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.

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.

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.

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.