Iron Deprivation Research Articles

404.3: Investigating Iron Limitation in Tolerance and Regulatory T Cells’ Dynamic Iron Requirements

Introduction: Pharmacological immunosuppression after transplantation increases morbidity and mortality, and thus alternative immunotherapies require investigation. Regulatory T cells (Tregs) are essential for maintaining immune tolerance and are great candidates for cellular therapy; understanding and modulating their function is therefore of formidable value. Iron restriction has been shown to impair effector T and B cell responses to vaccines and infections, but its effect on Tregs is largely unknown. A mutation in the transferrin receptor gene Tfrc (TfrcY20H/Y20H) leads to combined immunodeficiency in patients, caused by poor iron import via this transferrin receptor, but its effect on Tregs has not been studied. Methods: Mice receiving fully mismatched skin grafts were injected with a hepcidin mimetic to lower serum iron, and Tregs from peripheral blood, spleens, and graft-draining or contralateral lymph nodes were phenotyped via flow cytometry. Treg phenotype was also assessed in a systemic infection model in malaria-infected TfrcY20H/Y20H mice. To study cell-intrinsic iron limitation in Tregs, RAG1- mice with a fully mismatched skin allograft were adoptively transferred with CD4+CD25- conventional T cells (Tconvs) alongside CD4+CD25+ Tregs from either wildtype mice or iron-impaired TfrcY20H/Y20H mice. Mixed wildtype and TfrcY20H/Y20H bone marrow chimeras were created for analysis of these mice’s immune composition and Treg phenotype, both at resting state and during skin transplant rejection. Cell-extrinsic and intrinsic hypoferremia in Tregs and Tconvs was also studied in vitro with activation, proliferation, migration, and suppression assays. Results: Hepcidin mimetic injection impaired alloresponses in T cells and prolonged graft rejection, but Tregs seemed to be more resistant to iron limitation than Tconvs as shown by Tconv:Treg ratio, and Tregs even showed increased CD25 expression after hepcidin treatment. Similarly, TfrcY20H/Y20H mice had impaired immune responses to malaria but Tregs were preferentially less impaired and better activated than other cells such as Tconvs. However, TfrcY20H/Y20H Tregs were unable to control allorejection when adoptively transferred, and migration assays with TfrcY20H/Y20H Tregs suggest that this was not due to impaired migration to the graft. Mixed bone marrow chimeras are still in progress, but offer a direct comparison of wildtype and TfrcY20H/Y20H Tregs within the exact same environment at steady state and after allogenic challenge. Conclusions: Tregs and Tconvs often differ in their metabolism or response to the same stimulus, and pathways that affect them differentially show great promise in tipping the immune balance toward tolerance or immune activation. These results on preferential Treg resistance to iron deprivation highlight iron as a candidate for manipulating the Treg:Tconv ratio, which could have tremendous therapeutic applications.

Aroylhydrazone Glycoconjugate Prochelators Exploit Glucose Transporter 1 (GLUT1) to Target Iron in Cancer Cells.

Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.

Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans’ morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis.

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

SRNA-controlled iron sparing response in Staphylococci.

Staphylococcus aureus, a human opportunist pathogen, adjusts its metabolism to cope with iron deprivation within the host. We investigated the potential role of small non-coding RNAs (sRNAs) in dictating this process. A single sRNA, named here IsrR, emerged from a competition assay with tagged-mutant libraries as being required during iron starvation. IsrR is iron-repressed and predicted to target mRNAs expressing iron-containing enzymes. Among them, we demonstrated that IsrR down-regulates the translation of mRNAs of enzymes that catalyze anaerobic nitrate respiration. The IsrR sequence reveals three single-stranded C-rich regions (CRRs). Mutational and structural analysis indicated a differential contribution of these CRRs according to targets. We also report that IsrR is required for full lethality of S. aureus in a mouse septicemia model, underscoring its role as a major contributor to the iron-sparing response for bacterial survival during infection. IsrR is conserved among staphylococci, but it is not ortholog to the proteobacterial sRNA RyhB, nor to other characterized sRNAs down-regulating mRNAs of iron-containing enzymes. Remarkably, these distinct sRNAs regulate common targets, illustrating that RNA-based regulation provides optimal evolutionary solutions to improve bacterial fitness when iron is scarce.

Iron Deficiency and Overload Modulate the Inflammatory Responses and Metabolism of Alveolar Macrophages.

Alveolar macrophages (AM) are critical to defense against respiratory pathogens. This study evaluated cellular iron imbalance to immunometabolism in endotoxin-polarized porcine AMs (PAMs). PAMs collected from five 6-week-old pigs were treated with a basal media, iron chelator, or ferric ammonium citrate to maintain iron replete or induce iron deficiency or overload, respectively. After 24 h treatment, PAMs were challenged with saline or lipopolysaccharide (LPS) for 6 h. Cells were analyzed for gene, protein, and untargeted metabolome. Cytokines were determined in culture media. Data were assessed using two-way ANOVA. Treatments successfully induced iron deficiency and overload. The mRNA of DMT1 and ZIP14 was increased up to 300-fold by LPS, but unaffected by iron. Surprisingly, both iron deprivation and overload attenuated LPS-induced inflammation, showing less TNFα production and lower mRNA of pro- and anti-inflammatory cytokines than iron-replete PAMs. Forty-eight metabolites were altered by either or both main effects. LPS enhanced the glycolysis and polyol pathways. Iron deprivation disrupted the TCA cycle. Iron overload increased intracellular cholesterol. Interestingly, iron deprivation augmented, whereas iron overload diminished, LPS-induced itaconic acid production, which has anti-microbial and anti-inflammatory properties. Therefore, iron-deficient PAMs may be more resistant to intracellular pathogens which use PAMs as a conduit for infection.

The interplay between nitrogenated allelochemicals, mineral nutrition and metabolic profile in barley roots

AimsThe alkaloid hordenine is one of the major allelochemicals involved in the allelopathic ability of barley (Hordeum vulgare L.), whose biosynthesis and accumulation is preferentially located in roots. Hordenine appears to have been unintentionally favored during domestication in modern and cultivated barley cultivars at the expense of another alkaloid, gramine. In this study, we assessed the content of hordenine and its two precursors, N-methyltyramine (NMT) and tyramine, in roots and root exudates of the modern spring barley cv. Solist, and particularly how they are affected due to nutrient deficiencies.MethodsWe monitored the three metabolites during the early phases of barley growth i.e., up to 8 days, applying HPLC time-course and both target and untargeted metabolomic approaches. Barley plants were grown either in full nutrient solutions or in specific nutrient shortage conditions (N, S, P and Fe).ResultsResults confirmed a strong decrease of the allelochemical accumulation (hordenine and the two precursors) in roots and in root exudates during both 24 h and 8 days time-course experiments. Yet, the overall tyramine content was approximately tenfold lower compared to the other two compounds. In addition, plants subjected to nitrogen (-N), sulfur (-S), phosphorus (-P) and iron (-Fe) deprivation showed nutrient-dependent accumulation of hordenine, N-methyltyramine and tyramine, as well as of other secondary metabolites. Indeed, the synthesis of hordenine and N-methyltyramine was trigged under nutrient deficiencies.ConclusionsIn conclusion, this study highlighted the impact of nutrient availability on the growth-dependent accumulation patterns of all the three compounds investigated in modern barley roots.

Iron Deficiency Increases Phosphorylation of SP1 to Upregulate SPNS2 Expression in Hepatocellular Carcinoma.

The sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2) promotes tumor progression by modulating tumor immunity and enhancing tumor cells migration and invasion. Previously we found that iron deficiency in hepatocellular carcinoma upregulated SPNS2 expression to increase tumor metastasis. The present study aimed to identify the underlying mechanism of SPNS2 upregulation. Since the mRNA of SPNS2 was significantly increased, we used a transcription factor activity microarray to find the transcription factor responsible for this. The results showed that iron deprivation in hepatoma cells increased the transcriptional activities of 14 transcription factors while only 2 were decreased. Among these, 3 transcription factors, HIF1α, SP1, and YY1, were predicted to bind with the transcription promoter region of SPNS2. But only HIF1α and SP1 transcriptional activities on SPNS2 were increased by iron deficiency, and the increase of SP1 transcriptional activity was stronger than HIF1α. The protein level of HIF1α was increased by iron deficiency, while SP1 was not changed at the protein level but the phosphorylation level was increased. The inhibitor of HIF1α, PX478, and the inhibitor of SP1, Mithramycin A, reversed the increased mRNA and protein expressions of SPNS2 by iron deficiency, with a more significant effect by Mithramycin A. These results provided a comprehensive view of changes in transcriptional activities by iron deficiency and identified that SP1 was the main regulator of iron deficiency-inducing SPNS2 expression in hepatoma cells.

Iron-Deficiency in Atopic Diseases: Innate Immune Priming by Allergens and Siderophores.

Although iron is one of the most abundant elements on earth, about a third of the world's population are affected by iron deficiency. Main drivers of iron deficiency are beside the chronic lack of dietary iron, a hampered uptake machinery as a result of immune activation. Macrophages are the principal cells distributing iron in the human body with their iron restriction skewing these cells to a more pro-inflammatory state. Consequently, iron deficiency has a pronounced impact on immune cells, favoring Th2-cell survival, immunoglobulin class switching and primes mast cells for degranulation. Iron deficiency during pregnancy increases the risk of atopic diseases in children, while both children and adults with allergy are more likely to have anemia. In contrast, an improved iron status seems to protect against allergy development. Here, the most important interconnections between iron metabolism and allergies, the effect of iron deprivation on distinct immune cell types, as well as the pathophysiology in atopic diseases are summarized. Although the main focus will be humans, we also compare them with innate defense and iron sequestration strategies of microbes, given, particularly, attention to catechol-siderophores. Similarly, the defense and nutritional strategies in plants with their inducible systemic acquired resistance by salicylic acid, which further leads to synthesis of flavonoids as well as pathogenesis-related proteins, will be elaborated as both are very important for understanding the etiology of allergic diseases. Many allergens, such as lipocalins and the pathogenesis-related proteins, are able to bind iron and either deprive or supply iron to immune cells. Thus, a locally induced iron deficiency will result in immune activation and allergic sensitization. However, the same proteins such as the whey protein beta-lactoglobulin can also transport this precious micronutrient to the host immune cells (holoBLG) and hinder their activation, promoting tolerance and protecting against allergy. Since 2019, several clinical trials have also been conducted in allergic subjects using holoBLG as a food for special medical purposes, leading to a reduction in the allergic symptom burden. Supplementation with nutrient-carrying lipocalin proteins can circumvent the mucosal block and nourish selectively immune cells, therefore representing a new dietary and causative approach to compensate for functional iron deficiency in allergy sufferers.

An iron-chelating sulfonamide identified from Drosophila-based screening for antipathogenic discovery

ABSTRACT We exploited bacterial infection assays using the fruit fly Drosophila melanogaster to identify anti-infective compounds that abrogate the pathological consequences in the infected hosts. Here, we demonstrated that a pyridine-3-N-sulfonylpiperidine derivative (4a) protects Drosophila from the acute infections caused by bacterial pathogens including Pseudomonas aeruginosa. 4a did not inhibit the growth of P. aeruginosa in vitro, but inhibited the production of secreted toxins such as pyocyanin and hydrogen cyanide, while enhancing the production of pyoverdine and pyochelin, indicative of iron deprivation. Based on its catechol moiety, 4a displayed iron-chelating activity in vitro toward both iron (II) and iron (III), more efficiently than the approved iron-chelating drugs such as deferoxamine and deferiprone, concomitant with more potent antibacterial efficacy in Drosophila infections and unique transcriptome profile. Taken together, these results delineate a Drosophila–based strategy to screen for antipathogenic compounds, which interfere with iron uptake crucial for bacterial virulence and survival in host tissues.

Identification of a Novel Inhibitor of TfR1 from Designed and Synthesized Muriceidine A Derivatives.

The transferrin receptor 1 (TfR1) plays a key role in cellular iron uptake through its interaction with iron-bound Tf. TfR1 is often reported to be overexpressed in malignant cells, and this increase may be associated with poor prognosis in different types of cancer, which makes it an attractive target for antitumor therapy. The marine natural product Muriceidine A is a potent anticancer agent reported in our previous work. In this study, we designed and synthesized a series of Muriceidine A derivatives and described the systematic investigation into their cytotoxic activities against four tumor cells. Most of the derivatives showed stronger antitumor activity and we found that the introduction of electron-donating groups at position C-2 of unsaturated piperidine was beneficial to anticancer activity and unsaturated piperidine was responsible for the antiproliferative activity. Among these compounds, 12b (methyl at position C-2 of unsaturated piperidine) exhibited the strongest cytotoxicity against MDA-MB-231 cells. Further pharmacological research showed that 12b bound to Transferrin receptor 1 (TfR1) directly caused iron deprivation and ROS imbalance along with the degradations of several oncoproteins, especially FGFR1, through the proteasome pathway; thus, inducing cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells. Our findings indicate that 12b is a promising lead compound targeting TfR1 for triple negative breast cancer.

Local iron deficiency in the substantia nigra directly contributes to hyperlocomotion phenotypes

Brain iron is precisely regulated, and disrupted brain iron homeostasis is implicated in neuropsychological disease. Mounting evidence connects the iron status of the substantia nigra (SN) with locomotion-related neural symptomatology. Researchers in this field have long speculated that iron deficiency in the SN directly causes the high-locomotion symptoms observed in neuropsychiatric disorders. However, no direct experimental evidence of a causal relationship has been presented. To explore the relationship between iron deficiency in the SN and locomotion-related phenotypes, we stereotaxically injected the well-documented iron chelator, deferiprone (DFP) into the SN of mice to induce regional brain iron deprivation and subsequently performed behavioral tests. Altered expression of iron metabolism-related molecules was detected in the brain regions with interventions, and behavioral changes were observed. Targeted iron chelation effectively decreased the local iron content of the SN. Among the brain regions examined, only DFP injected into the SN resulted in the hyperlocomotion phenotype. Upon SN iron chelation, transferrin receptor (Tfr) expression was found to be upregulated. Conversely, viral vector-mediated SN-Tfr knockdown was sufficient to induce SN iron deficiency and mimic the hyperlocomotion phenotype. All locomotion changes had a significant negative correlation with iron alteration in the SN. Furthermore, SN iron disturbance also contributed to poor sleep efficiency. Thus, SN iron deficiency directly contributed to triggering both hyperlocomotion and sleep disturbances. This study offers a promising research and therapeutic direction for iron-linked neuropsychiatric diseases.

Changes in mRNA stability play an important role in the adaptation of yeast cells to iron deprivation

Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions.

Differential role of prolyl hydroxylases on mitochondrial function of HL-1 cells in a model of iron deficiency

Iron deficiency is observed in nearly half of the heart failure patients whilst closely correlated with mitochondrial dysfunction. Besides the structural roles in mitochondria, iron is also the cofactor of the hypoxia inducible factor (Hif) degradating enzyme, prolylhydroxylase, thereby Hif accumulation and its related metabolic effects commonly involve in iron deficiency. In this study, we used atrium derived HL-1 cells to investigate the effects of iron depletion on mitochondrial function under in vitro conditions. We aimed to discriminate the Hif dependent effects of iron deprivation on mitochondrial function to reveal the mechanisms leading to cardiac failure. For this purpose, HL-1 cells were either directly incubated with the iron chelating agent deferoxamine (DFO) or with dimethyloxalylglycine (DMOG, inhibitor of prolylhydroxylase). Mitochondrial function was evaluated by measuring cellular ATP content and mitochondrial potential (Ψ). According to our results, 48 h of DFO incubation affected cell viability and ATP production through further mechanisms additional to Hif-1α accumulation. Unlike DMOG group, DFO incubation did not disturb mitochondrial function probably due to its low permeability. Whether or not, prolyl hydroxylase inhibition without iron depletion may negatively affect mitochondrial function through Hif dependent mechanisms.

Deferasirox combination with eltrombopag shows anti-myelodysplastic syndrome effects by enhancing iron deprivation–related apoptosis

Iron overload (IO) affected the survival of patients with myelodysplastic syndrome (MDS). Deferasirox (DFX) is widely used in patients with MDS for iron chelation therapy, but is not suitable for...

Insights into the Antibacterial Mechanism of Action of Chelating Agents by Selective Deprivation of Iron, Manganese, and Zinc.

ABSTRACTBacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese, and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of 11 chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. The following four distinct effects were uncovered: (i) no apparent alteration in metal composition, (ii) depletion of manganese alongside reductions in iron and zinc levels, (iii) reduced zinc levels with a modest reduction in manganese, and (iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution; however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation.IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli. The chelators were studied both individually and in pairs, with the majority producing synergistic effects in combinations that maximize antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

Pharmacological Reduction of Mitochondrial Iron in AML Triggers a BAX/BAK Dependent Non-Canonical Cell Death Synergistic with Venetoclax

Although the BCL2 inhibitor venetoclax have been transformative in the management of AML, therapeutic resistance and relapse are frequently observed. In light of the urgent need to uncover novel therapeutic options in AML, we sought to study the potential role of ironomycin (AM5), a recently described small molecule that induces cell death through the sequestration of lysosomal iron.To evaluate the effects of ironomycin in AML, we chose a diverse panel of AML cell lines. These data showed a potent and dose-dependent effect, on proliferation, cell cycle progression and survival at a nanomolar range. In contrast to venetoclax, the cell death induced by ironomycin did not result in potent caspase activation or PARP1 cleavage. Neither the caspase inhibitor Z-VAD-fmk nor the necroptosis inhibitor necrostatin-1 did prevent cell death. Consistent with previous observations, we found that ironomycin accumulates in the lysosomes of AML cells leading to a sequestration of iron in this organelle but inhibitors of canonical ferroptosis, including ferrostatin-1 and liproxstatin-1 failed to prevent the activity of ironomycin.To gain greater insight into the molecular mechanism of ironomycin in AML cells, we performed a genome-wide positive-selection resistance screen under ironomycin selection pressure and collected several samples for sequencing. We found nine genes whose knock out conferred resistance to the drug. Interestingly, these data implicated key components of mitochondrial metabolic pathways, including phosphoglycolate phosphatase (PGP), a central phosphatase involved in glycolysis and pentose phosphate pathway (PPP) regulation and Hexokinase 2 (HK2), the first enzyme of glycolysis.Mass-spectrometry metabolomics analyses highlighted that ironomycin treatment significantly reduced key components of the TCA cycle and consequently the reducing agent nicotinamide adenine dinucleotide (NADH) and increased the intracellular concentration of amino acids. These data were corroborated with RNAseq showing a mitochondrial stress response mediated through the Activating Transcription Factor 4 (ATF4) and its paralog Activating Transcription Factor 5 (ATF5). As mitochondria are major hubs of iron utilization for oxidative respiration, we used Mass-spectrometry to measure mitochondrial iron load. We observed a rapid and dose-dependent decrease in mitochondrial iron after treatment mirroring the iron sequestration into the lysosomes and inducing the mitochondrial dysfunction.We next examined the ultrastructural appearance of mitochondria after ironomycin using transmission electron microscopy and observed a dramatic alteration of the structural integrity of mitochondria resulting in abnormal cristae, matrix density changes and mitochondrial membrane blebbing. In cells lacking BAX and BAK, the two main effectors of mitochondrial membrane permeabilization, structural changes and cell death were almost completely rescued but cell proliferation was still markedly affected, consistent with a BAX/BAK dependent cell death following mitochondrial iron deprivation. In vivo imaging confirmed that BAX activation occurred after 30 hours of treatment and preceded cell death, but we observed some major differences with canonical apoptosis induced by venetoclax. First, the structural alterations were clearly distinct. Next, delay between MOMP and cell death was significantly longer and caspase inhibitors weakly delayed cell death. Finally, BCL2 overexpression and P53 deletion did not rescue ironomycin cell death.These non-canonical features prompted us to assess the efficacy of the combination between ironomycin and venetoclax. In vitro experiment on AML cell lines found a high synergy between the two drugs. In vivo experiments on xenotransplanted mice confirmed the efficacy of the combination, which was associated with a significant increase in mice survival in comparison with the controls (Figure). Finally, primary AML samples from patients clinically resistant or refractory to venetoclax were sensitive to ironomycin in monotherapy and even more in combination with venetoclax.These results demonstrate that the novel mechanism of ironomycin action can be leveraged to resensitize AML cells to venetoclax and substitute for cytotoxic drugs as a more effective therapeutic combination in the salvage setting. [Display omitted] DisclosuresHuang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Wei: Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Novartis, Janssen, Amgen, Roche, Pfizer, Abbvie, Servier, BMS, Macrogenics, Agios, Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria.

The Loss of Liver Kinase B1 Accelerates Maturation Time in the Developing Erythrocyte

In the US, iron-restricted anemia contributes greatly to morbidity. The erythroid iron deprivation response, characterized by a pathway in which erythropoiesis is suppressed during iron restriction, underlies this anemia. In preliminary studies, liver kinase B1 (LKB1) was implicated as a potential key component in the erythroid iron deprivation response. Normal human CD34+ hematopoietic progenitor cells cultured for 3 days in erythroid medium with 100% or 10% transferrin saturations underwent immunoblot of whole cell lysates. Reproducibly, the levels of LKB1 did not change based on the transferrin saturation; however, immunofluorescence imaging showed a shift in subcellular localization when cells were subjected to low iron conditions.To assess the effects of LKB1 loss in the erythroid compartment, we used control and LKB1 conditional knockout (STK11 F/F; EpoR-Cre+) mice. In steady-state conditions, the loss of LKB1 does not confer a change in RBC count, though, there is a baseline increase in the number of reticulocytes, and a large increase in the level of serum Erythropoietin (Epo). In a model to precipitate anemia, these mice were challenged with intraperitoneal injection of phenylhydrazine (PHZ). It was found that LKB1 is dispensable for an appropriate response to this type of stress erythropoiesis.To assess the impact of LKB1 on maturation, we performed flow cytometric analysis using an ex vivo culture system of splenic erythroblasts. LKB1-deficient erythroid progenitors show increased percentages of more advanced cells as evidenced by the surface markers CD71 and Ter119 (the mouse analogue of human glycophorin A). Current studies are underway to assess if this change is due to signaling through the AMPK pathway. These studies will provide mechanistic detail of LKB1 function and activity on erythropoiesis, improving our understanding of programs involved in maturation of differentiation and lineage selection which may ultimately help to improve health outcomes and advance treatment for various types of anemias. DisclosuresNo relevant conflicts of interest to declare.

Comparative proteomic profiling reveals specific adaption of Vibrio anguillarum to oxidative stress, iron deprivation and humoral components of innate immunity

The gram-negative bacterium Vibrio (Listonella) anguillarum (VA) is the causative agent of vibriosis, a terminal hemorrhagic septicemia affecting the aquacultural industry across the globe. In the current study we used label-free quantitative proteomics to investigate how VA adapts to conditions that mimic defined aspects of vibriosis-related stress such as exposure to oxidative stress (H2O2), exposure to humoral factors of innate immunity through incubation with Atlantic salmon serum, and iron deprivation upon supplementation of 2,2′-dipyridyl (DIP) to the growth medium. We also investigated how regulation of virulence factors may be governed by the VA growth phase and availability of nutrients. All experimental conditions explored revealed stress-specific proteomic adaption of VA and only nine proteins were found to be commonly regulated in all conditions. A general observation made for all stress-related conditions was regulation of multiple metabolic pathways. Notably, iron deprivation and exposure to Atlantic salmon serum evoked upregulation of iron acquisition mechanisms. The findings made in the present study represent a source of potential virulence determinants that can be of use in the search for means to understand vibriosis. SignificanceVibriosis in fish and shellfish caused by V. anguillarum (VA) is responsible for large economic losses in the aquaculture sector across the globe. However, not much is known about the defense mechanism of this pathogen to percept and adapt to the imposed stresses during infection. Analyzing the response of VA to multiple host-related physiochemical stresses, the quantitative proteomic analysis of the present study indicates modulation of several virulence determinants and key defense networks of this pathogen. Our findings provide a theoretical basis to enhance our understanding of VA pathogenesis and can be employed to improve current intervention strategies to control vibriosis in aquaculture.

Sterol Composition Modulates the Response of Saccharomyces cerevisiae to Iron Deficiency.

Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway.