Yue-Ying Wang

Gain-of-function mutation of GATA-2 in acute myeloid transformation of chronic myeloid leukemia

Acquisition of additional genetic and/or epigenetic abnormalities other than the BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast crisis (BC). To gain insights into the underlying mechanisms of progression to BC, we screened DNA samples from CML patients during blast transformation for mutations in a number of transcription factor genes that are critical for myeloid–lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. A L359V substitution within zinc finger domain (ZF) 2 of GATA-2 was found in eight cases with myelomonoblastic features, whereas an in-frame deletion of 6 aa (Δ341–346) spanning the C-terminal border of ZF1 was detected in one patient at myeloid BC with eosinophilia. Further studies indicated that L359V not only increased transactivation activity of GATA-2 but also enhanced its inhibitory effects on the activity of PU.1, a major regulator of myelopoiesis. Consistent with the myelomonoblastic features of CML transformation with the GATA-2 L359V mutant, transduction of the GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring murine cells disturbed myelomonocytic differentiation/proliferation in vitro and in vivo, respectively. These data strongly suggest that GATA-2 mutations may play a role in acute myeloid transformation in a subset of CML patients.

C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice

The full-length AML1-ETO (AE) fusion gene resulting from t(8;21)(q22;q22) in human acute myeloid leukemia (AML) is not sufficient to induce leukemia in animals, suggesting that additional mutations are required for leukemogenesis. We and others have identified activating mutations of C-KIT in nearly half of patients with t(8;21) AML. To test the hypothesis that activating C-KIT mutations cooperate with AE to cause overt AML, we generated a murine transduction and transplantation model with both mutated C-KIT and AE. To overcome the intracellular transport block of human C-KIT in murine cells, we engineered hybrid C-KIT (HyC-KIT) by fusing the extracellular and transmembrane domains of the murine c-Kit in-frame to the intracellular signaling domain of human C-KIT. We showed that tyrosine kinase domain mutants HyC-KIT N822K and D816V, as well as juxtamembrane mutants HyC-KIT 571+14 and 557-558Del, could transform murine 32D cells to cytokine-independent growth. The protein tyrosine kinase inhibitor dasatinib inhibited the proliferation of 32D cells expressing these C-KIT mutants, with potency in the low nanomolar range. In mice, HyC-KIT N822K induced a myeloproliferative disease, whereas HyC-KIT 571+14 induces both myeloproliferative disease and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KIT N822K led to fatal AML. Our data have further enriched the two-hit model that abnormalities of both transcription factor and membrane/cytosolic signaling molecule are required in AML pathogenesis. Furthermore, dasatinib prolonged lifespan of mice bearing AE and HyC-KIT N822K-coexpressing leukemic cells and exerted synergic effects while combined with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

A distinct glucose metabolism signature of acute myeloid leukemia with prognostic value

Acute myeloid leukemia (AML) is a group of hematological malignancies with high heterogeneity. There is an increasing need to improve the risk stratification of AML patients, including those with normal cytogenetics, using molecular biomarkers. Here, we report a metabolomics study that identified a distinct glucose metabolism signature with 400 AML patients and 446 healthy controls. The glucose metabolism signature comprises a panel of 6 serum metabolite markers, which demonstrated prognostic value in cytogenetically normal AML patients. We generated a prognosis risk score (PRS) with 6 metabolite markers for each patient using principal component analysis. A low PRS was able to predict patients with poor survival independently of well-established markers. We further compared the gene expression patterns of AML blast cells between low and high PRS groups, which correlated well to the metabolic pathways involving the 6 metabolite markers, with enhanced glycolysis and tricarboxylic [corrected] acid cycle at gene expression level in low PRS group. In vitro results demonstrated enhanced glycolysis contributed to decreased sensitivity to antileukemic agent arabinofuranosyl cytidine (Ara-C), whereas inhibition of glycolysis suppressed AML cell proliferation and potentiated cytotoxicity of Ara-C. Our study provides strong evidence for the use of serum metabolites and metabolic pathways as novel prognostic markers and potential therapeutic targets for AML.

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

Significance Epigenetic modifications are required for the regulation of hematopoiesis. DNA methyltransferase 3A (DNMT3A), a critical epigenetic modifier responsible for de novo DNA methylation, was reported recently to be a frequently mutated gene in hematopoietic malignancies. However, the role of mutated DNMT3A in hematopoiesis remains largely unknown. Here we show that the Arg882 (R882) mutation of DNMT3A disrupts the normal function of this enzyme and results in chronic myelomonocytic leukemia (CMML) in mice. Meanwhile, the gene expression, DNA methylation, and protein–protein interaction assays suggest that DNMT3A R882 mutation drives CMML by disturbing the transcriptional expression/DNA methylation program and cell-cycle regulation of hematopoietic cells. This study may shed light on the function of DNMT3A mutant in myeloid leukemogenesis. The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.

Targeting of AML1-ETO in t(8;21) Leukemia by Oridonin Generates a Tumor Suppressor–Like Protein

Oridonin treats AML by generating a truncated version of the AML1-ETO oncoprotein that functions as a tumor suppressor. Herbal Fusion High-tech gadgets and designer medicines increasingly drive medical treatment. But newer isn’t always better, and there has been a revitalized push for “natural” and “herbal” remedies. Although evidence supporting some of these therapies is shaky at best, researchers have found solid scientific bases for others—isolating active compounds with well-established biological function. One such compound is oridonin. Originally identified as a component of the herb Isodon rubescens, oridonin selectively kills leukemic cells that express a particular oncoprotein. Now, Zhen et al. show us exactly how compound works. Acute myeloid leukemia (AML) is a cancer of myeloid cells in the blood and bone marrow. The AML1-ETO fusion protein—an oncoprotein that results from a chromosomal translocation found in a subset of AML patients—is cleaved as a result of oridonin exposure. In the current study, the authors demonstrated that oridonin has two functions within AML1-ETO+ AML cells. The drug bound and blocked components of the oxidative damage prevention system, glutathione and thioredoxin/thioredoxin reductase, which resulted in increased amounts of reactive oxygen species and activated caspase-3. Oridonin also specifically bound to AML1-ETO, causing it to be cleaved into a truncated version that acted as a tumor suppressor in the AML cells. These data show how a simple herb can contribute a lead compound for personalized therapy in AML1-ETO+ AML patients. Nearly 60% of acute myeloid leukemia (AML) patients with the t(8;21)(q22;q22) translocation fail to achieve long-term disease-free survival. Our previous studies demonstrated that oridonin selectively induces apoptosis of t(8;21) leukemia cells and causes cleavage of AML1-ETO oncoprotein resulting from t(8;21), but the underlying mechanisms remain unclear. We show that oridonin interacted with glutathione and thioredoxin/thioredoxin reductase to increase intracellular reactive oxygen species, which in turn activated caspase-3 in t(8;21) cells. Moreover, oridonin bound AML1-ETO, directing the enzymatic cleavage at aspartic acid 188 via caspase-3 to generate a truncated AML1-ETO (ΔAML1-ETO) and preventing the protein from further proteolysis. ΔAML1-ETO interacted with AML1-ETO and interfered with the trans-regulatory functions of remaining AML1-ETO oncoprotein, thus acting as a tumor suppressor that mediates the anti-leukemia effect of oridonin. Furthermore, oridonin inhibited the activity of c-Kit+ leukemia-initiating cells. Therefore, oridonin is a potential lead compound for molecular target–based therapy of leukemia.

Genomic landscape of CD34+ hematopoietic cells in myelodysplastic syndrome and gene mutation profiles as prognostic markers

Significance Myelodysplastic syndrome (MDS) represents a common hematopoietic disease, often in elderly patients, with heterogeneous clinical phenotypes and complex disease mechanisms. Here, we report on characteristic genome lesions, clonal architecture, and distinct tumor clone expansion patterns in a group of patients with refractory anemia with excess blasts, the MDS subtype with the highest propensity to acute myeloid leukemia. An integrative gene mutation analysis in 196 patients with different MDS subtypes allowed a regulatory network of mutually cooperative or exclusive molecules to be discovered among eight functional categories, whereas the combination of a panel of marker genes of prognostic value with the revised-International Prognostic Scoring System may provide a better stratification system for MDS. Myelodysplastic syndrome (MDS) includes a group of diseases characterized by dysplasia of bone marrow myeloid lineages with ineffective hematopoiesis and frequent evolution to acute myeloid leukemia (AML). Whole-genome sequencing was performed in CD34+ hematopoietic stem/progenitor cells (HSPCs) from eight cases of refractory anemia with excess blasts (RAEB), the high-risk subtype of MDS. The nucleotide substitution patterns were found similar to those reported in AML, and mutations of 96 protein-coding genes were identified. Clonal architecture analysis revealed the presence of subclones in six of eight cases, whereas mutation detection of CD34+ versus CD34− cells revealed heterogeneity of HSPC expansion status. With 39 marker genes belonging to eight functional categories, mutations were analyzed in 196 MDS cases including mostly RAEB (n = 89) and refractory cytopenia with multilineage dysplasia (RCMD) (n = 95). At least one gene mutation was detected in 91.0% of RAEB, contrary to that in RCMD (55.8%), suggesting a higher mutational burden in the former group. Gene abnormality patterns differed between MDS and AML, with mutations of activated signaling molecules and NPM1 being rare, whereas those of spliceosome more common, in MDS. Finally, gene mutation profiles also bore prognostic value in terms of overall survival and progression free survival.

Enhanced Fructose Utilization Mediated by SLC2A5 Is a Unique Metabolic Feature of Acute Myeloid Leukemia with Therapeutic Potential

Rapidly proliferating leukemic progenitor cells consume substantial glucose, which may lead to glucose insufficiency in bone marrow. We show that acute myeloid leukemia (AML) cells are prone to fructose utilization with an upregulated fructose transporter GLUT5, which compensates for glucose deficiency. Notably, AML patients with upregulated transcription of the GLUT5-encoding gene SLC2A5 or increased fructose utilization have poor outcomes. Pharmacological blockage of fructose uptake ameliorates leukemic phenotypes and potentiates the cytotoxicity of the antileukemic agent, Ara-C. In conclusion, this study highlights enhanced fructose utilization as a metabolic feature of AML and a potential therapeutic target.

Functional features of RUNX1 mutants in acute transformation of chronic myeloid leukemia and their contribution to inducing murine full-blown leukemia

The BCR-ABL fusion protein generated by t(9;22)(q34;q11) in chronic myeloid leukemia (CML) plays an essential role in the pathogenesis of the myeloproliferative disorder status at the chronic phase of the disease, but progression from the chronic phase to blast crisis (BC) is believed to require additional mutations. To explore the underlying mechanisms for BC, which is characterized by a blockage of blood cell differentiation, we screened several genes crucial to hematopoiesis and identified 10 types of mutations in RUNX1 among 11 of 85 (12.9%) patients with acute transformation of CML. Most of the mutations occurred in the runt homology domain, including H78Q, W79C, R139G, D171G, R174Q, L71fs-ter94, and V91fs-ter94. Further studies indicated that RUNX1 mutants not only exhibited decreased transactivation activity but also had an inhibitory effect on the WT RUNX1. To investigate the leukemogenic effect of mutated RUNX1, H78Q and V91fs-ter94 were transduced into 32D cells or BCR-ABL-harboring murine cells, respectively. Consistent with the myeloblastic features of advanced CML patients with RUNX1 mutations, H78Q and V91fs-ter94 disturbed myeloid differentiation and induced a BC or accelerated phase-like phenotype in mice. These results suggest that RUNX1 abnormalities may promote acute myeloid leukemic transformation in a subset of CML patients.

Conditional knockin of Dnmt3a R878H initiates acute myeloid leukemia with mTOR pathway involvement

Significance DNMT3A is a critical epigenetic modifier and tumor suppressor in the hematopoietic system. This gene is frequently mutated in hematopoietic malignancies, including acute myeloid leukemia (AML), with Dnmt3a R878H being the most common mutant. By using a conditional knockin approach, this study shows that Dnmt3a R878H is sufficient to initiate AML and recapitulate human leukemic features in mice. The leukemia-initiating cells are enriched in hematopoietic stem/progenitor cells. Through gene expression profiling, DNA methylation and histone modification analysis, and functional tests on important regulators for cell proliferation and differentiation in an animal model, this study has not only discovered mTOR pathway activation as a key player in the disease mechanism but also revealed the potential therapeutic effects of mTOR inhibition on DNMT3A mutation-related leukemia. DNMT3A is frequently mutated in acute myeloid leukemia (AML). To explore the features of human AML with the hotspot DNMT3A R882H mutation, we generated Dnmt3a R878H conditional knockin mice, which developed AML with enlarged Lin−Sca1+cKit+ cell compartments. The transcriptome and DNA methylation profiling of bulk leukemic cells and the single-cell RNA sequencing of leukemic stem/progenitor cells revealed significant changes in gene expression and epigenetic regulatory patterns that cause differentiation arrest and growth advantage. Consistent with leukemic cell accumulation in G2/M phase, CDK1 was up-regulated due to mTOR activation associated with DNA hypomethylation. Overexpressed CDK1-mediated EZH2 phosphorylation resulted in an abnormal trimethylation of H3K27 profile. The mTOR inhibitor rapamycin elicited a significant therapeutic response in Dnmt3aR878H/WT mice.

Valproic Acid Ameliorates Graft-versus-Host Disease by Downregulating Th1 and Th17 Cells

Graft-versus-host disease (GVHD) is the major complication after allogeneic bone marrow transplantation. Valproic acid (VPA) was described as a histone deacetylase inhibitor that had anti-inflammatory effects and reduced the production of proinflammatory cytokines in experimental autoimmune disease models. Using well-characterized mouse models of MHC-mismatched transplantation, we studied the effects of VPA on GVHD severity and graft-versus-leukemia (GVL) activity. Administration of VPA significantly attenuated the clinical severity of GVHD, the histopathology of GVHD-involved organs, and the overall mortality from GVHD. VPA downregulated Th1 and Th17 cell responses and cytokine production in vitro and in vivo, whereas its effect on GVHD was regulatory T cell independent. The effect of VPA was related to its ability to directly reduce the activity of Akt, an important regulator of T cell immune responses. Importantly, when mice received lethal doses of host-type acute leukemia cells, administration of VPA did not impair GVL activity and resulted in significantly improved leukemia-free survival. These findings reveal a unique role for VPA as a histone deacetylase inhibitor in reducing the donor CD4+ T cells that contribute to GVHD, which may provide a strategy to reduce GVHD while preserving the GVL effect.