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Background: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP+ pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG)n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation. Methods: DMPK (CTG)n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated. Results: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1. Conclusions: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden. [ABSTRACT FROM AUTHOR]

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors. [ABSTRACT FROM AUTHOR]

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented on several articles on adenosine triphosphate (ATP) and the use of biosensors based on fluorescence resonance energy transfer (FRET) that were published in the previous issues.

Simple Summary: The prognosis of children with relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor, and innovative treatments are needed. Most drugs approved for adults with AML over the last decade are not available or not licensed for use in children. Clinical trials in pediatric AML to assess the safety and/or efficacy of new drugs may take a long time to recruit given smaller patient numbers. The overarching aim of the Pediatric Acute Leukemia (PedAL) program, supported by the Leukemia and Lymphoma Society as part of its Dare to Dream Project, is to establish new standards of care for children with R/R AML via international collaboration in Europe, North America, and Asia-Pacific to accelerate clinical trial conduction, increase access to promising new therapies, and create a registry of uniformly-collected data. These efforts will facilitate biomarker-driven approaches of targeted therapies, of which an overview is provided in this manuscript, with the intent to obtain regulatory approvals. The prognosis of children with acute myeloid leukemia (AML) has improved incrementally over the last few decades. However, at relapse, overall survival (OS) is approximately 40–50% and is even lower for patients with chemo-refractory disease. Effective and less toxic therapies are urgently needed for these children. The Pediatric Acute Leukemia (PedAL) program is a strategic global initiative that aims to overcome the obstacles in treating children with relapsed/refractory acute leukemia and is supported by the Leukemia and Lymphoma Society in collaboration with the Children's Oncology Group, the Innovative Therapies for Children with Cancer consortium, and the European Pediatric Acute Leukemia (EuPAL) foundation, amongst others. In Europe, the study is set up as a complex clinical trial with a stratification approach to allocate patients to sub-trials of targeted inhibitors at relapse and employing harmonized response and safety definitions across sub-trials. The PedAL/EuPAL international collaboration aims to determine new standards of care for AML in a first and second relapse, using biology-based selection markers for treatment stratification, and deliver essential data to move drugs to front-line pediatric AML studies. An overview of potential treatment targets in pediatric AML, focused on drugs that are planned to be included in the PedAL/EuPAL project, is provided in this manuscript. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves. Here, we employ a patient-derived muscle cell model for DM1 to examine in detail the expression of MBNL RNA and protein variants during myogenic differentiation. This DM1 model consists of a panel of isogenic myoblast cell lines that either contain a pathogenic DMPK allele with a congenital mutation of 2600 triplets, or lack this expanded repeat through CRISPR/Cas9-mediated gene editing. We found that the temporal expression levels of MBNL1, MBNL2 and MBNL3 RNAs are not influenced by presence of the (CTG)2600 repeat during myogenesis in vitro. However, throughout myoblast proliferation and differentiation to myotubes a disproportionate inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8 occurs in cells with the (CTG)2600 repeat. As a consequence, a reduced quantity and imbalanced collection of splice variants of MBNL1 and MBNL2 accumulates in both the cytoplasm and the nucleus of DM1 myoblasts and myotubes. We thus propose that both the quantitative and qualitative changes in the intracellular partitioning of MBNL proteins are a pivotal cause of skeletal muscle problems in DM1, starting already in muscle progenitor cells. [ABSTRACT FROM AUTHOR]

Malignant cells infiltrating the bone marrow ( BM) interfere with normal cellular behaviour of supporting cells, thereby creating a malignant niche. We found that CXCR4-receptor expression was increased in paediatric precursor B-cell acute lymphoblastic leukaemia ( BCP- ALL) cells compared with normal mononuclear haematopoietic cells ( P < 0·0001). Furthermore, high CXCR4-expression correlated with an unfavourable outcome in BCP- ALL (5-year cumulative incidence of relapse ± standard error: 38·4% ± 6·9% in CXCR4-high versus 12% ± 4·6% in CXCR4-low expressing cases, P < 0·0001). Interestingly, BM levels of the CXCR4-ligand ( CXCL12) were 2·7-fold lower ( P = 0·005) in diagnostic BCP- ALL samples compared with non-leukaemic controls. Induction chemotherapy restored CXCL12 levels to normal. Blocking the CXCR4-receptor with Plerixafor showed that the lower CXCL12 serum levels at diagnosis could not be explained by consumption by the leukaemic cells, nor did we observe an altered CXCL12-production capacity of BM-mesenchymal stromal cells ( BM- MSC) at this time-point. We rather observed that a very high density of leukaemic cells negatively affected CXCL12-production by the BM- MSC while stimulating the secretion levels of granulocyte colony-stimulating factor (G- CSF). These results suggest that highly proliferative leukaemic cells are able to down-regulate secretion of cytokines involved in homing ( CXCL12), while simultaneously up-regulating those involved in haematopoietic mobilization (G- CSF). Therefore, interference with the CXCR4/ CXCL12 axis may be an effective way to mobilize BCP- ALL cells. [ABSTRACT FROM AUTHOR]

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages. [ABSTRACT FROM AUTHOR]

Abstract: Transformed cancer cells have an altered metabolism, characterized by a shift towards aerobic glycolysis, referred to as ‘the Warburg phenotype’. A change in flux through mitochondrial OXPHOS and cytosolic pathways for ATP production and a gain of capacity for biomass production in order to sustain the needs for altered growth and morphodynamics are typically involved in this global rewiring of cancer cell metabolism. Characteristically, these changes in metabolism are accompanied by enhanced uptake of nutrients like glucose and glutamine. Here we focus on the relationship between cell metabolism and cell dynamics, in particular the formation and function of invadopodia, specialized structures for focal degradation of the extracellular matrix. Since we recently found presence of enzymes that are active in glycolysis and associated pathways in invadopodia, we hypothesize that metabolic adaptation and invadopodia formation are linked processes. We give an overview on the background for this idea and show for the first time that extracellular matrix degradation by invadopodia can be differentially manipulated, without effects on cell proliferation, by use of metabolic inhibitors or changes in nutrient composition of cell culture media. We conclude that cell metabolism and carbohydrate availability, especially pyruvate, are involved in fuelling of invadopodia formation and activity. [Copyright &y& Elsevier]

The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM's most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1). [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1). For cellular delivery, ASOs were complexed into nanoparticles using the cationic cell-penetrating peptides nona-arginine and PepFect14 (PF14). Although both peptides facilitated uptake, only PF14 led to a dose-dependent correction of disease-typical abnormal splicing. In line with this observation, time-lapse confocal microscopy demonstrated that only PF14 mediated translocation of the ASOs to the nucleus, which is the main site of action. Through fluorescence lifetime imaging, we could distinguish intact oligonucleotide from free fluorophore, showing that PF14 also shielded the ASOs from degradation. Finally, we employed a combination of live-cell fluorescence correlation spectroscopy and immunofluorescence microscopy and demonstrated that intranuclear blocking-type oligonucleotide concentrations in the upper nanomolar range were required to dissolve nuclear muscleblind-like protein 1 foci, a hallmark of DM1. Our findings have important implications for the clinical use of ASOs in DM1 and provide a basis for further research on other types of ASOs. [ABSTRACT FROM AUTHOR]

Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323–330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation. [ABSTRACT FROM AUTHOR]

Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics. [ABSTRACT FROM AUTHOR]

Most relapses in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are not predicted using current prognostic features. Here, we determined the co-occurrence and independent prognostic relevance of 3 recently identified prognostic features: BCR-ABL1-like gene signature, deletions in IKZF1, and high CRLF2 messenger RNA expression (CRLF2-high). These features were determined in 4 trials representing 1128 children with ALL: DCOG ALL-8, ALL9, ALL10, and Cooperative ALL (COALL)-97/03. BCR-ABL1-like, IKZF1-deleted, and CRLF2-high cases constitute 33.7% of BCR-ABL1-negative, MLL wild-type BCP-ALL cases, of which BCR-ABL1-like and IKZF1 deletion (co)occurred most frequently. Higher cumulative incidence of relapse was found for BCR-ABL1-like and IKZF1-deleted, but not CRLF2-high, cases relative to remaining BCP-ALL cases, reflecting the observations in each of the cohorts analyzed separately. No relapses occurred among cases with CRLF2-high as single feature, whereas 62.9% of all relapses in BCR-ABL1-negative, MLL wild-type BCP-ALL occurred in cases with BCR-ABL1-like signature and/or IKZF1 deletion. Both the BCR-ABL1-like signature and IKZF1 deletions were prognostic features independent of conventional prognostic markers in a multivariate model, and both remained prognostic among cases with intermediate minimal residual disease. The BCR-ABL1-like signature and an IKZF1 deletion, but not CRLF2-high, are prognostic factors and are clinically of importance to identify high-risk patients who require more intensive and/or alternative therapies. [ABSTRACT FROM AUTHOR]

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of α-D-N-acetylglucosaminyl-2-O-sulfo-α-L-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a VH3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease. [ABSTRACT FROM AUTHOR]