IRF2BP2 counteracts the ATF7/JDP2 AP-1 heterodimer to prevent inflammatory overactivation in acute myeloid leukemia (AML) cells.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by abnormal proliferation and accumulation of immature myeloid cells in the bone marrow. Inflammation plays a crucial role in AML progression, but excessive activation of cell-intrinsic inflammatory pathways can also trigger cell death. IRF2BP2 is a chromatin regulator implicated in AML pathogenesis, although its precise role in this disease is not fully understood. In this study, we demonstrate that IRF2BP2 interacts with the AP-1 heterodimer ATF7/JDP2, which is involved in activating inflammatory pathways in AML cells. We show that IRF2BP2 is recruited by the ATF7/JDP2 dimer to chromatin and counteracts its gene-activating function. Loss of IRF2BP2 leads to overactivation of inflammatory pathways, resulting in strongly reduced proliferation. Our research indicates that a precise equilibrium between activating and repressive transcriptional mechanisms creates a pro-oncogenic inflammatory environment in AML cells. The ATF7/JDP2-IRF2BP2 regulatory axis is likely a key regulator of this process and may, therefore, represent a promising therapeutic vulnerability for AML. Thus, our study provides new insights into the molecular mechanisms underlying AML pathogenesis and identifies a potential therapeutic target for AML treatment.

KLF4-mediated upregulation of the NKG2D ligand MICA in acute myeloid leukemia: a novel therapeutic target identified by enChIP.

The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo. Video Abstract.

A Bioinformatics View on Acute Myeloid Leukemia Surface Molecules by Combined Bayesian and ABC Analysis.

"Big omics data" provoke the challenge of extracting meaningful information with clinical benefit. Here, we propose a two-step approach, an initial unsupervised inspection of the structure of the high dimensional data followed by supervised analysis of gene expression levels, to reconstruct the surface patterns on different subtypes of acute myeloid leukemia (AML). First, Bayesian methodology was used, focusing on surface molecules encoded by cluster of differentiation (CD) genes to assess whether AML is a homogeneous group or segregates into clusters. Gene expressions of 390 patient samples measured using microarray technology and 150 samples measured via RNA-Seq were compared. Beyond acute promyelocytic leukemia (APL), a well-known AML subentity, the remaining AML samples were separated into two distinct subgroups. Next, we investigated which CD molecules would best distinguish each AML subgroup against APL, and validated discriminative molecules of both datasets by searching the scientific literature. Surprisingly, a comparison of both omics analyses revealed that CD339 was the only overlapping gene differentially regulated in APL and other AML subtypes. In summary, our two-step approach for gene expression analysis revealed two previously unknown subgroup distinctions in AML based on surface molecule expression, which may guide the differentiation of subentities in a given clinical-diagnostic context.

IRF8 Is an AML-Specific Susceptibility Factor That Regulates Signaling Pathways and Proliferation of AML Cells.

Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients' prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs.

The Oncoprotein SKI Acts as A Suppressor of NK Cell-Mediated Immunosurveillance in PDAC.

Drugs targeting epigenetic mechanisms such as histone deacetylase inhibitors (HDACi) suppress tumor growth. HDACi also induce the expression of ligands for the cytotoxicity receptor NKG2D rendering tumors more susceptible to natural killer (NK) cell-dependent killing. The major acetylases responsible for the expression of NKG2D ligands (NKG2D-L) are CBP and p300. The role of the oncogene and transcriptional repressor SKI, an essential part of an HDAC-recruiting co-repressor complex, which competes with CBP/p300 for binding to SMAD3 in TGFß signaling, is unknown. Here we show that the siRNA-mediated downregulation of SKI in the pancreatic cancer cell lines Panc-1 and Patu8988t leads to an increased target cell killing by primary NK cells. However, the higher cytotoxicity of NK cells did not correlate with the induction of NKG2D-L. Of note, the expression of NKG2D-L and consequently NK cell-dependent killing could be induced upon LBH589 (LBH, panobinostat) or valproic acid (VPA) treatment irrespective of the SKI expression level but was significantly higher in pancreatic cancer cells upon genetic ablation of SKI. These data suggest that SKI represses the inducible expression of NKG2D-L. The combination of HDACi with NK cell-based immunotherapy is an attractive treatment option for pancreatic tumors, specifically for patients with high SKI protein levels.

MYB induces the expression of the oncogenic corepressor SKI in acute myeloid leukemia.

Acute myeloid leukemia (AML) arises through clonal expansion of transformed myeloid progenitor cells. The SKI proto-oncogene is highly upregulated in different solid tumors and leukemic cells, but little is known about its transcriptional regulation during leukemogenesis. MYB is an important hematopoietic transcription factor involved in proliferation as well as differentiation and upregulated in most human acute leukemias. Here, we find that MYB protein binds within the regulatory region of the SKI gene in AML cells. Reporter gene assays using MYB binding sites present in the SKI gene locus show MYB-dependent transcriptional activation. SiRNA-mediated depletion of MYB in leukemic cell lines reveals that MYB is crucial for SKI gene expression. Consistently, we observed a positive correlation of MYB and SKI expression in leukemic cell lines and in samples of AML patients. Moreover, MYB and SKI both were downregulated by treatment with histone deacetylase inhibitors. Strikingly, differentiation of AML cells induced by depletion of MYB is attenuated by overexpression of SKI. Our findings identify SKI as a novel MYB target gene, relevant for the MYB-induced differentiation block in leukemic cells.

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1.

SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression.

Immunosuppressive capacity of mesenchymal stem cells correlates with metabolic activity and can be enhanced by valproic acid.

BACKGROUND: Mesenchymal stem cells (MSCs) have entered the clinic as an Advanced Therapy Medicinal Product and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes, but until now reliable potency assays for the final MSC product are lacking. Because recent findings suggest superior therapeutic efficacy of freshly administered MSCs in comparison with frozen cells, we sought to correlate the T-cell suppressive capacity of MSCs with their metabolic activity. METHODS: Human MSCs were obtained from patients' bone fragments and were employed in coculture with peripheral blood mononuclear cells (PBMCs) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSCs was measured in real time in terms of aerobic glycolysis quantified by the extracellular acidification rate and mitochondrial respiration quantified by the oxygen consumption rate. RESULTS: We show that MSC-induced suppression of T-cell proliferation was highly dependent on individual healthy donors' lymphocytes. Moreover, coculture with PBMCs increased the glycolytic and respiratory activity of MSCs considerably in a PBMC donor-dependent manner. The twofold to threefold enhancement of cell metabolism was accompanied by higher T-cell suppressive capacities of MSCs. The cryoprotectant dimethyl sulfoxide decreased metabolic and immunosuppressive performances of MSCs while valproic acid (VPA) increased their glycolytic, respiratory and T-cell suppressive capacity. CONCLUSIONS: Functional fitness of MSCs can be determined by measuring metabolic activity and can be enhanced by exposure to VPA. Pretesting the increment of metabolic activity upon interaction of donor MSCs with patient T-cells provides a rational approach for an individualized potency assay prior to MSC therapy.

Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformants.

The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc-gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei. Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing Delta D transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa, gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation.