Overcoming Wnt–β-catenin dependent anticancer therapy resistance in leukaemia stem cells

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt–β-catenin and PI3K–Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate β-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt–β-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated β-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, β-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated β-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape

Mitochondria in protein homeostasis and aging

Loss of protein homeostasis (proteostasis) and mitochondrial dysfunction are hallmarks of both aging and age-associated neurodegenerative diseases. However, it has been unclear whether and how the loss of proteostasis and mitochondrial dysfunction are related. We discovered that structurally unstable proteins in the cytosol are imported into mitochondria for degradation in yeast and human cells. Mitochondrial import is required for timely dissolution and clearance of cytosolic aggregates. Defects in cytosolic chaperones, such as Hsp70s, lead to increased import of misfolded proteins into mitochondria, while compromised mitochondrial degradation, such as a point mutation in the proteolytic domain of Lon protease, causes delayed clearance of misfolded proteins in mitochondria. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol). The MAGIC pathway may be particularly relevant to aging and protein aggregation diseases. Recent studies found that aggregation-prone proteins constituting pathological inclusions in neurodegenerative diseases enter and accumulate in mitochondria in patients and disease models. However, how excessive misfolded proteins are managed within mitochondria remains unclear. We found that misfolded proteins that failed to be degraded in the mitochondrial matrix consolidated into solid-phase aggregates in yeast and human cells, which we term deposits of unfolded mitochondrial proteins (DUMP). Formation of DUMP occurs in mitochondria near endoplasmic reticulum-mitochondria contact sites and is regulated by mitochondrial proteins controlling the production of cytidine 5’ diphosphate diacylglycerol. DUMP formation is age dependent but accelerated by exogenous unfolded proteins. Key enzymes of the tricarboxylic acid cycle are components of native DUMP, and accelerated DUMP formation reduced their enzymatic activities. During cellular aging, DUMP are asymmetrically retained in the older cell, and the solid-phase condensation of misfolded proteins into DUMP is necessary for retaining damaged mitochondrial proteins in older cells. To systematically identify regulators of the MAGIC, we performed a genome-wide screen in yeast. Based on the phenotype, gene knockouts displaying either decreased or increased accumulation of misfolded proteins in mitochondria were isolated. Primary hits from different categories were further validated with different reporters and tailored secondary screens. Along with the known MAGIC regulator HSP104, many new regulators were revealed. Here, we focused on understanding functions of the highly conserved AMPK in MAGIC. We found that activation and inhibition of AMPK reduced and increased mitochondrial import of misfolded proteins, respectively, in both yeast and human cells. AMPK responds to metabolic states of cells and when the glucose level is low, AMPK is activated to reduce the MAGIC and promote mitochondrial respiration. Blocking the mitochondria-dependent clearance of P525L, a mutant form of FUS associated with Amyotrophic Lateral Sclerosis, by constitutive activation of AMPK, potentiated P525L toxicity in yeast cells. Taken together, our work provides an inventory of genes that affects the MAGIC, and the framework and tools to understand the pathway for future studies

RhTSG-6 inhibits IL-1β-induced extracellular matrix degradation and apoptosis by suppressing the p38, and JNK pathways in nucleus pulposus cells

Introduction. Intervertebral disc degeneration (IDD) is one of the major causes of low back pain (LBP) which seriously affects health and normal physical activity. Recombinant human tumor necrosis factor-a (TNF-a) induced protein 6 (rhTSG-6) has been reported to have therapeutic effects on a variety of inflammatory diseases, but the effect and mechanism of rhTSG-6 action in IDD are not fully understood. The present study was aimed to explore the functional role of rhTSG-6 in interleukin (IL)-1b-induced nucleus pulposus (NP) cell model. Materials and methods. Experimental human NP cells were isolated from the patients with idiopathic scoliosis and treated with culture medium containing IL-1b (10 ng/mL) for 24 hours to induce extracellular matrix degradation and apoptosis, simulating an IDD model in vitro. The viability of NP cells was analyzed by the CCK-8 assay. The relevant mRNA and protein levels were measured by RT-qPCR and western blot. The apoptosis of NP cells was determined by flow cytometry analysis and western blot. Results. Compared with the NP cells without IL-1b treatment, IL-1b caused approximately 70% reduction in the viability of NP cells, while RhTSG-6 partly increased the decrease of IL-1b on cell viabilities. Moreover, treatment with rhTSG-6 considerably attenuated the upregulation of extracellular matrix (ECM)-catabolic factors (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5), and increased the downregulation of ECM-anabolic factor (collagen II) in NP cells induced by IL-1b, indicating that ECM degradation was suppressed. Furthermore, rhTSG-6 also protected NP cells from IL-1b-induced apoptosis. Mechanically, rhTSG-6 inhibited the activation of members of mitogen-activated protein kinase (MAPK) pathway by blocking the phosphorylation of p38, c-Jun N-terminal kinase (JNK) and ERK in IL-1b-induced NP cells. Conclusions. RhTSG-6 can attenuate ECM degradation and apoptosis in IL-1b-induced NP cells by inhibiting the p38, JNK and ERK pathways, which may contribute to its potential application in the therapy of IDD

Neutron energy spectrum measurement of the Back-n white neutron source at CSNS

China Spallation Neutron Source (CSNS) has been built and commissioned successfully in 2018 which is implemented by impinging 1.6 GeV protons onto a tungsten target with 25 Hz repetition frequency. An associated white neutron beam line exploiting the back-streaming neutrons, named Back-n, with a flight path of 55 m in endstation 1 (#ES1) and 76 m in endstation 2 (#ES2) was built mainly for nuclear data measurements. The Back-n beam line started running since the beginning of 2018 and the neutron energy spectrum was measured at the very beginning since it is an essential information for feasibility study and analysis of any measurement. In this paper, the measurement of the neutron energy spectrum of Back-n #ES2 is reported. The first result of the neutron flux from 1 eV to 100 MeV measured by a fission chamber based on the 235U(n, f) reaction is presented. The total neutron flux normalized to the proton beam power of 100 kW is $7.03 \times 10^{6}$ neutrons/cm2/s. The results show a good agreement with that of the Monte Carlo simulation as well