Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus.

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

Mutation rates and adaptive variation among the clinically dominant clusters of Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is a multidrug-resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole-genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here, we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We performed phylogenetic inference using publicly available WGS from 483 Mab patient isolates. We implement a subsampling approach in combination with coalescent analysis to estimate the molecular clock rate along the long internal branches of the tree, indicating a faster long-term molecular clock rate compared to branches within phylogenetic clusters. We used ancestry simulation to predict the effects of clock rate variation on phylogenetic clustering and found that the degree of clustering in the observed phylogeny is more easily explained by a clock rate slowdown than by transmission. We also find that phylogenetic clusters are enriched in mutations affecting DNA repair machinery and report that clustered isolates have lower spontaneous mutation rates in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organism's mutation rate and that this manifests as phylogenetic clustering. These results challenge the model that phylogenetic clustering in Mab is explained by person-to-person transmission and inform our understanding of transmission inference in emerging, facultative pathogens.

Biotin-dependent cell envelope remodelling is required for Mycobacterium abscessus survival in lung infection.

Mycobacterium abscessus is an emerging pathogen causing lung infection predominantly in patients with underlying structural abnormalities or lung disease and is resistant to most frontline antibiotics. As the pathogenic mechanisms of M. abscessus in the context of the lung are not well-understood, we developed an infection model using air-liquid interface culture and performed a transposon mutagenesis and sequencing screen to identify genes differentially required for bacterial survival in the lung. Biotin cofactor synthesis was required for M. abscessus growth due to increased intracellular biotin demand, while pharmacological inhibition of biotin synthesis prevented bacterial proliferation. Biotin was required for fatty acid remodelling, which increased cell envelope fluidity and promoted M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodelling plays a critical role in pathogenic adaptation to the lung niche, suggesting that biotin synthesis and fatty acid metabolism might provide therapeutic targets for treatment of M. abscessus infection.

Methionine synthase is essential for cancer cell proliferation in physiological folate environments.

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.

Mycobacteriophages as Genomic Engineers and Anti-infective Weapons.

Mycobacterium abscessus (Mab) is an emerging pathogen that is highly tolerant to current antibiotic therapies, and the current standard of care has a high failure rate. Mycobacteriophages represent a promising alternative treatment that have the potential to kill Mab with few side effects. However, the repertoire of phages that infect Mab is limited, and little is understood about the determinants of phage susceptibility in mycobacteria. Two studies from the Hatfull group (R. M. Dedrick, B. E. Smith, R. A. Garlena, D. A. Russell, et al., mBio 12:e03431-20, 2021, https://doi.org/10.1128/mBio.03431-20, and R. M. Dedrick, H. G. Aull, D. Jacobs-Sera, R. A. Garlena, et al., mBio 12:e03441-20, 2021, https://doi.org/10.1128/mBio.03441-20) shed new light on the natural phage complement of Mab and provide some of the first insights into what factors might drive susceptibility to these phages. These studies not only lay the groundwork for therapeutic development of more effective phage therapy in Mab but also provide a foothold for studying how mobile elements such as phages and plasmids impact Mab biology and evolution.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Deoxycytidine Release from Pancreatic Stellate Cells Promotes Gemcitabine Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analogue gemcitabine is among the most effective therapies to treat PDAC, however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. Conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protected PDAC cells from gemcitabine toxicity. The protective effect of PSC-conditioned media was mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibited the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogues on cancer cells. These results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies. SIGNIFICANCE: This study provides important new insight into mechanisms that contribute to gemcitabine resistance in PDAC and suggests new avenues for improving gemcitabine efficacy.

Increased PHGDH expression promotes aberrant melanin accumulation.

BACKGROUND: Copy number gain of the D-3-phosphoglycerate dehydrogenase (PHGDH) gene, which encodes the first enzyme in serine biosynthesis, is found in some human cancers including a subset of melanomas. METHODS: In order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDHtetO allele that allows tissue-specific, doxycycline-inducible PHGDH expression, and we analyzed the phenotype of mice with a ubiquitous increase in PHGDH expression. RESULTS: Tissues and cells derived from PHGDHtetO mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDHtetO mice reveals the presence of melanin granules in early anagen hair follicles, despite the fact that melanin synthesis is closely coupled to the hair follicle cycle and does not normally begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The aberrant presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in early anagen skin. CONCLUSIONS: These data suggest increased PHGDH expression impacts normal melanocyte biology, but PHGDH expression alone is not sufficient to cause cancer.

Determinants of nutrient limitation in cancer.

Proliferation requires that cells accumulate sufficient biomass to grow and divide. Cancer cells within tumors must acquire a variety of nutrients, and tumor growth slows or stops if necessary metabolites are not obtained in sufficient quantities. Importantly, the metabolic demands of cancer cells can be different from those of untransformed cells, and nutrient accessibility in tumors is different than in many normal tissues. Thus, cancer cell survival and proliferation may be limited by different metabolic factors than those that are necessary to maintain noncancerous cells. Understanding the variables that dictate which nutrients are critical to sustain tumor growth may identify vulnerabilities that could be used to treat cancer. This review examines the various cell-autonomous, local, and systemic factors that determine which nutrients are limiting for tumor growth.

Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability.

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.

Increased Serine Synthesis Provides an Advantage for Tumors Arising in Tissues Where Serine Levels Are Limiting.

Tumors exhibit altered metabolism compared to normal tissues. Many cancers upregulate expression of serine synthesis pathway enzymes, and some tumors exhibit copy-number gain of the gene encoding the first enzyme in the pathway, phosphoglycerate dehydrogenase (PHGDH). However, whether increased serine synthesis promotes tumor growth and how serine synthesis benefits tumors is controversial. Here, we demonstrate that increased PHGDH expression promotes tumor progression in mouse models of melanoma and breast cancer, human tumor types that exhibit PHGDH copy-number gain. We measure circulating serine levels and find that PHGDH expression is necessary to support cell proliferation at lower physiological serine concentrations. Increased dietary serine or high PHGDH expression is sufficient to increase intracellular serine levels and support faster tumor growth. Together, these data suggest that physiological serine availability restrains tumor growth and argue that tumors arising in serine-limited environments acquire a fitness advantage by upregulating serine synthesis pathway enzymes.

Isolation and Quantification of Metabolite Levels in Murine Tumor Interstitial Fluid by LC/MS.

Cancer is a disease characterized by altered metabolism, and there has been renewed interest in understanding the metabolism of tumors. Even though nutrient availability is a critical determinant of tumor metabolism, there has been little systematic study of the nutrients directly available to cancer cells in the tumor microenvironment. Previous work characterizing the metabolites present in the tumor interstitial fluid has been restricted to the measurement of a small number of nutrients such as glucose and lactate in a limited number of samples. Here we adapt a centrifugation-based method of tumor interstitial fluid isolation readily applicable to a number of sample types and a mass spectrometry-based method for the absolute quantitation of many metabolites in interstitial fluid samples. In this method, tumor interstitial fluid (TIF) is analyzed by liquid chromatography-mass spectrometry (LC/MS) using both isotope dilution and external standard calibration to derive absolute concentrations of targeted metabolites present in interstitial fluid. The use of isotope dilution allows for accurate absolute quantitation of metabolites, as other methods of quantitation are inadequate for determining nutrient concentrations in biological fluids due to matrix effects that alter the apparent concentration of metabolites depending on the composition of the fluid in which they are contained. This method therefore can be applied to measure the absolute concentrations of many metabolites in interstitial fluid from diverse tumor types, as well as most other biological fluids, allowing for characterization of nutrient levels in the microenvironment of solid tumors.

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.

Serine Synthesis via PHGDH Is Essential for Heme Production in Endothelial Cells.

The role of phosphoglycerate dehydrogenase (PHGDH), a key enzyme of the serine synthesis pathway (SSP), in endothelial cells (ECs) remains poorly characterized. We report that mouse neonates with EC-specific PHGDH deficiency suffer lethal vascular defects within days of gene inactivation, due to reduced EC proliferation and survival. In addition to nucleotide synthesis impairment, PHGDH knockdown (PHGDHKD) caused oxidative stress, due not only to decreased glutathione and NADPH synthesis but also to mitochondrial dysfunction. Electron transport chain (ETC) enzyme activities were compromised upon PHGDHKD because of insufficient heme production due to cellular serine depletion, not observed in other cell types. As a result of heme depletion, elevated reactive oxygen species levels caused EC demise. Supplementation of hemin in PHGDHKD ECs restored ETC function and rescued the apoptosis and angiogenesis defects. These data argue that ECs die upon PHGDH inhibition, even without external serine deprivation, illustrating an unusual importance of serine synthesis for ECs.

When cancer needs what's non-essential.

The non-essential amino acids serine and glycine are critical for proliferative metabolism. A study in Nature now finds that dietary serine and glycine deprivation inhibits growth of some tumours. Whether this dietary intervention is effective depends on both the oncogenic context and tumour tissue of origin.

The importance of serine metabolism in cancer.

Serine metabolism is frequently dysregulated in cancers; however, the benefit that this confers to tumors remains controversial. In many cases, extracellular serine alone is sufficient to support cancer cell proliferation, whereas some cancer cells increase serine synthesis from glucose and require de novo serine synthesis even in the presence of abundant extracellular serine. Recent studies cast new light on the role of serine metabolism in cancer, suggesting that active serine synthesis might be required to facilitate amino acid transport, nucleotide synthesis, folate metabolism, and redox homeostasis in a manner that impacts cancer.