Monocarboxylate transporter 1 blockade with AZD3965 inhibits lipid biosynthesis and increases tumour immune cell infiltration.

BACKGROUND: Monocarboxylate transporter 1 (MCT1) is a regulator of cell metabolism and a therapeutic target for cancer treatment. Understanding the changes in tumour function accompanying MCT1 inhibition will better characterise the anti-tumour effects of MCT1 inhibitors, potentially enabling the identification of pharmacodynamic biomarkers for the clinical development of these agents. METHODS: We assessed the impact of the MCT1 inhibitor AZD3965 on tumour metabolism and immune cell infiltration as key determinants of tumour biological function in the MCT1-dependent Raji B cell lymphoma model. RESULTS: Treatment of Raji xenograft-bearing severe combined immunodeficiency mice with AZD3965 led to inhibition of tumour growth paralleled with a decrease in tumour choline, as detected by non-invasive in vivo proton nuclear magnetic resonance spectroscopy. This effect was attributed to inhibition of phosphocholine de novo synthesis following decreased choline kinase α protein and messenger RNA expression that correlated with the AZD3965-induced build-up in intracellular lactate. These changes were concomitant with increased tumour immune cell infiltration involving dendritic and natural killer cells. CONCLUSIONS: Our data provide new insights into the metabolic and cellular changes that occur in the tumour microenvironment following MCT1 blockade, which may contribute to the anti-tumour activity of AZD3965 and could have potential as pharmacodynamic biomarkers of MCT1 inhibition.

Increased inflammatory lipid metabolism and anaplerotic mitochondrial activation follow acquired resistance to vemurafenib in BRAF-mutant melanoma cells.

BACKGROUND: BRAF inhibitors, such as vemurafenib, have shown efficacy in BRAF-mutant melanoma treatment but acquired-resistance invariably develops. Unveiling the potential vulnerabilities associated with vemurafenib resistance could provide rational strategies for combinatorial treatment. METHODS: This work investigates the metabolic characteristics and vulnerabilities of acquired resistance to vemurafenib in three generated BRAF-mutant human melanoma cell clones, analysing metabolic profiles, gene and protein expression in baseline and nutrient withdrawal conditions. Preclinical findings are correlated with gene expression analysis from publicly available clinical datasets. RESULTS: Two vemurafenib-resistant clones showed dependency on lipid metabolism and increased prostaglandin E2 synthesis and were more responsive to vemurafenib under EGFR inhibition, potentially implicating inflammatory lipid and EGFR signalling in ERK reactivation and vemurafenib resistance. The third resistant clone showed higher pyruvate-carboxylase (PC) activity indicating increased anaplerotic mitochondrial metabolism, concomitant with reduced GLUT-1, increased PC protein expression and survival advantage under nutrient-depleted conditions. Prostaglandin synthase (PTGES) expression was inversely correlated with melanoma patient survival. Increases in PC and PTGES gene expression were observed in some patients following progression on BRAF inhibitors. CONCLUSIONS: Altogether, our data highlight heterogeneity in metabolic adaptations during acquired resistance to vemurafenib in BRAF-mutant melanoma, potentially uncovering key clinically-relevant mechanisms for combinatorial therapeutic targeting.

Lentiviral Hematopoietic Stem Cell Gene Therapy Rescues Clinical Phenotypes in a Murine Model of Pompe Disease.

Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.

MCT1 Inhibitor AZD3965 Increases Mitochondrial Metabolism, Facilitating Combination Therapy and Noninvasive Magnetic Resonance Spectroscopy.

Monocarboxylate transporters (MCT) modulate tumor cell metabolism and offer promising therapeutic targets for cancer treatment. Understanding the impact of MCT blockade on tumor cell metabolism may help develop combination strategies or identify pharmacodynamic biomarkers to support the clinical development of MCT inhibitors now in clinical trials. In this study, we assessed the impact of the MCT1 inhibitor AZD3965 on cancer cell metabolism in vitro and in vivo Exposing human lymphoma and colon carcinoma cells to AZD3965 increased MCT4-dependent accumulation of intracellular lactate, inhibiting monocarboxylate influx and efflux. AZD3965 also increased the levels of TCA cycle-related metabolites and 13C-glucose mitochondrial metabolism, enhancing oxidative pyruvate dehydrogenase and anaplerotic pyruvate carboxylase fluxes. Increased mitochondrial metabolism was necessary to maintain cell survival under drug stress. These effects were counteracted by coadministration of the mitochondrial complex I inhibitor metformin and the mitochondrial pyruvate carrier inhibitor UK5099. Improved bioenergetics were confirmed in vivo after dosing with AZD3965 in mouse xenograft models of human lymphoma. Our results reveal new metabolic consequences of MCT1 inhibition that might be exploited for therapeutic and pharmacodynamic purposes. Cancer Res; 77(21); 5913-24. ©2017 AACR.

Detecting human melanoma cell re-differentiation following BRAF or heat shock protein 90 inhibition using photoacoustic and magnetic resonance imaging.

Targeted therapies specific to the BRAF-MEK-ERK signaling pathway have shown great promise in the treatment of malignant melanoma in the last few years, with these drugs now commonly used in clinic. Melanoma cells treated using these agents are known to exhibit increased levels of melanin pigment and tyrosinase activity. In this study we assessed the potential of non-invasive imaging approaches (photoacoustic imaging (PAI) and magnetic resonance imaging (MRI)) to detect melanin induction in SKMEL28 human melanoma cells, following inhibition of Hsp90 and BRAF signaling using 17-AAG and vemurafenib, respectively. We confirmed, using western blot and spectrophotometry, that Hsp90 or BRAF inhibitor-induced melanoma cell differentiation resulted in an upregulation of tyrosinase and melanin expression levels, in comparison to control cells. This post-treatment increase in cellular pigmentation induced a significant increase in PAI signals that are spectrally identifiable and shortening of the MRI relaxation times T 1 and [Formula: see text]. This proof-of-concept study demonstrates the potential of MRI and PAI for detecting the downstream cellular changes induced by Hsp90 and BRAF-MEK-targeted therapies in melanoma cells with potential significance for in vivo imaging.

The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate-Lactate Exchange in BRAF-Mutant Human Melanoma Cells.

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. 1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAFWT cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. Mol Cancer Ther; 15(12); 2987-99. ©2016 AACR.

Identifying division symmetry of mouse embryonic stem cells: negative impact of DNA methyltransferases on symmetric self-renewal.

Cell division is a process by which a mother cell divides into genetically identical sister cells, although sister cells often display considerable diversity. In this report, over 350 sister embryonic stem cells (ESCs) were isolated through a microdissection method, and then expression levels of 48 key genes were examined for each sister cell. Our system revealed considerable diversities between sister ESCs at both pluripotent and differentiated states, whereas the similarity between sister ESCs was significantly elevated in a 2i (MEK and GSK3b inhibitors) condition, which is believed to mimic the ground state of pluripotency. DNA methyltransferase 3a/3b were downregulated in 2i-grown ESCs, and the loss of DNA methyltransferases was sufficient to generate nearly identical sister cells. These results suggest that DNA methylation is a major cause of the diversity between sister cells at the pluripotent states, and thus demethylation per se plays an important role in promoting ESC's self-renewal.