Targeting amino acid metabolism in cancer.

Metabolic rewiring is a characteristic hallmark of cancer cells. This phenomenon sustains uncontrolled proliferation and resistance to apoptosis by increasing nutrients and energy supply. However, reprogramming comes together with vulnerabilities that can be used against tumor and can be applied in targeted therapy. In the last years, the genetic background of tumors has been identified thoroughly and new therapies targeting those mutations tested. Nevertheless, we propose that targeting the phenotype of cancer cells could be another way of treatment aiming to avoid drug resistance and non-responsiveness of cancer patients. Amino acid metabolism is part of the altered processes in cancer cells. Amino acids are building blocks and also sensors of signaling pathways regulating main biological processes. In this comprehensive review, we described four amino acids (asparagine, arginine, methionine, and cysteine) which have been actively investigated as potential targets for anti-tumor therapy. Asparagine depletion is successfully used for decades in the treatment of acute lymphoblastic leukemia and there is a strong implication to apply it to other types of tumors. Arginine auxotrophic tumors are great candidates for arginine-starvation therapy. Higher requirement for essential amino acids such as methionine and cysteine point out promising targetable weaknesses of cancer cells.

PTEN/PI3K/Akt pathway alters sensitivity of T-cell acute lymphoblastic leukemia to L-asparaginase.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment. L-asparaginase (ASNase) is a key therapy component in pediatric T-ALL and lower sensitivity of leukemia cells to this drug negatively influences overall treatment efficacy and outcome. PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

Loss of hexokinase 1 sensitizes ovarian cancer to high-dose metformin.

BACKGROUND: Hexokinases (HKs) are well-studied enzymes catalyzing the first step of glycolysis. However, non-canonical regulatory roles of HKs are still incompletely understood. Here, we hypothesized that HKs comprise one of the missing links between high-dose metformin and the inhibition of the respiratory chain in cancer. METHODS: We tested the isoenzyme-specific regulatory roles of HKs in ovarian cancer cells by examining the effects of the deletions of HK1 and HK2 in TOV-112D ovarian adenocarcinoma cells. We reverted these effects by re-introducing wild-type HK1 and HK2, and we compared the HK1 revertant with the knock-in of catalytically dead HK1 p.D656A. We subjected these cells to a battery of metabolic and proliferation assays and targeted GC×GC-MS metabolomics. RESULTS: We found that the HK1 depletion (but not the HK2 depletion) sensitized ovarian cancer cells to high-dose metformin during glucose starvation. We confirmed that this newly uncovered role of HK1 is glycolysis-independent by the introduction of the catalytically dead HK1. The expression of catalytically dead HK1 stimulated similar changes in levels of TCA intermediates, aspartate and cysteine, and in glutamate as were induced by the HK2 deletion. In contrast, HK1 deletion increased the levels of branched amino acids; this effect was completely eliminated by the expression of catalytically dead HK1. Furthermore, HK1 revertants but not HK2 revertants caused a strong increase of NADPH/NADP ratios independently on the presence of glucose or metformin. The HK1 deletion (but not HK2 deletion) suppressed the growth of xenotransplanted ovarian cancer cells and nearly abolished the tumor growth when the mice were fed the glucose-free diet. CONCLUSIONS: We provided the evidence that HK1 is involved in the so far unknown glycolysis-independent HK1-metformin axis and influences metabolism even in glucose-free conditions.

Metabolic profile of leukemia cells influences treatment efficacy of L-asparaginase.

BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.

Assessment of the Metabolic Profile of Primary Leukemia Cells.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Altered Metabolism of Leukemic Cells: New Therapeutic Opportunity.

The cancer metabolic program alters bioenergetic processes to meet the higher demands of tumor cells for biomass production, nucleotide synthesis, and NADPH-balancing redox homeostasis. It is widely accepted that cancer cells mostly utilize glycolysis, as opposed to normal cells, in which oxidative phosphorylation is the most employed bioenergetic process. Still, studies examining cancer metabolism had been overlooked for many decades, and it was only recently discovered that metabolic alterations affect both the oncogenic potential and therapeutic response. Since most of the published works concern solid tumors, in this comprehensive review, we aim to summarize knowledge about the metabolism of leukemia cells. Leukemia is a malignant disease that ranks first and fifth in cancer-related deaths in children and adults, respectively. Current treatment has reached its limits due to toxicity, and there has been a need for new therapeutic approaches. One of the possible scenarios is improved use of established drugs and another is to introduce new druggable targets. Herein, we aim to describe the complexity of leukemia metabolism and highlight cellular processes that could be targeted therapeutically and enhance the effectiveness of current treatments.

Development of engineered yeast for biosorption of beer haze-active polyphenols.

Compared to most other alcoholic beverages, the shelf life of beer is much more limited due to its instability in the bottle. That instability is most likely to appear as turbidity (haze), even sedimentation, during storage. The haze in beer is mostly caused by colloidal particles formed by interactions between proteins and polyphenols within the beer. Therefore, beers are usually stabilized by removing at least one of these components. We developed and constructed a Saccharomyces cerevisiae strain with a proline-rich QPF peptide attached to the cell wall, using the C-terminal anchoring domain of α-agglutinin. The QPF peptide served to bind polyphenols during fermentation and, thus, to decrease their concentration. Strains displaying QPF were able to bind about twice as much catechin and epicatechin as a control strain displaying only the anchoring domain. All these experiments were done with model solutions. Depending on the concentration of yeast, uptake of polyphenols was 1.7-2.5 times higher. Similarly, the uptake of proanthocyanidins was increased by about 20 %. Since the modification of yeasts with QPF did not affect their fermentation performance under laboratory conditions, the display of QPF appears to be an approach to increase the stability of beer.

Surface-engineered Saccharomyces cerevisiae displaying α-acetolactate decarboxylase from Acetobacter aceti ssp xylinum.

OBJECTIVES: To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour. RESULTS: We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation. CONCLUSIONS: Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

Accumulation of Ag and Cu in Amanita strobiliformis and characterization of its Cu and Ag uptake transporter genes AsCTR2 and AsCTR3.

Macrofungi can accumulate in their sporocarps remarkably high concentrations of Cu and Ag. We have previously demonstrated that the non-essential Ag is in the ectomycorrhizal, Ag-hyperaccumulating Amanita strobiliformis sequestered by 3.4-kDa metallothioneins (MTs) produced as AsMT1a, 1b and 1c isoforms. Here, we describe two populations of wild-grown A. strobiliformis sporocarps, which showed certain correlation between the concentrations of accumulated Ag (284 ± 64 and 67 ± 15 mg kg(-1)) and Cu (76 ± 13 and 30 ± 12 mg kg(-1)), suggesting that an overlap may exist in the cell biology of Ag and Cu in this species. Metal speciation analysis revealed that the intracellular Cu in the sporocarps of both populations was, like Ag, associated with the 3.4-kDa MTs. A search of A. strobiliformis transcriptome for sequences encoding proteins of the Cu transporter (CTR) family identified four AsCTR cDNAs, which were, like AsMT1s, confirmed in both populations. The predicted AsCTR proteins showed homology to vacuolar (AsCTR1 and AsCTR4) and plasma membrane (AsCTR2 and AsCTR3) CTRs. Heterologous expression of AsCTR2, AsCTR3 and their translational fusions with green fluorescent protein (GFP) in Cu uptake-deficient S. cerevisiae indicated that both AsCTRs are functional Cu and Ag uptake transporters: recombinant genes complemented growth defects and increased Cu and Ag uptake rates in yeasts and the GFP-tagged protein localized to the cell periphery. Site directed mutagenesis revealed the importance of the conserved-among-CTRs M-X3-M motif for the AsCTR2- and AsCTR3-mediated transport of both Cu and Ag. These results provide the first evidence that fungal CTRs can recognize Ag for transport.

Characterization of three distinct metallothionein genes of the Ag-hyperaccumulating ectomycorrhizal fungus Amanita strobiliformis.

Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to AsMT1a, 1b, and 1c isogenes, the fungus has two other MT genes: AsMT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; AsMT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of AsMT1s and AsMT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; AsMT2 showed a moderate upregulation in the presence of Cd. Expression of AsMTs in the metal-sensitive Saccharomyces cerevisiae revealed that all AsMTs confer increased Cd tolerance (AsMT3 proved the most effective) and that, unlike AsMT1 and AsMT2, AsMT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing AsMT1a. Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (AsMT1s) and Zn (AsMT3).

Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8.

The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters.