Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia.

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.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome.

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.

TET2 is essential for survival and hematopoietic stem cell homeostasis.

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.

Bmi1 is essential for leukemic reprogramming of myeloid progenitor cells.

The polycomb group (PcG) proteins, particularly Bmi1, have an essential role in maintaining the self-renewing capacity of leukemic stem cells (LSCs). Although one of their major targets in LSCs is known to be the Ink4a/Arf tumor suppressor gene locus, the role of PcG proteins in the leukemic reprogramming of target cells into LSCs is not well characterized. In this study, Bmi1(-/-) granulocyte/macrophage progenitors (GMPs) were transformed with the leukemic fusion gene MLL-AF9. Although Bmi1 was not essential to the immortalization of GMPs in vitro, Bmi1(-/-) cells showed enhanced differentiation and retained less LSCs. A number of genes were derepressed in the absence of Bmi1 including potential tumor suppressor genes. Transplantation assays demonstrated that Bmi1 was indispensable for the development of leukemia in vivo and deletion of both the Ink4a and Arf genes only partially restored the leukemogenic capacity of Bmi1(-/-) LSCs. Of note, the complementation of immortalized Bmi1(-/-)Ink4a-Arf(-/-) GMPs with Bmi1 failed to restore the expression of the majority of deregulated genes and leukemogenic activity in vivo. These findings indicate that Bmi1 is essential for the faithful reprogramming of myeloid progenitors into LSCs and unveil that leukemic fusion genes require PcG proteins exerting an effect in concert to establish LSC-specific transcriptional profiles, which confer full leukemogenic activity on LSCs.

Array-based genomic resequencing of human leukemia.

To identify oncogenes in leukemias, we performed large-scale resequencing of the leukemia genome using DNA sequence arrays that determine approximately 9 Mbp of sequence corresponding to the exons or exon-intron boundaries of 5648 protein-coding genes. Hybridization of genomic DNA from CD34-positive blasts of acute myeloid leukemia (n=19) or myeloproliferative disorder (n=1) with the arrays identified 9148 nonsynonymous nucleotide changes. Subsequent analysis showed that most of these changes were also present in the genomic DNA of the paired controls, with 11 somatic changes identified only in the leukemic blasts. One of these latter changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in a total of 9 out of 286 cases of leukemia. Our experiments also showed a somatic change responsible for an Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which resulted in a loss of DNA methylation activity of >50%. Our data have thus shown a unique profile of gene mutations in human leukemia.

The six-minute walk test and body weight-walk distance product in healthy Brazilian subjects

We assessed the 6-min walk distance (6MWD) and body weight x distance product (6MWw) in healthy Brazilian subjects and compared measured 6MWD with values predicted in five reference equations developed for other populations. Anthropometry, spirometry, reported physical activity, and two walk tests in a 30-m corridor were evaluated in 134 subjects (73 females, 13-84 years). Mean 6MWD and 6MWw were significantly greater in males than in females (622 ± 80 m, 46,322 ± 10,539 kg.m vs 551 ± 71 m, 36,356 ± 8,289 kg.m, P < 0.05). Four equations significantly overestimated measured 6MWD (range, 32 ± 71 to 137 ± 74 m; P < 0.001), and one significantly underestimated it (-36 ± 86 m; P < 0.001). 6MWD significantly correlated with age (r = -0.39), height (r = 0.44), body mass index (r = -0.24), and reported physical activity (r = 0.25). 6MWw significantly correlated with age (r = -0.21), height (r = 0.66) and reported physical activity (r = 0.25). The reference equation devised for walk distance was 6MWDm = 622.461 - (1.846 x Ageyears) + (61.503 x Gendermales = 1; females = 0); r2 = 0.300. In an additional group of 85 subjects prospectively studied, the difference between measured and the 6MWD predicted with the equation proposed here was not significant (-3 ± 68 m; P = 0.938). The measured 6MWD represented 99.6 ± 11.9 percent of the predicted value. We conclude that 6MWD and 6MWw variances were adequately explained by demographic and anthropometric attributes. This reference equation is probably most appropriate for evaluating the exercise capacity of Brazilian patients with chronic diseases.

The six-minute walk test and body weight-walk distance product in healthy Brazilian subjects.

We assessed the 6-min walk distance (6MWD) and body weight x distance product (6MWw) in healthy Brazilian subjects and compared measured 6MWD with values predicted in five reference equations developed for other populations. Anthropometry, spirometry, reported physical activity, and two walk tests in a 30-m corridor were evaluated in 134 subjects (73 females, 13-84 years). Mean 6MWD and 6MWw were significantly greater in males than in females (622 +/- 80 m, 46,322 +/- 10,539 kg.m vs 551 +/- 71 m, 36,356 +/- 8,289 kg.m, P < 0.05). Four equations significantly overestimated measured 6MWD (range, 32 +/- 71 to 137 +/- 74 m; P < 0.001), and one significantly underestimated it (-36 +/- 86 m; P < 0.001). 6MWD significantly correlated with age (r = -0.39), height (r = 0.44), body mass index (r = -0.24), and reported physical activity (r = 0.25). 6MWw significantly correlated with age (r = -0.21), height (r = 0.66) and reported physical activity (r = 0.25). The reference equation devised for walk distance was 6MWDm = 622.461 - (1.846 x Ageyears) + (61.503 x Gendermales = 1; females = 0); r2 = 0.300. In an additional group of 85 subjects prospectively studied, the difference between measured and the 6MWD predicted with the equation proposed here was not significant (-3 +/- 68 m; P = 0.938). The measured 6MWD represented 99.6 +/- 11.9% of the predicted value. We conclude that 6MWD and 6MWw variances were adequately explained by demographic and anthropometric attributes. This reference equation is probably most appropriate for evaluating the exercise capacity of Brazilian patients with chronic diseases.

Plk1 regulates liver tumor cell death by phosphorylation of TAp63.

We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21(Cip1/WAF1) and 14-3-3sigma, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.

Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression.

The proto-oncoproteins ETS1 and growth factor independent-1 (GFI1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant transformation. Here, we report that ETS1 and GFI1 interact and affect gene expression through their cross-talk. Co-immunoprecipitation analyses and glutathione-S-transferase pull-down assays revealed that ETS1 bound directly to GFI1 via its Ets domain, and GFI1 bound to ETS1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reporters showed that GFI1 repressed ETS1-mediated transcriptional activation and ETS1 repressed GFI1-mediated transcriptional activation, in a dose-dependent manner. However, in the Bax promoter where the Ets- and Gfi-binding sites (EBS and GBS) are adjacent, ETS1 and GFI1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the Bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses confirmed that an ETS1-GFI1 complex formed on the Bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against ETS1 and/or GFI1 enhanced endogenous Bax gene expression. Our results suggest that the interaction between ETS1 and GFI1 facilitates their binding to specific sites on the Bax promoter and represses Bax expression in vivo.

Polycomb gene product Bmi-1 regulates stem cell self-renewal.

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSCs). However, the role of each component of PcG complex in HSCs and the impact of forced expression of PcG genes on stem cell self-renewal remain to be elucidated. To address these issues, we performed both loss-of-function and gain-of-function analysis on various PcG proteins. Expression analysis revealed that not only Bmi-1 but also other PcG genes are predominantly expressed in HSCs. Loss-of-function analyses, however, demonstrated that absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal, indicating a central role for Bmi-1, but not the other components, in the maintenance of HSC self-renewal. Over-expression analysis of PcG genes also confirmed an important role of Bmi-1 in HSC self-renewal. Our findings indicate that the expression level of Bmi-1 is the critical determinant for the self-renewal capacity of HSCs. These findings uncover novel aspects of stem cell regulation exerted through epigenetic modifications by the PcG proteins.

Disease-associated fibronectin matrix fragments trigger anoikis of human primary ligament cells: p53 and c-myc are suppressed.

Inflammation in periodontal disease is characterized by the breakdown of the extracellular matrix. This study shows that an inflammation-associated matrix breakdown fragment of fibronectin (FN) induces anoikis of human periodontal ligament (PDL) cells. This 40 kDa fragment was identified in human inflammatory crevicular fluid and is associated with disease status. Previously, we reported that a similar recombinant FN fragment triggered apoptosis of PDL cells by an alternate apoptotic signaling pathway that requires transcriptional downregulation of p53 and c-myc. Thus, to determine whether the physiologically relevant 40 kDa fragment triggers apoptosis in these cells, the 40 kDa fragment was generated and studied for its apoptotic properties. The 40 kDa fragment induces apoptosis of PDL cells, and preincubation of cells with intact vitronectin, FN, and to a limited extent collagen I, rescue this apoptotic phenotype. These data suggest that the 40 kDa fragment prevents PDL cell spreading, thereby inducing anoikis. The signaling pathway also involves a downregulation in p53 and c-myc, as determined by Western blotting and real time quantitative PCR. These data indicate that an altered FN matrix as is elaborated in inflammation induces anoikis of resident cells and thus may contribute to disease progression.

The effect of high sugar intake on the development of periradicular lesions in rats with type 2 diabetes.

Diabetes mellitus is associated with depression of natural defenses against infection and increases the risk of periodontal disease. However, the effects of diabetes on periradicular tissue, which differs structurally from periodontal tissue, are not known. In this study, we evaluated the effects of type 2 diabetes on the development of periradicular lesions after exposure of the pulp in the left mandibular first molar through the occlusal surface in rats. GK rats with spontaneous non-insulin-dependent diabetes mellitus and Wistar rats (controls) received a normal laboratory diet and either water or a 30% sucrose solution. At both 2 and 4 weeks after pulp exposure, histologic analysis showed that alveolar bone resorption was most severe and the periradicular lesions were largest in diabetic rats given the sucrose solution. These results suggest that the metabolic conditions produced by type 2 diabetes enhance the development of periradicular lesions in rats.

Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia.

Primary familial and congenital polycythemia (PFCP) is a disorder characterized by an increased number of erythrocytes despite normal blood oxygen pressure and a normal serum erythropoietin (EPO) level. Recent studies revealed that erythroid progenitor cells from certain individuals with PFCP express various forms of EPO receptor (EPOR) truncated at the terminal carboxyl site (EPOR-TTC(PFCP)). EPOR-TTC(PFCP) can transmit EPO-mediated proliferative signals more efficiently than can full-length EPOR (EPOR-F), at least partly because of defective recruitment of SHP-1 phosphatase to these receptors. In agreement with previous studies, Ba/F3 transfectants expressing EPOR-TTC(PFCP) showed higher proliferative responses to EPO. In those transfectants, we found that EPOR-TTC(PFCP) was expressed more abundantly on the cell surface than was EPOR-F. This tendency was confirmed by a transient-expression experiment using COS7 cells. Since expression levels of EPOR protein were not significantly different among these transfectants, differences in cell surface expression were likely dependent on post-translational mechanism(s). In addition to defective recruitment of SHP-1 to EPOR-TTC(PFCP), more efficient transport and expression on the cell surface appear to serve as mechanisms responsible for increased EPO-responsiveness of erythroid progenitor cells in PFCP.

Molecular cloning and characterization of mouse Tspan-3, a novel member of the tetraspanin superfamily, expressed on resting dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells and play an essential role for triggering T-cell-mediated immune responses. In search for novel cell surface molecules expressed on DCs involved in T cell priming by representational differential analysis, we identified a mouse homologue of Tspan-3 (mTspan-3), a novel member of the tetraspanin superfamily. The mTspan-3 consists of four hydrophobic, putative transmembrane regions, forming a small and a large extracellular loop, with short intracellular amino and carboxil tails. Although the mTspan-3 is expressed on a variety of immune cell types including resting DCs, its expression on DCs is downregulated during activation induced by cross-linking CD40 with anti-CD40 monoclonal antibody. These results suggest that mTspan-3 may be involved in the function of DCs in association with T cell stimulation.

Fractalkine shares signal sequence with TARC: gene structures and expression profiles of two chemokine genes.

In the process of cloning the gene (Scyd1) encoding the mouse CX3C chemokine fractalkine, we identified a novel cDNA that encodes a chimeric molecule termed fracTARC. This molecule is a variant form of the mouse CC chemokine, TARC (for thymus- and activation-regulated chemokine), bearing the fractalkine signal sequence instead of its own. Analysis of the genomic organization of the two genes revealed that Scyd1 and Scya17, encoding TARC, are tightly linked on chromosome 8 and that fracTARC is generated by alternative splicing of the two genes. Among tissues in which Scyd1 mRNA is expressed, fracTARC mRNA is selectively expressed in brain and kidney, indicating that fracTARC mRNA is generated by tissue-specific alternative splicing under the control of the Scyd1 promoter. On the other hand, Scya17 and the fracTARC gene are reciprocally expressed in thymus, brain, lung, and kidney and are never expressed in the same tissue. These expression profiles indicate that tissue specificity of Scya17 is precisely regulated by two independent mechanisms, one by transcription from its own promoter and the other from the promoter of Scyd1 followed by tissue-specific alternative splicing. These data provide evidence for a novel mechanism that controls gene expression of two independent genes of the same family. Such a mechanism may also operate in other genes that are tightly linked on the same chromosome.

c-Myc is a critical target for c/EBPalpha in granulopoiesis.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.