Reduction of senescent fibro-adipogenic progenitors in progeria-aged muscle by senolytics rescues the function of muscle stem cells.

BACKGROUND: Fibro-adipogenic progenitors (FAPs) in the muscles have been found to interact closely with muscle progenitor/stem cells (MPCs) and facilitate muscle regeneration at normal conditions. However, it is not clear how FAPs may interact with MPCs in aged muscles. Senolytics have been demonstrated to selectively eliminate senescent cells and generate therapeutic benefits on ageing and multiple age-related disease models. METHODS: By studying the muscles and primary cells of age matched WT mice and Zmpste24-/- (Z24-/- ) mice, an accelerated ageing model for Hutchinson-Gilford progeria syndrome (HGPS), we examined the interaction between FAPs and MPCs in progeria-aged muscle, and the potential effect of senolytic drug fisetin in removing senescent FAPs and improving the function of MPCs. RESULTS: We observed that, compared with muscles of WT mice, muscles of Z24-/- mice contained a significantly increased number of FAPs (2.4-fold; n > =6, P < 0.05) and decreased number of MPCs (2.8-fold; n > =6, P < 0.05). FAPs isolated from Z24-/- muscle contained about 44% SA-ß-gal+ senescent cells, in contrast to about 3.5% senescent cells in FAPs isolated from WT muscle (n > =6, P < 0.001). The co-culture of Z24-/- FAPs with WT MPCs resulted in impaired proliferation and myogenesis potential of WT MPCs, with the number of BrdU positive proliferative cells being reduced for 3.3 times (n > =6, P < 0.001) and the number of myosin heavy chain (MHC)-positive myotubes being reduced for 4.5 times (n > =6, P < 0.001). The treatment of the in vitro co-culture system of Z24-/- FAPs and WT MPCs with the senolytic drug fisetin led to increased apoptosis of Z24-/- FAPs (14.5-fold; n > =6, P < 0.001) and rescued the impaired function of MPCs by increasing the number of MHC-positive myotubes for 3.1 times (n > =6, P < 0.001). Treatment of Z24-/- mice with fisetin in vivo was effective in reducing the number of senescent FAPs (2.2-fold, n > =6, P < 0.05) and restoring the number of muscle stem cells (2.6-fold, n > =6, P < 0.05), leading to improved muscle pathology in Z24-/- mice. CONCLUSIONS: These results indicate that the application of senolytics in the progeria-aged muscles can be an efficient strategy to remove senescent cells, including senescent FAPs, which results in improved function of muscle progenitor/stem cells. The senescent FAPs can be a potential novel target for therapeutic treatment of progeria ageing related muscle diseases.

Aberrant RhoA activation in macrophages increases senescence-associated secretory phenotypes and ectopic calcification in muscular dystrophic mice.

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Effect of Oral Losartan on Orthobiologics: Implications for Platelet-Rich Plasma and Bone Marrow Concentrate-A Rabbit Study.

Recent efforts have focused on customizing orthobiologics, such as platelet-rich plasma (PRP) and bone marrow concentrate (BMC), to improve tissue repair. We hypothesized that oral losartan (a TGF-ß1 blocker with anti-fibrotic properties) could decrease TGF-ß1 levels in leukocyte-poor PRP (LP-PRP) and fibrocytes in BMC. Ten rabbits were randomized into two groups (N = 5/group): osteochondral defect + microfracture (control, group 1) and osteochondral defect + microfracture + losartan (losartan, group 2). For group 2, a dose of 10mg/kg/day of losartan was administrated orally for 12 weeks post-operatively. After 12 weeks, whole blood (WB) and bone marrow aspirate (BMA) samples were collected to process LP-PRP and BMC. TGF-ß1 concentrations were measured in WB and LP-PRP with multiplex immunoassay. BMC cell populations were analyzed by flow cytometry with CD31, CD44, CD45, CD34, CD146 and CD90 antibodies. There was no significant difference in TGF-ß1 levels between the losartan and control group in WB or LP-PRP. In BMC, the percentage of CD31+ cells (endothelial cells) in the losartan group was significantly higher than the control group (p = 0.008), while the percentage of CD45+ cells (hematopoietic cells-fibrocytes) in the losartan group was significantly lower than the control group (p = 0.03).

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle-derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24-/- (Z24-/- ) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin-induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F-actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei-induced cGAS-Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24-/- mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.

CRISPR/Cas9-Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors.

Although the lack of dystrophin expression in muscle myofibers is the central cause of Duchenne muscular dystrophy (DMD), accumulating evidence suggests that DMD may also be a stem cell disease. Recent studies have revealed dystrophin expression in satellite cells and demonstrated that dystrophin deficiency is directly related to abnormalities in satellite cell polarity, asymmetric division, and epigenetic regulation, thus contributing to the manifestation of the DMD phenotype. Although metabolic and mitochondrial dysfunctions have also been associated with the DMD pathophysiology profile, interestingly, the role of dystrophin with respect to stem cells dysfunction has not been elucidated. In the past few years, editing of the gene that encodes dystrophin has emerged as a promising therapeutic approach for DMD, although the effects of dystrophin restoration in stem cells have not been addressed. Herein, we describe our use of a clustered regularly interspaced short palindromic repeats/Cas9-based system to correct the dystrophin mutation in dystrophic (mdx) muscle progenitor cells (MPCs) and show that the expression of dystrophin significantly improved cellular properties of the mdx MPCs in vitro. Our findings reveal that dystrophin-restored mdx MPCs demonstrated improvements in cell proliferation, differentiation, bioenergetics, and resistance to oxidative and endoplasmic reticulum stress. Furthermore, our in vivo studies demonstrated improved transplantation efficiency of the corrected MPCs in the muscles of mdx mice. Our results indicate that changes in cellular energetics and stress resistance via dystrophin restoration enhance muscle progenitor cell function, further validating that dystrophin plays a role in stem cell function and demonstrating the potential for new therapeutic approaches for DMD. Stem Cells 2019;37:1615-1628.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes.

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

PURPOSE: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. EXPERIMENTAL DESIGN: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. RESULTS: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. CONCLUSIONS: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins.

Protein phosphatase 2A regulatory subunits are starting to reveal their functions in plant metabolism and development.

Canonical protein phosphatase 2A (PP2A) consists of a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to confer substrate specificity and cellular localization to the PP2A complex, and are generally divided into three non-related families in plants, i.e., B55, B' and B''. The two Arabidopsis B55 subunits (α and ß) interact with nitrate reductase (NR) in the bimolecular fluorescence complementation assay in planta, and are necessary for rapid activation of NR. Interestingly, knockout of all four B55 alleles is probably lethal, because a homozygous double knockout (pp2a-b55αß) could not be found. The B55 subunits, therefore, appear to have essential functions that cannot be replaced by other regulatory B subunits. A double mutant (pp2a-b'αß) of two close B' homologs show severely impaired fertility, pointing to the essential role also of B' subunits in plant development.

Protein phosphatase 2A B55 and A regulatory subunits interact with nitrate reductase and are essential for nitrate reductase activation.

Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55ß. Interestingly, the homozygous double mutant (b55α × b55ß) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bß and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.

Diversity in subcellular targeting of the PP2A B'eta subfamily members.

Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase comprising a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to be responsible for substrate specificity and localization of the PP2A complex. In plants, three families of B subunits exist, i.e. B (B55), B', and B''. Here, we report differential subcellular targeting within the Arabidopsis B'eta subfamily, which consists of the close homologs B'eta, B'theta, B'gamma and B'zeta. Phenotypes of corresponding knockouts were observed, and particularly revealed delayed flowering for the B'eta knockout. The B' subunits were linked to fluorescent tags and transiently expressed in various tissues of onion, tobacco and Arabidopsis. B'eta and B'gamma targeted the cytosol and nucleus. B'zeta localized to the cytoplasm and partly co-localized with mitochondrial markers when the N-terminus was free. Provided its C-terminus was free, the B'theta subunit targeted peroxisomes. The importance of the C-terminal end for peroxisomal targeting was further confirmed by truncation of the C-terminus. The results revealed that the closely related B' subunits are targeting different organelles in plants, and exemplify the usage of the peptide serine-serine-leucine as a PTS1 peroxisomal signaling peptide.