An aptamer-mediated base editing platform for simultaneous knock-in and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in murine tumor transplantation models.

Pancreatic ductal adenocarcinoma (PDA) is aggressive cancer characterized by rapid progression, metastatic recurrence, and highly resistant to treatment. PDA cells exhibit aerobic glycolysis, or the Warburg effect, which reduces the flux of pyruvate into mitochondria. As a result, more glycolytic metabolites are shunted to pathways for the production of building blocks (e.g., ribose) and reducing agents (e.g., NADPH) for biosynthesis that are necessary for cell proliferation. In addition, PDA cells are highly addicted to glutamine for both maintaining biosynthetic pathways and achieving redox balance. Mitochondrial uncoupling facilitates proton influx across the mitochondrial inner membrane without generating ATP, leading to a futile cycle that consumes glucose metabolites and glutamine. We synthesized a new mitochondrial uncoupler MB1-47 and tested its effect on cancer cell metabolism and the anticancer activity in pancreatic cancer cell models and murine tumor transplantation models. MB1-47 uncouples mitochondria in the pancreatic cancer cells, resulting in: (1) the acceleration of pyruvate oxidation and TCA turnover; (2) increases in AMP/ATP and ADP/AMP ratios; and (3) a decrease in the synthesis rate of nucleotides and sugar nucleotides. Moreover, MB1-47 arrests cell cycle at G0-G1 phase, reduces clonogenicity, and inhibits cell growth of murine and human pancreatic cancer cells. In vivo studies showed that MB1-47 inhibits tumor growth in murine tumor transplantation models, and inhibits the hepatic metastasis when tumor cells were transplanted intrasplenically. Our results provide proof of concept for a potentially new strategy of treating PDA, and a novel prototype experimental drug for future studies and development.

Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System.

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-pointTM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Niclosamide piperazine prevents high-fat diet-induced obesity and diabetic symptoms in mice.

PURPOSE: Obesity and type 2 diabetes (T2D) have become the major public health challenges globally. Mitochondrial uncoupling, which reduces intracellular lipid loads and corrects the underlying cause of insulin resistance, has emerged as a promising anti-obese and anti-diabetic intervention. Niclosamide is an anthelmintic drug approved by the US FDA with the mechanism of action that uncouples mitochondria of parasitic worms. Recently, niclosamide ethanolamine salt (NEN) was found to be a safe and effective hepatic mitochondrial uncoupler for the prevention and treatment of obesity and T2D in mouse models. The striking features of NEN prompt us to examine the anti-obese and anti-diabetic efficacy of other salt forms of niclosamide, with the ultimate goal to identify a suitable salt formulation for future clinical development. Here, we report the study with niclosamide piperazine salt (NPP), another salt form of niclosamide with documented safety profile. METHODS: Mitochondrial uncoupling activity of NEN and NPP were determined by oxygen consumption assay with Seahorse XF24e Analyzer, as well as by mitochondrial membrane potential measurement in cultured cells. The in vivo anti-diabetic and anti-obesity activities were determined in C57BL/6J mice fed high-fat diet (HFD) or HFD containing 2000 ppm. NPP for 11 weeks. RESULTS: Niclosamide piperazine salt showed a comparable mitochondrial uncoupling activity to NEN. Oral administration of NPP significantly reduced HFD-induced obesity, hyperglycemia and hepatic steatosis, and sensitized the insulin responses in mice. CONCLUSIONS: Niclosamide piperazine salt may hold the promise to become an alternative to NEN as a drug lead for the treatment of obesity and T2D. No level of evidence Animal study.

Effect of mitochondrial uncouplers niclosamide ethanolamine (NEN) and oxyclozanide on hepatic metastasis of colon cancer.

Metabolism of cancer cells is characterized by aerobic glycolysis, or the Warburg effect. Aerobic glycolysis reduces pyruvate flux into mitochondria, preventing a complete oxidation of glucose and shunting glucose to anabolic pathways essential for cell proliferation. Here we tested a new strategy, mitochondrial uncoupling, for its potential of antagonizing the anabolic effect of aerobic glycolysis and for its potential anticancer activities. Mitochondrial uncoupling is a process that facilitates proton influx across the mitochondrial inner membrane without generating ATP, stimulating a futile cycle of acetyl- CoA oxidation. We tested two safe mitochondrial uncouplers, NEN (niclosamide ethanolamine) and oxyclozanide, on their metabolic effects and anti-cancer activities. We used metabolomic NMR to examine the effect of mitochondrial uncoupling on glucose metabolism in colon cancer MC38 cells. We further tested the anti-cancer effect of NEN and oxyclozanide in cultured cell models, APCmin/+ mouse model, and a metastatic colon cancer mouse model. Using a metabolomic NMR approach, we demonstrated that mitochondrial uncoupling promotes pyruvate influx to mitochondria and reduces various anabolic pathway activities. Moreover, mitochondrial uncoupling inhibits cell proliferation and reduces clonogenicity of cultured colon cancer cells. Furthermore, oral treatment with mitochondrial uncouplers reduces intestinal polyp formation in APCmin/+ mice, and diminishes hepatic metastasis of colon cancer cells transplanted intrasplenically. Our data highlight a unique approach for targeting cancer cell metabolism for cancer prevention and treatment, identified two prototype compounds, and shed light on the anti-cancer mechanism of niclosamide.

New methods for monitoring mitochondrial biogenesis and mitophagy in vitro and in vivo.

Removal of damaged mitochondria through mitophagy is critical for maintaining cellular homeostasis and functions. Increasing evidence implicates mitophagy in red blood cell differentiation, neurodegeneration, macrophage-mediated inflammation, ischemia, adipogenesis, drug-induced tissue injury, and cancer. Considerable progress has been made toward understanding the biochemical mechanisms involved in mitophagy regulation. However, few reliable assays to monitor and quantify mitophagy have been developed, particularly in vivo. In this review, we summarize the recent development of three assays, MitoTimer, mt-Keima and mito-QC, for monitoring and quantifying mitophagy in cells and in animal tissues. We also discuss the advantages and limitations of these three assays when using them to monitor and quantify mitophagy. Impact statement Removal of damaged mitochondria through mitophagy is critical for maintaining cellular homeostasis and functions. However, reliable quantitative assays to monitor mitophagy, particularly in vivo, are just emerging. This review will summarize the current novel quantitative assays to monitor mitophagy in vivo.

A role for ABCG2 beyond drug transport: Regulation of autophagy.

The ABC drug transporters, including ABCG2, are well known for their ability to efflux a wide spectrum of chemotherapeutic agents, thereby conferring a multidrug-resistant phenotype. However, studies over the past several years suggest that the ABC transporters may play additional role(s) in cell survival in the face of stress inducers that are not ABCG2 substrates (i.e., nutrient deprivation, ionizing radiation, rapamycin). The mechanism by which this occurs is largely unknown. In the present study, using several cancer cell lines and their ABCG2-overexpressing sublines, we show that cells overexpressing ABCG2 were more resistant to these stressors. This resistance was associated with an elevated level of autophagy flux, as measured by a higher rate of SQSTM1/p62 degradation and greater accumulation of LC3-II when compared to parental cells. Knockdown of ABCG2 reduced autophagic activity in resistant cells to a level similar to that observed in parental cells, confirming that the enhanced autophagy was ABCG2-dependent. Moreover, using cell viability, apoptosis, and clonogenic assays, we demonstrated that the ABCG2-expressing cells were more resistant to amino acid starvation and radiation-induced cell death. Importantly, knockdown of the critical autophagy factors ATG5 and ATG7 greatly reduced cell survival, verifying that enhanced autophagy was critical for this effect. Taken together, these data indicate that autophagy induced by various stressors is enhanced/accelerated in the presence of ABCG2, resulting in delayed cell death and enhanced cell survival. This defines a new role for this transporter, one with potential clinical significance.

Niclosamide ethanolamine-induced mild mitochondrial uncoupling improves diabetic symptoms in mice.

Type 2 diabetes (T2D) has reached an epidemic level globally. Most current treatments ameliorate the hyperglycemic symptom of the disease but are not effective in correcting its underlying cause. One important causal factor of T2D is ectopic accumulation of lipids in metabolically sensitive organs such as liver and muscle. Mitochondrial uncoupling, which reduces cellular energy efficiency and increases lipid oxidation, is an appealing therapeutic strategy. The challenge, however, is to discover safe mitochondrial uncouplers for practical use. Niclosamide is an anthelmintic drug approved by the US Food and Drug Administration that uncouples the mitochondria of parasitic worms. Here we show that niclosamide ethanolamine salt (NEN) uncouples mammalian mitochondria at upper nanomolar concentrations. Oral NEN increases energy expenditure and lipid metabolism in mice. It is also efficacious in preventing and treating hepatic steatosis and insulin resistance induced by a high-fat diet. Moreover, it improves glycemic control and delays disease progression in db/db mice. Given the well-documented safety profile of NEN, our study provides a potentially new and practical pharmacological approach for treating T2D.

The bioluminescent imaging of spontaneously occurring tumors in immunocompetent ODD-luciferase bearing transgenic mice.

The imaging of spontaneously occurring tumors in mice poses many technical and logistical problems. Recently a mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase was ubiquitously expressed in all tissues. Hypoxic stress leads to the accumulation of ODD-luciferase in the tissues of this mouse model which can be identified by noninvasive bioluminescence measurement. Crossing this transgenic mouse with tumorigenic mice yields solid tumors with hypoxic cores that may be successfully imaged and characterized using the technique described herein.

Autophagy in adipose tissue biology.

Obesity, which predisposes individuals to type II diabetes and cardiovascular diseases, results from accumulation of white adipose tissue (WAT). WAT comprises mainly white adipocytes that have a unique cellular structure in which almost the entire intracellular space is occupied by one single lipid droplet. The cytoplasm envelopes this lipid droplet and occupies negligible space. Differentiation of WAT, or adipogenesis, requires dramatic cytoplasmic reorganization, including a dynamic change in mitochondrial mass. Autophagy is a major cytoplasmic degradation pathway and a primary pathway for mitochondrial degradation. Recent studies indicate that autophagy is implicated in adipogenesis. In this review, we summarize our current knowledge on autophagy in adipose tissue biology, with the emphasis on its role in mitochondrial degradation. Adipose tissue is a central component for whole-body energy homeostasis regulation. Advancement in this research area may provide novel venues for the intervention of obesity and obesity related diseases.

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation.

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP(7) and IP(8); and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP(4) and IP(5). We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.

Mitochondrial complex III: Tuner of autophagy.

Using chemical approaches, Ma et al. in this issue of Chemistry & Biology identify mitochondrial complex III as a specific positive regulator of autophagy. This study brings us a step closer to understanding the mechanism by which basal autophagy is coupled to cellular energy flux.

Vaccinia virus leads to ATG12­ATG3 conjugation and deficiency in autophagosome formation.

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12­ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Use of the ODD-luciferase transgene for the non-invasive imaging of spontaneous tumors in mice.

BACKGROUND: In humans, imaging of tumors provides rapid, accurate assessment of tumor growth and location. In laboratory animals, however, the imaging of spontaneously occurring tumors continues to pose many technical and logistical problems. Recently a mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase was ubiquitously expressed in all tissues. Hypoxic stress leads to the accumulation of ODD-luciferase in the tissues of this mouse model which can be identified by non-invasive bioluminescence measurement. Since solid tumors often contain hypoxic regions, we performed proof-of-principle experiments testing whether this transgenic mouse model may be used as a universal platform for non-invasive imaging analysis of spontaneous solid tumors. METHODS AND MATERIALS: ODD-luciferase transgenic mice were bred with MMTV-neu/beclin1+/- mice. Upon injection of luciferin, bioluminescent background of normal tissues in the transgenic mice and bioluminescent signals from spontaneously mammary carcinomas were measured non-invasively with an IVIS Spectrum imaging station. Tumor volumes were measured manually and the histology of tumor tissues was analyzed. CONCLUSION: Our results show that spontaneous mammary tumors in ODD-luciferase transgenic mice generate substantial bioluminescent signals, which are clearly discernable from background tissue luminescence. Moreover, we demonstrate a strong quantitative correlation between the bioluminescent tumor contour and the volume of palpable tumors. We further demonstrate that shrinkage of the volume of spontaneous tumors in response to chemotherapeutic treatment can be determined quantitatively using this system. Finally, we show that the growth and development of spontaneous tumors can be monitored longitudinally over several weeks. Thus, our results suggest that this model could potentially provide a practical, reliable, and cost-effective non-invasive quantitative method for imaging spontaneous solid tumors in mice.

Autophagic degradation of mitochondria in white adipose tissue differentiation.

Recent work has revealed that autophagy plays a significant role in the process of white adipocyte differentiation. In both in vitro and in vivo model systems, autophagy inactivation by targeted deletion of essential autophagy genes results in alterations in white adipocyte structure. In both models, postdifferentiation cells exhibit atypical morphology, with many small lipid droplets and large numbers of mitochondria, rather than the single large lipid droplet and relatively few mitochondria observed in normal white adipocytes. The role of autophagy as the primary means of the degradation of mitochondria has long been studied, and it is likely that a deficiency in the degradation of mitochondria contributes to the unusual phenotypes observed in mice with autophagy-deficient adipose tissue, including reduced adiposity, resistance to diet-induced obesity, and increased insulin sensitivity. What is not yet known is whether the process of mitochondria-specific autophagy, often referred to as "mitophagy," is specifically induced during adipogenesis or if a general increase in the nonspecific autophagic degradation of mitochondria plays a role in normal adipose differentiation. Despite remaining questions, these findings not only establish the critical role of autophagy in white adipose tissue development, but also suggest that the manipulation of autophagy in adipose tissue may provide novel therapeutic opportunities for metabolic diseases.

Autophagy and adipogenesis: implications in obesity and type II diabetes.

Obesity is a direct result of the accumulation of white adipose tissue (WAT). In this study, the role of autophagy in the differentiation of white adipose tissue was studied by deleting the autophagy-related 7 (atg7) gene from adipose tissue in mice. This deletion results in a striking phenotype at the cellular, tissue and whole-organism levels. Adipose tissue deposits in the mutant mice are much smaller in mass than those observed in their wild-type counterparts, and mutant adipocytes exhibit unusual morphological characteristics including multilocular lipid droplets and greatly increased numbers of mitochondria. The knockout mice are noticeably slimmer than their wild-type littermates, despite parity in food and water consumption. The mutant mice also exhibit higher basal physical activity levels and an array of metabolic changes revealed through blood tests. Most importantly, these mice show resistance to high-fat diet-induced obesity and markedly increased sensitivity to insulin. These findings establish a new function for autophagy and provide a new model system for use in the search for treatments for obesity and type II diabetes.

Autophagy and the degradation of mitochondria.

The cellular process of macromolecular degradation known as macroautophagy has long been known to play a role in the elimination of mitochondria. Over the past decade, much progress has been made in the development of systems by which the nature and mechanism of mitochondria degradation may be studied. Recent findings imply that the degradation of mitochondria via autophagy may be more specific and more tightly regulated than originally thought, and have led to designation of this specific type of autophagy as "mitophagy". In this review we provide a brief history of the development of mitophagy models and their associated discoveries.

Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis.

White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, approximately 50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of beta-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect.

Targeted deletion of autophagy-related 5 (atg5) impairs adipogenesis in a cellular model and in mice.

Mammalian white adipocytes have a unique structure in which nearly the entire cell volume is occupied by a single large lipid droplet, while the surrounding cytoplasm occupies minimal space. The massive cytoplasmic remodeling processes involved in the formation of this unique cellular structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation of cytoplasmic components. Here, we investigated the functional role of atg5, a gene encoding an essential protein required for autophagy, in adipocyte differentiation in a cellular model and in mice. Massive autophagy was activated when wild-type primary mouse embryonic fibroblasts (MEFs) were induced for adipocyte differentiation. Importantly, the autophagy deficient primary atg5(-/-) MEFs exhibited dramatically reduced efficiency in adipogenesis. Time-lapse microscopy revealed that atg5(-/-) MEFs initially appeared to differentiate normally; however, a majority of the differentiating atg5(-/-) cells ultimately failed to undergo further morphological transformation and eventually died, likely through apoptosis. Consistent with these in vitro results, histological analysis revealed that the atg5(-/-) late-stage embryos and neonatal pups had much less subcutaneous perilipin A-positive adipocytes. Consistently, when treated with chloroquine, a functional inhibitor of autophagy, wild-type MEFs exhibited drastically reduced efficiency of adipocyte differentiation. Taken together, these findings demonstrated that Atg5 is involved in normal adipocyte differentiation, suggesting an important role of autophagy in adipogenesis.