IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation.

BACKGROUND: R140Q mutation in isocitrate dehydrogenase 2 (IDH2) promotes leukemogenesis. Targeting IDH2/R140Q yields encouraging therapeutic effects in the clinical setting. However, therapeutic resistance occurs in 12% of IDH2/R140Q inhibitor treated patients. The IDH2/R140Q mutant converted TF-1 cells to proliferate in a cytokine-independent manner. This study investigated the signaling pathways involved in TF-1(R140Q) cell proliferation conversion as alternative therapeutic strategies to improve outcomes in patients with acute myeloid leukemia (AML) harboring IDH2/R140Q. METHODS: The effects of IDH2/R140Q mutation on TF-1 cell survival induced by GM-CSF withdrawal were evaluated using flow cytometry assay. The expression levels of apoptosis-related proteins, total or phosphorylated STAT3/5, ERK, and AKT in wild-type TF-1(WT) or TF-1(R140Q) cells under different conditions were evaluated using western blot analysis. Cell viability was tested using MTT assay. The mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-11 in TF-1(WT) and TF-1(R140Q) cells were quantified via RT-PCR. The secretion levels of GM-CSF, OSM, and LIF were determined using ELISA. RESULTS: Our results showed that STAT3 and STAT5 exhibited aberrant constitutive phosphorylation in TF-1(R140Q) cells compared with TF-1(WT) cells. Inhibition of STAT3/5 phosphorylation suppressed the cytokine-independent proliferation of TF-1(R140Q) cells. Moreover, the autocrine GM-CSF, LIF and OSM levels increased, which is consistent with constitutive STAT5/3 activation in TF-1(R140Q) cells, as compared with TF-1(WT) cells. CONCLUSIONS: The autocrine cytokines, including GM-CSF, LIF, and OSM, contribute to constitutive STAT3/5 activation in TF-1(R140Q) cells, thereby modulating IDH2/R140Q-mediated malignant proliferation in TF-1 cells. Targeting STAT3/5 phosphorylation may be a novel strategy for the treatment of AML in patients harboring the IDH2/R140Q mutation. Video Abstract.

Impaired tissue homing by the Ikzf3N159S variant is mediated by interfering with Ikaros function.

AIOLOS, encoded by IKZF3, is a member of the IKZF family of proteins that plays an important role in regulating late B-cell differentiation. Human individuals heterozygous for the AIOLOS p.N160S variant displayed impaired humoral immune responses as well as impaired B and T cell development. We have previously reported that a mouse strain harboring an Ikzf3N159S allele that corresponds to human IKZF3N160S recapitulated immune-deficient phenotypes, such as impaired B cell development and loss of CD23 expression. In this study, we investigated the effect of the Ikzf3N159S variant and found that B1a cell development was impaired in Ikzf3N159S/N159S mice. In addition, CD62L expression was severely decreased in both B and T lymphocytes by the Ikzf3N159S mutation, in a dose-dependent manner. Mixed bone marrow chimera experiments have revealed that most immunodeficient phenotypes, including low CD62L expression, occur in intrinsic cells. Interestingly, while Ikzf3N159S/N159S lymphocytes were still present in the spleen, they were completely outcompeted by control cells in the lymph nodes, suggesting that the capacity for homing or retention in the lymph nodes was lost due to the Ikzf3N159S mutation. The homing assay confirmed severely decreased homing abilities to lymph nodes of Ikzf3N159S/N159S B and T lymphocytes but selective enrichment of CD62L expressing Ikzf3N159S/N159S lymphocytes in lymph nodes. This finding suggests that impaired CD62L expression is the major reason for the impaired homing capacity caused by the Ikzf3N159S mutation. Interestingly, an excess amount of Ikaros, but not Aiolos, restored CD62L expression in Ikzf3N159S/N159S B cells. Together with the loss of CD62L expression due to Ikaros deficiency, the AiolosN159S mutant protein likely interferes with Ikaros function through heterodimerization, at least in activating the Sell gene encoding CD62L expression. Thus, our results revealed that AiolosN159S causes some immunodeficient phenotypes via the pathogenesis referred to as the heterodimeric interference as observed for AiolosG158R variant.

T and B cell abnormalities, pneumocystis pneumonia, and chronic lymphocytic leukemia associated with an AIOLOS defect in patients.

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy.

Natural attenuation mechanism of hexavalent chromium in a wetland: Zoning characteristics of abiotic and biotic effects.

Natural wetland has great retention effect on Cr(VI) migration due to its abiotic and biotic reduction abilities, however, the zoning characteristics of dominating reduction mechanism along Cr(VI) pollution plume in wetland is still unclear. In this study, a Cr(VI) contaminated natural wetland was explored to investigate the distributions of Cr and Fe in groundwater and sediment, and their relationship with microorganisms according to metagenomics, aiming to reveal the natural attenuation mechanism of Cr(VI) from the perspective of zoning characteristics of abiotic and biotic effects. The wetland was divided into contaminated zone, transition zone and uncontaminated zone according to the contamination states of groundwater and sediment. At the upstream of contaminated zone, Cr(VI) concentration in groundwater was as high as 26.7 mg L-1, which has significant inhibition effect on microbial growth, and thus chemical reduction of Cr(VI) by natural organic matters (NOMs) dominated in this area, leading to the increasing of H/C and O/C ratios of NOMs because of the oxidation of aromatic moieties. At the downstream of contaminated zone, Cr(VI) concentration in groundwater decreased to less than 4.46 mg L-1 resulting from dilution and attenuation, but the microbial community was altered substantially, chromate resistant bacteria with ChrA, ChrR, NemA and AzoR genes were enriched, such as Sphingomonas, Mesorhizobium and Comamonadaceae, and thus the direct microbial reduction of Cr(VI) dominated in this area. While at the transition zone, which is located at the front edge of the pollution plume, Cr(VI) could only reached in this area intermittently, and the microbial community remained similar to that of the uncontaminated zone, dominated by Chloroflexi and Acidobateria phylum with dissimilatory ferric iron reduction capacity, and thus Cr(VI) was indirectly reduced by Fe2+ intermediately in this area.

Synergistic/antagonistic effects and mechanisms of Cr(VI) adsorption and reduction by Fe(III)-HA coprecipitates.

Widespread Fe(III)-humic acid (HA) coprecipitates (FHCs) have substantial impacts on the adsorption and reduction of Cr(VI) in soils and sediments, but whether this process is equal to the sum of their individual components remains unknown. In this study, ferrihydrite (Fh)- and HA-like FHCs (C/Fe<3 and C/Fe>3, respectively) were synthesized by controlling the initial C/Fe ratios (0.5-18) to explore the potential synergistic/antagonistic effects during the adsorption and reduction of Cr(VI). According to the results, antagonistic effects on Cr(VI) adsorption (5%-80%) were observed on Fh- and HA-like FHCs, where the antagonistic intensity increased with increasing HA proportions, respectively caused by the more serious occupation of adsorption sites and the stronger electrostatic repulsion to Cr(VI). Notably, significant synergistic reduction effects (5%-650%) occurred on Fh-like FHCs were found to be achieved by the activation of low-molecular HA (0.1-0.3 kDa) with primary/secondary hydroxylic groups, which might be induced by the inductive effect of Fh on complexed HA molecules according to density-functional theory (DFT) calculation. While slight antagonistic reduction effects (2%-45%) by HA-like FHCs were attributed to the decreasing accessibility of Cr(VI) to reductive phenolic groups, which might be blocked within FHC particles or complexed with Fe(III) ions through cation bridges.

New insight into the removal of Cd(II) from aqueous solution by diatomite.

Diatomite is an economical and environmentally friendly adsorbent, and its use has been applied widely for the treatment of water contaminated by heavy metals. Despite this, the mechanism for the removal of the heavy metal Cd(II) remains unclear. In this work, we explored the adsorption mechanism of Cd(II) by diatomite using batch experiment, and characterized the diatomite using scanning electron microscopy, energy-dispersive spectrometry, specific surface area, and pore size distribution analysis. Our results showed that, under the experimental conditions, the kinetic adsorption approached equilibrium within 5 min, and the Sips isotherm model was most suitable for data fitting. EDS characterization of the Cd-loaded diatomite indicated that Cd(II) was adsorbed onto the diatomite. Furthermore, desorption experiments showed that Ca2+ and Mg2+ in the diatomite caused an ion exchange interaction, and this was primarily responsible for Cd(II) adsorption. Moreover, we found that its contribution to the whole adsorption reaction could reach 80%, while the remainder of Cd(II) was probably trapped in the microporous structure of the diatomite. Additionally, our data indicated that the adsorption mechanism did not change significantly after regeneration. These results have provided special insight into the deep understanding of the mechanism of Cd(II) adsorption by diatomite, and could provide theoretical support and guidance for further development and application of diatomite in the treatment of Cd(II)-contaminated water. Graphical abstract.

Molecular structure-reactivity correlations of humic acid and humin fractions from a typical black soil for hexavalent chromium reduction.

Different soil humus fractions are structurally distinct from each other molecularly, however, the relationship between their microscopic molecular structures and the macroscopic reduction of Cr(VI) is still unknown, especially for the humin fraction. In this study, different humus fractions (HA, humic acid; HMi, humin linked to iron oxides; HMc, humin linked to clay; and HMr, humin residue) were sequentially extracted from a typical black soil and well characterized. It was found that HA, HMi and HMc were the same type of humus with similar molecular structures, while HMr was structurally different from the other fractions with a high cellulose content. The removal rate of Cr(VI) in solution decreased with progressive humus fractionation, namely, HA > HMi > HMc > HMr. Based on the two-dimensional correlation spectroscopic analysis (2DCOS) of the FTIR data, the changing functional groups of all humus fractions during reacting with Cr(VI) followed a similar order: carboxyl > phenol > hydroxyl > methyl > methylene. According to the correlation analysis, Cr(VI) reduction rates by different humus fractions were mainly determined by the content of phenol (R2 = 0.99) instead of carboxyl (R2 = 0.28). Except for HMr, the Cr(VI) reduction rates of different humus fractions were also positively correlated with surface and bulk polarity (R2 = 0.98 and 0.99) but not with aromaticity or aliphaticity (R2 = 0.21).

Collagen-based three-dimensional culture microenvironment promotes epithelial to mesenchymal transition and drug resistance of human ovarian cancer in vitro.

Ovarian cancer (OvCa) is a leading cause of mortality from gynecologic malignancy due to its disseminated peritoneal metastasis. The tumor microenvironment dominates epithelial-mesenchymal transition (EMT) development and impacts cancer metastasis as well as mediates drug resistance. Tumour cell interaction with the collagen I matrix is critical in OvCa development. To better understand the role of the collagen matrix and the underlying mechanisms in the early stage of OvCa invasion, we developed a three-dimensional (3D) culture model in vitro by embedding OvCa cells within collagen I to recreate the architecture of a solid tumour. Our results showed that tumour spheroids formed in the 3D collagen model displayed good viability and decreased growth rates, which partly recapitulated the growth behavior of in vivo tumour cells. Collagen I enhanced the OvCa cell motility/invasion capability by up-regulating the expression of MMPs and α5ß1 integrin. Moreover, highly invasive OvCa cells in collagen showed the overexpression of mesenchymal markers (N-cadherin, vimentin and fibronectin) and transcriptional factors (Snail and Slug). EMT-associated TGF-ß1/Smad4 and Wnt5b/ß-catenin signaling pathways were significantly up-regulated accordingly. Additionally, a remarkably enhanced drug resistance to chemotherapeutics was also detected in the 3D cultures. Collectively, the bioengineered 3D collagen models could recapitulate the in vivo tumour-like microenvironment and reflect some biological characteristics of human OvCa more accurately. The collagen I matrix promoted local invasion via EMT and enhanced the multidrug resistance in OvCa. This system might serve as a comprehensive in vitro model to better understand the manifold mechanisms of OvCa metastasis and also provide a robust tool for screening new anti-ovarian cancer therapeutics.