DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming. METHODS: Ultra-High Performance Liquid Chromatography-High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells. RESULTS: DNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells. CONCLUSIONS: Taken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

RNA silencing of GM-CSF in CAR-T cells reduces the secretion of multiple inflammatory cytokines.

Chimeric antigen receptor T (CAR-T) cell therapy has become a research hotspot in the field of hematological malignancies. However, CAR-T cell therapy can lead to immunotherapy-associated side effects including cytokine release syndrome and neurotoxicity. Gene depletion of GM-CSF in CAR-T cells was found preventive against adverse effects, but additional transfections were required to produce CAR-T cells. In this study, we interrupted GM-CSF expression in CAR-T cells by inserting the GM-CSF shRNA-expression cassette in the CAR vector. Reduction of GM-CSF in CAR-T cells could decrease the level of several proinflammatory cytokines without hampering the killing capacity. The manufacture of GM-CSF knockdown CAR-T cells does not require complicated transfections, which makes it more practical and feasible for clinical application.

Endothelial lipase promotes acute myeloid leukemia progression through metabolic reprogramming.

Metabolic reprogramming occurs in the clonal evolution of acute myeloid leukemia (AML), which contributes to cell survival under metabolic stress and the development of drug resistance. Leukemic cells exhibit various metabolic profiles, which involve multiple metabolic pathways due to the heterogeneity of AML. However, studies on metabolic targets for AML treatment are mostly focused on glycolysis at present. In this work, we established conditional knock-in AML mouse models harboring Dnmt3aR878H/WT, NrasG12D/WT, and both of the mutations, respectively. Transcriptomic analysis of Gr1+ cells from bone marrow was performed afterward to screen interested metabolic pathways and target genes. Candidate genes were studied using the CRISPR/Cas9 technique, quantitative real-time RT-PCR, and flow cytometric analyses. We revealed that multiple metabolic pathways were affected in AML mice, including lipid metabolism. Endothelial lipase (LIPG) was obviously upregulated in leukemic cells from AML mice with Dnmt3a mutation. We performed knockout of LIPG in OCI-AML3 cells carrying DNMT3A R882C mutation by using the CRISPR/Cas9 technique. Depletion of LIPG led to proliferation inhibition, apoptosis, damage of antioxidant capacity, and myeloid differentiation in OCI-AML3 cells. LIPG might serve as a potential metabolic target for the treatment of AML with abnormal lipid metabolism.

Combination of Expanded Allogeneic NK Cells and T Cell-Based Immunotherapy Exert Enhanced Antitumor Effects.

Immunotherapies based on immune checkpoint blockade, neoantigen-reactive tumor-infiltrating lymphocytes and T cell receptor-engineered T cells (TCR-T) have achieved favorable clinical outcomes in tumor treatment. However, sustained immune response and tumor regression have been observed only in a few patients due to immune escape. Natural killer (NK) cells can mediate direct tumor lysis and target cancer cells with low or no expression of human leukocyte antigen class I (HLA-I) that are no longer recognized by T cells during immune escape. Therefore, the combination of T cell-based immunotherapy and NK cell therapy is a promising strategy for improving antitumor response and response rate. However, allogeneic NK cells for adoptive cell therapy have been limited by both the required cell number and quality. Here, we developed an efficient manufacturing system that relies on genetically modified K562 cells for the expansion of high-quality NK cells derived from peripheral blood mononuclear cells. NK cells with the optimal expansion and activity were identified by comparing the different culture systems. Furthermore, we demonstrated that the cooperation of NK cells with tumor-reactive T cells or with NY-ESO-1-specific TCR-T cells further enhanced tumors lysis, especially against tumors with downregulated HLA-I expression. The advantages of HLA-mismatch and non-rejection by other allogeneic immune cells demonstrated the potential of "off-the-shelf" NK cells with the capacity to target tumors for immunotherapy. Our results indicate that the combination strategy based on T cell and allogeneic NK cell immunotherapy might have potential for overcoming the barrier of immune incompetence caused by HLA-I downregulation.

Therapeutic Potential of TNFα and IL1ß Blockade for CRS/ICANS in CAR-T Therapy via Ameliorating Endothelial Activation.

Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) strongly hampered the broad clinical applicability of chimeric antigen receptor T cell (CAR-T) therapy. Vascular endothelial activation has been suggested to contribute to the development of CRS and ICANS after CAR-T therapy. However, therapeutic strategies targeting endothelial dysfunction during CAR-T therapy have not been well studied yet. Here, we found that tumor necrosis factor α (TNFα) produced by CAR-T cells upon tumor recognition and interleukin 1ß (IL1ß) secreted by activated myeloid cells were the main cytokines in inducing endothelial activation. Therefore, we investigated the potential effectiveness of TNFα and IL1ß signaling blockade on endothelial activation in CAR-T therapy. The blockade of TNFα and IL1ß with adalimumab and anti-IL1ß antibody respectively, as well as the application of focal adhesion kinase (FAK) inhibitor, effectively ameliorated endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Moreover, adalimumab and anti-IL1ß antibody exerted synergistic effect on the prevention of endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Our results indicate that TNFα and IL1ß blockade might have therapeutic potential for the treatment of CAR-T therapy-associated CRS and neurotoxicity.

Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization.

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and ß-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

Gene mutational pattern and expression level in 560 acute myeloid leukemia patients and their clinical relevance.

BACKGROUND: Cytogenetic aberrations and gene mutations have long been regarded as independent prognostic markers in AML, both of which can lead to misexpression of some key genes related to hematopoiesis. It is believed that the expression level of the key genes is associated with the treatment outcome of AML. METHODS: In this study, we analyzed the clinical features and molecular aberrations of 560 newly diagnosed non-M3 AML patients, including mutational status of CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1. RESULTS: Certain gene expression levels were associated with the cytogenetic aberration of the disease, especially for MECOM, MEIS1 and BAALC. FLT3, C-KIT and NRAS mutations contained conversed expression profile regarding MEIS1, WT1, GATA2 and BAALC expression, respectively. FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group. Higher MECOM and MEIS1 gene expression levels showed a significant impact on complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis, respectively; and an additive effect could be observed. By systematically integrating gene mutational status results and gene expression profile, we could establish a more refined system to precisely subdivide AML patients into distinct prognostic groups. CONCLUSIONS: Gene expression abnormalities contained important biological and clinical informations, and could be integrated into current AML stratification system.