👤 Regina Ebert

The prognostic impact of PICALM::MLLT10 status in childhood leukaemia is not well described. Ten International Berlin Frankfurt Münster-affiliated study groups and the Children's Oncology Group collaborated in this multicentre retrospective study. The presence of the PICALM::MLLT10 fusion gene was confirmed by fluorescence in situ hybridization and/or RNA sequencing at participating sites. Ninety-eight children met the study criteria. T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML) predominated 55 (56%) and 39 (40%) patients, respectively. Most patients received a chemotherapy regimen per their disease phenotype: 58% received an ALL regimen, 40% an AML regimen and 1% a hybrid regimen. Outcomes for children with PICALM::MLLT10 ALL were reasonable: 5-year event-free survival (EFS) 67% and 5-year overall survival (OS) 76%, but children with PICALM::MLLT10 AML had poor outcomes: 5-year EFS 22% and 5-year OS 26%. Haematopoietic stem cell transplant (HSCT) did not result in a significant improvement in outcomes for PICALM::MLLT10 AML: 5-year EFS 20% for those who received HSCT versus 23% for those who did not (p = 0.6) and 5-year OS 37% versus 36% (p = 0.7). In summary, this study confirms that PICALM::MLLT10 AML is associated with a dismal prognosis and patients cannot be salvaged with HSCT; exploration of novel therapeutic options is warranted. Show less

Though immune checkpoint inhibition has recently shown encouraging clinical efficacy in mesothelioma, most patients do not respond. Combining immune checkpoint inhibition with radiotherapy presents an attractive option for improving treatment responses owing to the various immunomodulatory effects of radiation on tumors. However, the ideal dosing and scheduling of combined treatment remains elusive, as it is poorly studied in mesothelioma. The present study characterizes the dose- and time-dependent changes to expression of various immune markers and cytokines important to antitumor responses following irradiation of mesothelioma cell lines. Two murine (AB1, AE17) and two human (BYE, JU77) mesothelioma cell lines were treated with titrated gamma-radiation doses (1-8 Gy) and the expression of MHC class-I, MHC class-II and PD-L1 was measured over a series of post-irradiation timepoints (1-72 hours) by flow cytometry. Levels of cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α, and GM-CSF were measured by multiplex immunoassay in murine cell lines following 8 Gy radiation. Following irradiation, a dose-dependent upregulation of MHC-I and PD-L1 was observed on three of the four cell lines studied to varying extents. For all cell lines, the increase in marker expression was most pronounced 72 hours after radiation. At this timepoint, increases in levels of cytokines IFN-β, MCP-1 and IL-6 were observed following irradiation with 8 Gy in AB1 but not AE17, reflecting patterns in marker expression. Overall, this study establishes the dose- and time-dependent changes in immune marker expression of commonly studied mesothelioma cell lines following radiation and will inform future study into optimal dosing and scheduling of combined radiotherapy and immune checkpoint inhibition for mesothelioma. Show less

Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.5) causes perturbations in the expression patterns of specific genes in the developing heart in the acute period (1-3 days) following the binge episode. To test this hypothesis and identify strong candidate ethanol-sensitive target genes of interest, we adapted a mouse binge alcohol model that is associated with a high incidence of congenital heart defects as described below. Pregnant mice were administered a single dose of alcohol (2.5 g/kg in saline) or control (saline alone) via oral gavage. To evaluate the impact of maternal binge alcohol on cardiac gene expression profiles, we isolated embryonic hearts from both groups (n = 5/group) at 24, 48, and 72 h post-gavage for transcriptomic analyses. RNA was extracted and evaluated using quantitative RNA-sequencing (RNA-Seq) methods. To identify a cohort of binge-altered cardiac genes, we set the threshold for change at >2.0-fold difference with adjusted p < 0.05 versus control.  RNA-Seq analysis of cardiac gene expression revealed that of the 17 genes that were altered within the first 48 h post-binge, with the largest category consisting of transcription factors (Alx1, Alx4, HoxB7, HoxD8, and Runx2), followed by signaling molecules (Adamts18, Dkk2, Rtl1, and Wnt7a). Furthermore, multiple comparative and pathway analyses suggested that several of the candidate genes identified through differential RNA-Seq analysis may interact through certain common pathways. To investigate this further, we performed gene-specific qPCR analyses for three representative candidate targets: Runx2, Wnt7a, and Mlxipl. Notably, only Wnt7a showed significantly (p < 0.05) decreased expression in response to maternal binge alcohol in the qPCR assays. These findings identify Wnt7a and a short list of potential other candidate genes and pathways for further study, which could provide mechanistic insights into how maternal binge alcohol consumption produces congenital cardiac malformations. Show less

Melanocortin 4 receptor (MC4R) mutation is the most common cause of known monogenic obesity in humans. Unexpectedly, humans and rodents with MC4R deficiency do not develop hyperglycaemia despite chronic obesity and insulin resistance. To explain the underlying mechanisms for this phenotype, we determined the role of MC4R in glucose homeostasis in the presence and absence of obesity in mice. We used global and hypothalamus-specific MC4R-deficient mice to investigate the brain regions that contribute to glucose homeostasis via MC4R. We performed oral, intraperitoneal and intravenous glucose tolerance tests in MC4R-deficient mice that were either obese or weight-matched to their littermate controls to define the role of MC4R in glucose regulation independently of changes in body weight. To identify the integrative pathways through which MC4R regulates glucose homeostasis, we measured renal and adrenal sympathetic nerve activity. We also evaluated glucose homeostasis in adrenaline (epinephrine)-deficient mice to investigate the role of adrenaline in mediating the effects of MC4R in glucose homeostasis. We employed a graded [ We found that obese and weight-matched MC4R-deficient mice exhibited improved glucose tolerance due to elevated glucosuria, not enhanced beta cell function. Moreover, MC4R deficiency selectively in the paraventricular nucleus of the hypothalamus (PVH) is responsible for reducing the renal threshold for glucose as measured by graded [ These findings define a previously unrecognised function of hypothalamic MC4R in glucose reabsorption mediated by adrenaline and renal GLUT2. Taken together, our findings indicate that elevated glucosuria due to low sympathetic tone explains why MC4R deficiency does not cause hyperglycaemia despite inducing obesity and insulin resistance. Graphical abstract. Show less

The process of restenosis is based on the interplay of various mechanical and biological processes triggered by angioplasty-induced vascular trauma. Early arterial recoil, negative vascular remodeling, and neointimal formation therefore limit the long-term patency of interventional recanalization procedures. The most serious of these processes is neointimal hyperplasia, which can be traced back to 4 main mechanisms: endothelial damage and activation; monocyte accumulation in the subintimal space; fibroblast migration; and the transformation of vascular smooth muscle cells. A wide variety of animal models exists to investigate the underlying pathophysiology. Although mouse models, with their ease of genetic manipulation, enable cell- and molecular-focused fundamental research, and rats provide the opportunity to use stent and balloon models with high throughput, both rodents lack a lipid metabolism comparable to humans. Rabbits instead build a bridge to close the gap between basic and clinical research due to their human-like lipid metabolism, as well as their size being accessible for clinical angioplasty procedures. Every different combination of animal, dietary, and injury model has various advantages and disadvantages, and the decision for a proper model requires awareness of species-specific biological properties reaching from vessel morphology to distinct cellular and molecular features. Show less

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition. Show less

Although metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples. We compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells. We found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression - notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells. We report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages. Show less

Bisphosphonates play an important role in the treatment of metabolic bone diseases such as osteoporosis. In addition to their anti-resorptive activity by triggering osteoclast apoptosis, nitrogen-containing bisphosphonates (N-BP) may also influence osteogenic differentiation, which might rely on their capacity to inhibit the mevalonate pathway. In vascular endothelial cells inhibition of this pathway by cholesterol-lowering statins activates the MEK5/ERK5 mitogen-activated protein kinase cascade, which plays an important role in cellular differentiation, apoptosis or inflammatory processes. Here we evaluated whether N-BP may also target the MEK5/ERK5 pathway and analysed the consequences of ERK5 activation on osteogenic differentiation. We show that N-BP dose-dependently activate ERK5 in primary human endothelial cells and osteoblasts. The mechanism likely involves farnesyl pyrophosphate synthase inhibition and subsequent functional inhibition of the small GTPase Cdc42 since siRNA-mediated knockdown of both genes could reproduce N-BP-induced ERK5 activation. ERK5 activation resulted in regulation of several bone-relevant genes and was required for calcification and osteogenic differentiation of bone marrow-derived mesenchymal stems cells as evident by the lack of alkaline phosphatase induction and alizarin-red S staining observed upon ERK5 knockdown or upon differentiation initiation in presence of a pharmacological ERK5 inhibitor. Our data provide evidence that N-BP activate the MEK5/ERK5 cascade and reveal an essential role of ERK5 in osteogenic differentiation and mineralization of skeletal precursors. Show less

Self-renewal is a hallmark of both hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs); therefore, the identification of mechanisms that are required for LSC, but not HSC, function could provide therapeutic opportunities that are more effective and less toxic than current treatments. Here, we employed an in vivo shRNA screen and identified jumonji domain-containing protein JMJD1C as an important driver of MLL-AF9 leukemia. Using a conditional mouse model, we showed that loss of JMJD1C substantially decreased LSC frequency and caused differentiation of MLL-AF9- and homeobox A9-driven (HOXA9-driven) leukemias. We determined that JMJD1C directly interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program. In contrast, loss of JMJD1C led to only minor defects in blood homeostasis and modest effects on HSC self-renewal. Together, these data establish JMJD1C as an important mediator of MLL-AF9- and HOXA9-driven LSC function that is largely dispensable for HSC function. Show less