A novel image-based high throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease

Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs (Adult Polyglucosan Body Disease (APBD), Tarui and Lafora diseases) are caused by intracellular accumulation of insoluble inclusions, called polyglucosan bodies (PB), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid-Schiff's (PAS) stained structures, and quantified. We screened the DIVERSet-CL 10,084 compound library using this assay in high throughput format and discovered 11 dose-dependent and 8 non dose-dependent PB-reducing hits. ~70% of the hits appear to act through reducing glycogen synthase (GS) activity which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1 (PP1). Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders

Guaiacol as a drug candidate for treating adult polyglucosan body disease

Adult polyglucosan body disease (APBD) is a late-onset disease caused by intracellular accumulation of polyglucosan bodies, formed due to glycogen-branching enzyme (GBE) deficiency. To find a treatment for APBD, we screened 1,700 FDA-approved compounds in fibroblasts derived from APBD-modeling GBE1-knockin mice. Capitalizing on fluorescent periodic acid–Schiff reagent, which interacts with polyglucosans in the cell, this screen discovered that the flavoring agent guaiacol can lower polyglucosans, a result also confirmed in APBD patient fibroblasts. Biochemical assays showed that guaiacol lowers basal and glucose 6-phosphate–stimulated glycogen synthase (GYS) activity. Guaiacol also increased inactivating GYS1 phosphorylation and phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. Guaiacol treatment in the APBD mouse model rescued grip strength and shorter lifespan. These treatments had no adverse effects except making the mice slightly hyperglycemic, possibly due to the reduced liver glycogen levels. In addition, treatment corrected penile prolapse in aged GBE1-knockin mice. Guaiacol’s curative effects can be explained by its reduction of polyglucosans in peripheral nerve, liver, and heart, despite a short half-life of up to 60 minutes in most tissues. Our results form the basis to use guaiacol as a treatment and prepare for the clinical trials in APBD

Radiofrequency treatment alters cancer cell phenotype

The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment

Mesenchymal Stromal Cells Derived from the Bone Marrow of Acute Lymphoblastic Leukemia Patients Show Altered BMP4 Production: Correlations with the Course of Disease

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated