Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

MCT4-dependent lactate secretion suppresses antitumor immunity in LKB1-deficient lung adenocarcinoma.

Inactivating STK11/LKB1 mutations are genomic drivers of primary resistance to immunotherapy in KRAS-mutated lung adenocarcinoma (LUAD), although the underlying mechanisms remain unelucidated. We find that LKB1 loss results in enhanced lactate production and secretion via the MCT4 transporter. Single-cell RNA profiling of murine models indicates that LKB1-deficient tumors have increased M2 macrophage polarization and hypofunctional T cells, effects that could be recapitulated by the addition of exogenous lactate and abrogated by MCT4 knockdown or therapeutic blockade of the lactate receptor GPR81 expressed on immune cells. Furthermore, MCT4 knockout reverses the resistance to PD-1 blockade induced by LKB1 loss in syngeneic murine models. Finally, tumors from STK11/LKB1 mutant LUAD patients demonstrate a similar phenotype of enhanced M2-macrophages polarization and hypofunctional T cells. These data provide evidence that lactate suppresses antitumor immunity and therapeutic targeting of this pathway is a promising strategy to reversing immunotherapy resistance in STK11/LKB1 mutant LUAD.

Effectors Enabling Adaptation to Mitochondrial Complex I Loss in Hürthle Cell Carcinoma.

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.

There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.

Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors.

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called "CRISPR-plus" (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.