SummarySince the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.

Highlights d Proline synthesis through PYCR1 is increased in low-oxygen conditions d PYCR1 activity in hypoxia supports TCA cycle function through NADH oxidation d PYCR1 is required for maintenance of hypoxic tumor regions

Mutations in succinate dehydrogenase (SDH) lead to the development of tumors in a restricted subset of cell types, including chromaffin cells and paraganglia.The molecular basis for this specificity is currently unknown. We show that loss of SDH activity in a chromaffin cell model does not perturb complex I function, retaining the ability to oxidize NADH within the electron transport chain. This activity supports continued oxidation of substrates within the tricarboxylic acid (TCA) cycle. However, due to the block in the TCA cycle at SDH, the high glutamine oxidation activity is only maintained through an efflux of succinate. We also show that although the mitochondria of SDH-deficient cells are less active per se, their higher mass per cell results in an overall respiratory rate that is comparable with wild-type cells. Finally, we observed that when their mitochondria are uncoupled, SDH-deficient cells are unable to preserve their viability, suggesting that the mitochondrial metabolic network is unable to compensate when exposed to additional stress. We therefore show that in contrast to models of SDH deficiency based on epithelial cells, a chromaffin cell model retains aspects of metabolic "health," which could form the basis of cell specificity of this rare tumor type. | KĽUČKOVÁ et al | MATERIALS AND METHODS | Cell culture and chemicalsPreviously characterized immortalized mouse chromaffin cell lines deficient in Sdhb (Sdhb −/− CL6 and CL8) as well K E Y W O R D S electron transport chain, metabolism, mitochondria, pheochromocytoma, succinate dehydrogenase

T cells demonstrate impaired function in Multiple Myeloma (MM), but suppressive mechanisms in the bone marrow microenvironment remain poorly defined. We observe that bone marrow CD8+ T-cell function is decreased in MM patients compared to controls, and also is consistently lower within bone marrow samples than matched peripheral blood. These changes are accompanied by decreased mitochondrial mass and markedly elevated long-chain fatty acid uptake. In vitro modelling confirmed that uptake of bone marrow lipids suppresses CD8+ T function, which is impaired in autologous bone marrow plasma, but rescued by lipid removal. Analysis of single-cell RNA-sequencing data identified expression of fatty acid transport protein 1 (FATP1) in bone marrow CD8+ T cells in MM, and FATP1 blockade also rescued CD8+ T-cell function, thereby identifying this as a novel target to augment T cell activity in MM. Finally, analysis of samples from treated patient cohorts identified that CD8+ T cell metabolic dysfunction resolves in treatment-responsive but not relapsed MM patients and is associated with substantial T cell functional restoration.

Background Multiple Myeloma (MM) is a plasma cell malignancy that develops in the bone marrow. Function of T lymphocytes is impaired in patients with MM and the bone marrow microenvironment is described as hostile for T cell activity. Precise suppressive mechanisms within the bone marrow microenvironment remain poorly defined but will impact efficacy of bispecific T cell engager and chimeric antigen receptor (CAR) T cell therapies. Methods In this study T cell phenotype, function and metabolic activity were analysed within paired bone marrow aspirate and peripheral blood samples from 72 patients across the spectrum of MM, including individuals with premalignant and asymptomatic disease, alongside age-matched controls. This permitted assessment of effects of disease stage and the bone marrow microenvironment. The bone marrow microenvironment was also modelled in vitro using autologous plasma co-culture systems. Results Bone marrow CD8+ T cell function decreased with MM development and was consistently lower within bone marrow samples than matched peripheral blood. These changes were accompanied by decreased mitochondrial mass, which correlated tightly with T cell function. Conversely, long-chain fatty acid uptake and peroxidation was markedly elevated in bone marrow CD8+ T cells. In vitro modelling confirmed uptake of bone marrow lipids suppresses CD8+ T function, which was impaired in autologous bone marrow plasma, but rescued by both lipid removal and inhibition of lipid peroxidation. Analysis of single-cell RNA-sequencing data identified expression of fatty acid transport protein 1 (FATP1) in bone marrow CD8+ T cells in MM, and FATP1 blockade also rescued CD8+ T cell function. Finally, analysis of samples from treated patient cohorts identified CD8+ T cell metabolic dysfunction resolves in treatment-responsive but not relapsed MM patients and is associated with substantial functional restoration. Conclusions CD8+ T cells are functionally impaired within the MM bone marrow microenvironment. This is accompanied by decreased mitochondrial mass but elevated uptake of long-chain fatty acids. Blockade of FATP1 restores CD8+ T cell function in presence of BM lipids and may therefore represent a novel therapeutic target to augment their activity in the bone marrow in MM and improve efficacy of T cell-directed therapies.