Akt inhibitor, downregulated EZH2 expression at the mRNA and protein levels via interference with the Rb-E2F pathway, while EZH1 was compensatively upregulated to maintain H3K27me3 modifications. Consistent with these results, the dual EZH2/EZH1 inhibitor, UNC1999, but not the selective EZH2 inhibitor, GSK126, synergistically enhanced TAS-117-induced cytotoxicity and provoked myeloma cell apoptosis. RNA-seq analysis revealed the activation of the FOXO signaling pathway after TAS-117 treatment. FOXO3/4 mRNA and their downstream targets were upregulated with the enhanced nuclear localization of FOXO3 protein after TAS-117 treatment. ChIP assays confirmed the direct binding of FOXO3 to EZH1 promoter, which was enhanced by TAS-117 treatment. Moreover, FOXO3 knockdown repressed EZH1 expression. Collectively, the present results reveal some molecular interactions between Akt signaling and epigenetic modulators, which emphasize the benefits of targeting PRC2 full activity and the Akt pathway as a therapeutic option for multiple myeloma.

Setdb1 maintains hematopoietic stem and progenitor cells by restricting the ectopic activation of nonhematopoietic genes.

Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2 The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs.

Depletion of Sf3b1 impairs proliferative capacity of hematopoietic stem cells but is not sufficient to induce myelodysplasia.

Numerous studies have recently reported mutations involving multiple components of the messenger RNA (mRNA) splicing machinery in patients with myelodysplastic syndrome (MDS). SF3B1 is mutated in 70% to 85% of refractory anemia with ringed sideroblasts (RARS) patients and is highly associated with the presence of RARS, although the pathological role of SF3B1 mutations in MDS-RARS has not been elucidated yet. Here, we analyzed the function of pre-mRNA splicing factor Sf3b1 in hematopoiesis. Sf3b1(+/-) mice maintained almost normal hematopoiesis and did not develop hematological malignancies during a long observation period. However, Sf3b1(+/-) cells had a significantly impaired capacity to reconstitute hematopoiesis in a competitive setting and exhibited some enhancement of apoptosis, but they did not show any obvious defects in differentiation. Additional depletion of Sf3b1 with shRNA in Sf3b1(+/-) hematopoietic stem cells (HSCs) severely compromised their proliferative capacity both in vitro and in vivo. Finally, we unexpectedly found no changes in the frequencies of sideroblasts in either Sf3b1(+/-) erythroblasts or cultured Sf3b1(+/-) erythroblasts expressing shRNA against Sf3b1. Our findings indicate that the level of Sf3b1 expression is critical for the proliferative capacity of HSCs, but the haploinsufficiency for Sf3b1 is not sufficient to induce a RARS-like phenotype.

Histone lysine methyltransferase SUV39H1 is a potent target for epigenetic therapy of hepatocellular carcinoma.

Histone H3 lysine 9 trimethylation (H3K9me3) is associated with transcriptional repression and regulated by histone lysine methyltransferases such as SUV39H1 and ESET. However, the functional roles of these enzymes in hepatocellular carcinoma (HCC) remain uncertain. In this study, we conducted loss-of-function assays for HCC cells. SUV39H1 knockdown but not ESET knockdown reduced H3K9me3 levels and impaired HCC cell growth and sphere formation. The pharmacological inhibition of SUV39H1 by chaetocin resulted in cell growth inhibition and inducing cellular apoptosis in culture and xenograft subcutaneous tumors. Real-time polymerase chain reaction analysis indicated high levels of SUV39H1 expression in 24 of 42 (57.1%) HCC surgical samples compared with corresponding nontumor tissues. Immunohistochemistry identified high levels of H3K9me3 and ESET proteins in 23 (54.8%) and 29 (69.0%) tumor tissues, respectively. However, these proteins' expressions were only observed in biliary epithelial cells and periportal hepatocytes of nontumor tissues. Expression levels of SUV39H1 but not those of ESET were significantly correlated with H3K9me3 levels. The cumulative HCC recurrence rate was significantly higher for patients with elevated SUV39H1 expression and H3K9me3 levels. In conclusion, our data indicate that elevated SUV39H1 expression and high levels of H3K9me3 have important roles in HCC development and progression. Therefore, the pharmacological inhibition of SUV39H1 may be a promising therapeutic approach for HCC treatment.

Histone H3.3 variant plays a critical role on zygote-to-oocyst development in malaria parasites.

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Genetic deletion and pharmacologic inhibition of E3 ubiquitin ligase HOIP impairs the propagation of myeloid leukemia.

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.

Mesenchymal loss of p53 alters stem cell capacity and models human soft tissue sarcoma traits.

Soft tissue sarcomas (STSs) are a heterogeneous group of tumors that originate from mesenchymal cells. p53 is frequently mutated in human STS. In this study, we found that the loss of p53 in mesenchymal stem cells (MSCs) mainly causes adult undifferentiated soft tissue sarcoma (USTS). MSCs lacking p53 show changes in stem cell properties, including differentiation, cell cycle progression, and metabolism. The transcriptomic changes and genetic mutations in murine p53-deficient USTS mimic those seen in human STS. Furthermore, single-cell RNA sequencing revealed that MSCs undergo transcriptomic alterations with aging-a risk factor for certain types of USTS-and that p53 signaling decreases simultaneously. Moreover, we found that human STS can be transcriptomically classified into six clusters with different prognoses, different from the current histopathological classification. This study paves the way for understanding MSC-mediated tumorigenesis and provides an efficient mouse model for sarcoma studies.

Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and ß-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease.

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.

CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture.

Isolation of long-term hematopoietic stem cell (HSC) is possible by utilizing flow cytometry with multiple cell surface markers. However, those cell surface phenotypes do not represent functional HSCs after in vitro culture. Here we show that cultured HSCs express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSCs were divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. The result was same when HSCs were cultured in an efficient expansion medium containing polyvinyl alcohol. Chemically induced endoplasmic reticulum (ER) stress signal increased the CD244+ subpopulation, whereas ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which was correlated to improved reconstitution output. These data suggest CD244 is a potent marker to exclude non-functional HSCs after in vitro culture thereby useful to elucidate mechanism of functional decline of HSCs during ex vivo treatment.

Immunoglobulin superfamily member 8 maintains myeloid leukemia stem cells through inhibition of ß-catenin degradation.

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 ß-catenin degradation. At a molecular level, we found that activation of ß-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.

Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells.

Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.

Insufficiency of non-canonical PRC1 synergizes with JAK2V617F in the development of myelofibrosis.

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.