Epigenetic activation of O-linked ß-N-acetylglucosamine transferase overrides the differentiation blockage in acute leukemia.

BACKGROUND: Overriding the differentiation blockage in acute myeloid leukemia (AML) is the most successful mode-of-action in leukemia therapy - now curing the vast majority of patients with acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA)-based regimens. Similar approaches in other leukemia subtypes, such as IDH1/2-mutated AML, are under active investigation. We herein present successful release of the differentiation blockage upon treatment with the natural (-)-Δ9-Tetrahydrocannabinol isomer dronabinol in vitro and in vivo. METHODS: Cellular maturation and differentiation were followed in two patients employing whole genome methylation profiling, proteome analyses, NGS deep sequencing and multispectral imaging flow cytometry. For functional studies lentiviral OGT knock-down in vitro and ex vivo cell models were created to evaluate proliferative, apoptotic and differentiating effects of OGT in acute leukemia. FINDINGS: In here, we provide molecular evidence that dronbinol is capable to override the differentiation blockage of acute leukemia blasts at the state of the leukemia-initiating clone. We further identify the O-linked ß-N-acetyl glucosamine (O-GlcNAc) transferase (OGT) to be crucial in this process. OGT is a master regulator enzyme adding O-GlcNAc to serine or threonine residues in a multitude of target proteins. Aberrant O-GlcNAc modification is implicated in pathologies of metabolic, neurodegenerative and autoimme diseases as well as cancers. We provide evidence that dronabinol induces transcription of OGT via epigenetic hypomethylation of the transcription start site (TSS). A lentiviral OGT-knock out approach proves the central role of OGT exerting antileukemic efficacy via a dual-mechanism of action: High concentrations of dronabinol result in induction of apoptosis, whereas lower concentrations drive cellular maturation. Most intriguingly, overriding of the differentiation blockage of acute leukemia blasts is validated in vivo following two patients treated with dronabinol. INTERPRETATION: In conclusion, we provide evidence for overcoming the differentiation blockage in acute leukemia in subentities beyond promyelocytic and IDH1/2-mutated leukemia and thereby identify O-GlcNAcylation as a novel (drugable) field for future leukemia research. FUNDING: Unrestricted grant support by the IZKF Program of the Medical Faculty Tübingen (MMS) and Brigitte Schlieben-Lange Program as well as the Margarete von Wrangell Program of the Ministry of Science, Research and the Arts, Baden-Württemberg, Germany (KKS) and Athene Program of the excellence initiative University of Tübingen (KKS).

Crossbreeding applied to systems of beef cattle production to improve performance traits and carcass quality.

Crossbreeding represents an important technique to improve growth, beef quality and adaptability in beef production systems in tropical countries. The aim of this study was to evaluate sire and dam breed effects on performance and carcass traits of crossbred cattle produced in a tropical environment. Heifers and steers were evaluated during the pre-weaning, the post-weaning (n = 173) and the finishing phase (n = 123). Animals were produced by mating Nellore (N_N), Angus × Nellore (A_N) and Caracu × Nellore (C_N) dams with Braford, Charbray and Caracu sires. After weaning, animals were raised grazing on Marandu grass for 12 months; thereafter they were housed in individual pens and finished in a feedlot, receiving a total mixed ration. Ultrasound carcass evaluations were performed to determine ribeye area (R_A), backfat thickness (B_T) and rump fat thickness (R_T). A_N progeny were heavier at birth than N_N (P < 0.05), and Braford progeny had greater birth BW than Caracu (P < 0.05). Greater weaning BW was observed in the A_N and C_N offspring compared to N_N (P < 0.01). Greater average daily gain during the post-weaning period was verified in the N_N progeny compared to C_N (P < 0.05). No dam or sire breed effects were observed for BW at the end of the post-weaning period (P > 0.05). Progeny of N_N cows had greater B_T (P < 0.05) and R_T (P < 0.01) at the end of the post-weaning period in relation to C_N. Greater R_A was observed in the Caracu progeny than in the Braford (P < 0.05), which showed greater R_T than the Charbray progeny at the end of the post-weaning period (P < 0.05). No dam or sire breed effects were verified for final BW at the feedlot or for feed efficiency traits (P > 0.05). A_N progeny were superior in final B_T compared to C_N (P < 0.01), and Braford progeny had greater R_T at the end of finishing than Charbray (P = 0.05). The use of crossbred dams allows an increase in productivity until weaning, but this is not maintained in the post-weaning and finishing periods. The use of Braford sires produces similar growth performance in the different stages of the production system to those seen with Charbray and Caracu sires but generates animals with higher fat thickness at the end of finishing, which may improve carcass quality and commercial value.

Mucus organisation is shaped by colonic content; a new view.

The colonic mucus barrier is commonly described as a continuous double layer covering the epithelium, separating the microbiota from the intestinal tissue. This model is currently considered valid throughout the colon. The colon is characterised by regional anatomo-functional specificities such as presence and consistency of contents and location. In this study, we characterised the organisation of the colonic mucus barrier in proximal and distal colon of rodents by histological and FISH staining, taking into account aforementioned specificities. By using longitudinal sections and imaging extensive areas of tissue with and without colonic contents, we have obtained a spatiotemporal overview of mucus organisation in the colon. We describe for the first time that the colonic mucus layer covers the faeces instead of the epithelium in the distal colon. This faecal mucus layer confines the microbiota to the faeces and prevents it from remaining in empty distal colon. In the proximal colon, the mucus did not form a separating layer between bacteria and epithelium. We conclude that the organisation of colonic mucus is reliant on the presence of the colonic content, and the location within the colon. Our findings reopen the discussion on the nature of the colonic mucus barrier.

Genotypic and allelic frequencies of gene polymorphisms associated with meat tenderness in Nellore beef cattle.

The objectives of this study were to characterize the allelic and genotypic frequencies of polymorphisms in the µ-calpain and calpastatin genes, and to assess their association with meat tenderness and animal growth in Nellore cattle. We evaluated 605 Nellore animals at 24 months of age, on average, at slaughter. The polymorphisms were determined for the molecular markers CAPN316, CAPN530, CAPN4751, CAPN4753, and UOGACAST1. Analyses of meat tenderness at 7, 14, and 21 days of maturation were performed in samples of longissimus thoracis obtained between the 12th and 13th rib and sheared using a Warner Bratzler Shear Force. Significant effects were observed for meat tenderness at days 7, 14, and 21 of maturation for the marker CAPN4751, at day 21 for the marker CAPN4753, and at days 14 and 21 for the marker UOGCAST1. For genotypic combinations of markers, the results were significant for the combination CAPN4751/UOGCAST1 in the three maturation periods and CAPN4753/UOGCAST1 at days 14 and 21 of maturation.

Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts.

The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide 'proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo.

Durable responses to ibrutinib in patients with relapsed CLL after allogeneic stem cell transplantation.

Ibrutinib, a recently approved inhibitor of Bruton's tyrosine kinase (BTK), has shown great efficacy in patients with high-risk CLL. Nevertheless, there are few data regarding its use in patients who relapsed after allogeneic stem cell transplantation (alloSCT). We report clinical data from five CLL patients treated with ibrutinib for relapse after first or even second allogeneic transplantation. Additionally, we performed analyses on cytokine levels and direct measuring of CD4 Th1 and CD4 Th2 cells to evaluate possible clinically relevant immunomodulatory effects of ibrutinib. All patients achieved partial responses including one minimal residual disease (MRD)-negative remission. Within 1 year of follow-up, no relapse was observed. One patient died of severe pneumonia while on ibrutinib treatment. Beside this, no unexpected adverse events were observed. Flow cytometry and analyses of T cell-mediated cytokine levels (IL10 and TNFα) did not reveal substantial changes in T-cell distribution in favor of a CD4 Th1 T-cell shift in our patients. No acute exacerbation of GvHD was reported. In conclusion, these results support further evaluation of ibrutinib in CLL patients relapsing after alloSCT.

Clofarabine salvage therapy before allogeneic hematopoietic stem cell transplantation in patients with relapsed or refractory AML: results of the BRIDGE trial.

In patients with relapsed or refractory (r/r) acute myeloid leukemia (AML), long-term disease control can only be achieved by allogeneic hematopoietic stem cell transplantation (HSCT). We studied the safety and efficacy of clofarabine-based salvage therapy. The study was designed as phase II, multicenter, intent-to-transplant (ITT) study. A total of 84 patients with r/r AML were enrolled. All patients received at least one cycle of CLARA (clofarabine 30 mg/m(2) and cytarabine 1 g/m(2), days 1-5). Chemo-responsive patients with a donor received HSCT in aplasia after first CLARA. Generally, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine (4 × 30 mg/m(2)) and melphalan (140 mg/m(2)). The median patient age was 61 years (range 40-75). On day 15 after start of CLARA, 26% of patients were in a morphologically leukemia-free state and 79% exposed a reduction in bone marrow blasts. Overall, 67% of the patients received HSCT within the trial. The primary end point, defined as complete remission after HSCT, was achieved by 60% of the patients. According to the ITT, overall survival at 2 years was 43% (95% confidence interval (CI), 32-54%). The 2-year disease-free survival for transplanted patients was 52% (95% CI, 40-69%). Clofarabine-based salvage therapy combined with allogeneic HSCT in aplasia shows promising results in patients with r/r AML.

Visual body-scores selection and its influence on body size and ultrasound carcass traits in Nellore cattle.

Genetic parameters, genetic trends, and genetic progress of carcass traits were estimated for 12,447 Nellore individuals from different Brazilian herds. The following carcass traits were analyzed: visual body scores of conformation (CONF), precocity (PREC), and muscularity (MUSC); rump width (RW) and rump length (RL), body size (frame); and ultrasound evaluation of ribeye area (REA), backfat thickness (BFT), and rump fat thickness (RFT). Heritability estimates (± SE) for CONF, PREC, and MUSC were 0.23 ± 0.02, 0.33 ± 0.03, and 0.31 ± 0.02, respectively. The heritability estimates (± SE) for RW, RL, and frame were 0.25 ± 0.05, 0.16 ± 0.04, and 0.31 ± 0.03, respectively. Ultrasound of the REA, BFT, and RFT presented mean heritability estimates (± SE) of 0.38 ± 0.03, 0.44 ± 0.04, and 0.47 ± 0.04, respectively. The visual body score of CONF had high genetic correlations with RW, RL, and frame (0.74 ± 0.09, 0.82 ± 0.08, and 0.83 ± 0.04, respectively), indicating a high association of CONF with the measurements related to body structure. The REA had no correlation with BFT and RFT (-0.09 ± 0.04 and -0.03 ± 0.04, respectively) and showed only a moderate genetic correlation with MUSC (0.39 ± 0.05). The BFT and RFT had moderate genetic correlations with PREC (0.36 ± 0.07 and 0.29 ± 0.07, respectively) and no correlation with frame (-0.02 ± 0.03 and 0.05 ± 0.05, respectively), suggesting that selection for frame had no effect on the subcutaneous fat content of the carcass. Low genetic trends and low genetic progress were obtained for REA (0.026 cm, 0.02%), BFT (0.0031 mm, 0.05%), and RFT (0.0013 mm, 0.02%), with no significant values of genetic progress detected throughout the studied period. The CONF, PREC, and MUSC presented high genetic trends (0.030 points, 0.030 points, and 0.029 points, respectively) and high genetic progress (0.60%, 0.56%, and 0.59%, respectively), indicating a significant genetic progress for these traits throughout the evaluated period. Carcass traits evaluated by ultrasound and visual body scores are eligible for selection and can be used in genetic improvement programs in Nellore cattle; however, visual body score selection for carcass traits can result in slower genetic progress for carcass quality when compared to selection performed by ultrasound.

Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts.

Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future.

Genome-wide association with residual body weight gain in Bos indicus cattle.

Weight gain is a key performance trait for beef cat-tle; however, attention should be given to the production costs for better profitability. Therefore, a feed efficiency trait based on per-formance can be an interesting approach to improve performance without increasing food costs. To identify candidate genes and ge-nomic regions associated with residual body weight gain (RWG), we conducted a genome-wide association study (GWAS) with 720 Nellore cattle using the GRAMMAR-Gamma association test. We identified 30 significant single nucleotide polymorphisms (SNPs), especially on chromosomes 2, 8, 12, and 17. Several genes and quantitative train loci (QTLs) present in the regions identified were appointed; we highlight DMRT2 (doublesex and mab-3 related tran-scription factor 2), IFFO2 (intermediate filament family orphan 2), LNX2 (ligand of numb-protein X 2), MTIF3 (mitochondrial transla-tional initiation factor 3), and TRNAG-CCC (transfer RNA glycine anticodon CCC). The metabolic pathways that can explain part of the phenotypic variation in RWG are related to oxidative stress and muscle control.

A genomewide association mapping study using ultrasound-scanned information identifies potential genomic regions and candidate genes affecting carcass traits in Nellore cattle.

The aim of this study was to identify candidate genes and genomic regions associated with ultrasound-derived measurements of the rib-eye area (REA), backfat thickness (BFT) and rumpfat thickness (RFT) in Nellore cattle. Data from 640 Nellore steers and young bulls with genotypes for 290 863 single nucleotide polymorphisms (SNPs) were used for genomewide association mapping. Significant SNP associations were explored to find possible candidate genes related to physiological processes. Several of the significant markers detected were mapped onto functional candidate genes including ARFGAP3, CLSTN2 and DPYD for REA; OSBPL3 and SUDS3 for BFT; and RARRES1 and VEPH1 for RFT. The physiological pathway related to lipid metabolism (CLSTN2, OSBPL3, RARRES1 and VEPH1) was identified. The significant markers within previously reported QTLs reinforce the importance of the genomic regions, and the other loci offer candidate genes that have not been related to carcass traits in previous investigations.

NFATc1 as a therapeutic target in FLT3-ITD-positive AML.

Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

Sire effects on carcass and meat quality traits of young Nellore bulls.

Meat quality is being increasingly demanded by consumers in recent years. Several factors can affect meat quality, ranging from animal traits such as breed and genetic heritage to pre- and post-slaughter processes. This study investigated the influence of Nellore bulls on carcass and meat quality traits. We used 475 young uncastrated males, the progeny of 54 bulls, to evaluate characteristics of the following carcass traits: hot carcass weight, rib-eye area, and fat thickness. We also evaluated the following beef quality traits: marbling, color, drip loss, cooking loss, and shear force at 0, 7, and 14 days of aging. Bulls had a significant influence (P≤0.05) on rib-eye area, fat thickness, marbling, drip loss at 14 days of aging and color at all aging periods. Based on these results, the use of bulls with high breeding values for these traits can provide important advances in carcass traits and meat quality in breeding programs of Nellore cattle that are raised in tropical conditions.

Costimulation improves the killing capability of T cells redirected to tumor cells expressing low levels of CD33: description of a novel modular targeting system.

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

PIK3R1 mutations in SHORT syndrome.

SHORT syndrome (OMIM 269880) is a rare autosomal-dominant disorder characterized by short stature, hyperextensibility of joints, hernias, ocular depression, ophthalmic anomalies (Rieger anomaly, posterior embryotoxon, glaucoma), teething delay, partial lipodystrophy, insulin resistance and facial dysmorphic signs. Heterozygous mutations in PIK3R1 were recently identified in 14 families with SHORT syndrome. Eight of these families had a recurrent missense mutation (c.1945C>T; p.Arg649Trp). We report on two unrelated patients with typical clinical features of SHORT syndrome and additional problems such as pulmonary stenosis and ectopic kidney. Analysis of PIK3R1 revealed the mutation c.1945C>T; p.Arg649Trp de novo in both patients. These two patients not only provide additional evidence that PIK3R1 mutations cause SHORT syndrome, but also broaden the clinical spectrum of this syndrome and further confirm that the amino acid exchange c.1945C>T; p.Arg649Trp is a hotspot mutation in this gene.

PDK1 controls upstream PI3K expression and PIP3 generation.

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.