ALBERT

Beschreibung: 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.

Beschreibung: Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-transretinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5′-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I–binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA).TRIM25encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I–mediated antiviral signaling. We show that RIG-I could bindTRIM25mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability ofTRIM25transcripts. RIG-I could increase the transcriptional expression ofTRIM25by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I–induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.

Beschreibung: Background: The present prognosis for AML mainly rely on the cyto- and molecular genetic features. However, the bone marrow microenvironment also plays a critical role in the development and progression of the disease, and the immune signature within the bone marrow microenvironment of AML remains unknown, and should be clarified. Patients and methods: Here, we applied the robust algorithm CIBERSORT method to generate the distribution and frequency of 22 immune subsets in the bone marrow of AML from public datasets. The associations between the overall survival and proportions of putative marrow immune cell types were analyzed to generate an immune risk score in a public training cohort according to LASSO cox regression model and validated in other validation cohorts. Gene Set Enrichment Analysis was adopted to analyze the immune risk score associated biological phenotypes. Results: Data from GSE37642 (n = 531) were used for immune model estimation, and samples from GSE425 (n =81) and TCGA (n =111) were served as external cohorts for validation. We first derived the 22 immune subsets through CIBERSORT, and found that monocytes, resting mast cells, eosinophils, CD8+T cells, resting NK cells, resting dendritic cells and resting memory CD4+ T cells were the most represented cell fractions within AML bone marrow microenvironment. LASSO cox regression was then applied to identify a total of 10 immune subsets to generate the immune risk score that may predict the survival prognosis of AML patients. This model has an excellent reproducibility, with the area under curve of the immune risk score at 5- year OS of 0.65, 0.84 and 0.74 in the training, external 1 and external 2 cohort. Patients with low immune risk score had a significant survival advantage than those with high immune risk score (5-year OS: 39.2 vs 19.6%, P 〈 0.001 for the training cohort; 77.4 vs 20.1%, P = 0.004 for the external cohort 1; 27.2 vs 0.0%, P = 0.006 for the external cohort 2). After adjusting the confounding variables of the classic ELN risk category and other clinical factors, the multivariable analysis revealed that immune risk score remained an independent prognostic factor for OS in all the three cohorts (HR: 1.34, 5.42 and 2.16 for the training, external cohort1 and 2 cohorts; all P 〈 0.05). Next, we explored the related biological phenotypes to immune risk score and found that the immune risk score was significantly associated with the markers of immune checkpoint (HMGB1, CEACAM1, LAG3 and CTLA-4), cell proliferation (NOTCH1, LYN, TSC2, SETBP1, TGFBR2, MAP3K14, TP53, NFKB2 and FOXO1), DNA repairment (RAD50 and BRCA1), epigenetic regulation and transcriptional regulation (SRSF6, DNMT3, TLE1). Furthermore, GSEA analysis indicated a significant enrichment of active immune-related pathways in low immune risk group. Finally, we noted that the immune risk score consistently increased follow the increased cytogenetic risk level. Conclusions: The study identified a novel immune prognostic category of AML, which may be used as another prognostic tool that complements the existing genetic risk classification. Figure Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: Acute myeloid leukemia (AML) is a type of cancer that consists of a group of hematological malignancies with high heterogeneity. AML patients with DNMT3A mutations often have a poor prognosis. Metabolic alterations have long been recognized in cancer cells. Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change the cellular metabolism, priming cells for malignancy. It is well known that 2-hydroxyglutarate (2-HG) is an oncometabolite resulting from mutations of the isocitrate dehydrogenase 1 and 2 (IDH1and IDH2) genes and isa strong prognostic predictor independent of other clinical and molecular features. Alpha-ketoglutarate-dependent dioxygenase encodedby the TET2 gene could catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), thereby regulating DNA methylation and inducing hematopoietic malignancies. Methods: Metabolomic profiles of all serum samples were achieved using the GC-TOF MS platform. CCK-8 assays were used to examine cell viability and cytotoxic activity of 2-ketoisocaproic acid (2-KA) to leukemia cells. Methyl-Cellulose colony-forming cell assays and flow cytometry were performed to assess cell proliferation and cell differentiation. The pIpC was administrated in the Dnmt3aR878H/WTMx1Cre+mice at the time of weaning (4 weeks old) to activate transgene expression in vivo. The expressions of genes/proteins were detected by RNA sequencing, quantitative RT-PCR and western blot. Results: We report a metabolomics study with a total of 171 newly diagnosed AML patients and identified a distinct leucine metabolic signature in AML patients with DNMT3A mutations. The prognostic value of 2-KA was demonstrated in cytogenetically normal AML patients, and a low 2-KA level predicted a poor overall patient survival. We further compared the gene expression patterns of the blast cells in bone marrow (BM) between AML groups with and without a DNMT3A mutation; these patterns correlated well with those of the leucine metabolic pathways and exhibited enhanced ACAA1 and decreased ALDH7A1 gene expression in DNMT3A-mutated leukemic cells. In vitro results demonstrated that a high 2-KA level could inhibit the proliferation of DNMT3A-mutated cells but did not affect the differentiation of these leukemic cells. Conclusions:Our data suggest that 2-KA isa unique feature of AML with a DNMT3A mutation and might be a metabolic marker of early-stage hematopoietic malignances.This study also suggests that ACAA1 and ALDH7A1 were aberrantly expressed in DNMT3A mutant leukemic cells and may be potential targetsfor AML therapy. We also hypothesized that a combination of the metabolic inhibitor and chemotherapeutic agent may be a possible treatment strategy for DNMT3A-mutated AML. Disclosures No relevant conflicts of interest to declare.

Beschreibung: The HOXA gene family is associated with various cancer types. However, the role of HOXA genes in acute myeloid leukemia (AML) have not been comprehensively studied. We compared the transcriptional expression, survival data, and network analysis of HOXA-associated signaling pathways in patients with AML using the ONCOMINE, GEPIA, LinkedOmics, cBioPortal, and Metascape databases. We observed that HOXA2-10 mRNA expression levels were significantly upregulated in AML and that high HOXA1-10 expression was associated with poor AML patient prognosis. The HOXA genes were altered in ~18% of the AML samples, either in terms of amplification, deep deletion, or elevated mRNA expression. The following pathways were modulated by HOXA gene upregulation: GO:0048706: embryonic skeletal system development; R-HSA-5617472: activation of HOX genes in anterior hindbrain development during early embryogenesis; GO:0060216: definitive hemopoiesis; hsa05202: transcriptional mis-regulation in cancer; and GO:0045638: negative regulation of myeloid cell differentiation, and they were significantly regulated due to alterations affecting the HOXA genes. This study identified HOXA3-10 genes as potential AML therapeutic targets and prognostic markers.

Beschreibung: Background: Myelodysplastic syndrome (MDS) was characterized as ineffective hematopoiesis, increased transformation to acute myeloid leukemia (AML), and accompanied by immune system dysfunction. However, the immune signature of MDS remains elusive. Methods: The clinical data (age, sex, international prognostic score system (IPSS), hemoglobin, blast, RBC transfusion dependence, and corresponding subject-level survival) as well as expression profiles of MDS (CD34+ cells) were obtained from Gene Expression Omnibus (GEO: GSE 58831; GSE 114922). A robust prognosis model of immune genes was constructed by the least absolute shrinkage and selection operator (LASSO) regression analysis. Survival analysis for prognostic model was carried out through the Kaplan-Meier curve and Log-rank test. The receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to assess the accuracy of prognostic models. Immune score for different subtype were calculated further by single sample gene set enrichment analysis (ssGSEA). Result: A novel robust immune gene prognostic model indicate that subtype with lower risk score were longer overall survival (OS) than subtype with higher risk score in training cohort (Figure1 A, C). The model was further verified by the validation cohort (Figure1 B, D). The multivariate Cox regression analysis demonstrated the model was an independent prognostic factor for OS prediction with hazard ratios of 56.694 (95% CIs: 9.038−355.648), 3.009 (95% CIs: 1.042−8.692) both in train cohort and external validation cohort respectively (Figure1 G, H). The AUC of 5- year were 0.92 (95% CIs: 0.86 - 0.97) and 0.7 (95% CIs: 0.51 - 0.89) for OS respectively in training cohort and validation cohort (Figure1 E, F). Furthermore, ssGSEA showed higher risk score subtype was significantly associated with higher immune score of check point, human leukocyte antigen (HLA), T cell co-inhibition and type I interferon (IFN) response (Figure1 K-N), which indicating that the poor outcome might be caused by tumor-associated immune response dysfunction partly. Conclusion: We constructed a robust immune gene prognostic model, which have a potential prognostic value for MDS patients and may provide evidence for personalized immunotherapy. Figure Disclosures No relevant conflicts of interest to declare.