Organoids for Functional Precision Medicine in Advanced Pancreatic Cancer.

BACKGROUND & AIMS: Patient-derived organoids (PDOs) are promising tumor avatars that could enable ex vivo drug tests to personalize patients' treatments in the frame of functional precision oncology. However, clinical evidence remains scarce. This study aims to evaluate whether PDOs can be implemented in clinical practice to benefit patients with advanced refractory pancreatic ductal adenocarcinoma (PDAC). METHODS: During 2021 to 2022, 87 patients were prospectively enrolled in an institutional review board-approved protocol. Inclusion criteria were histologically confirmed PDAC with the tumor site accessible. A panel of 25 approved antitumor therapies (chemogram) was tested and compared to patient responses to assess PDO predictive values and map the drug sensitivity landscape in PDAC. RESULTS: Fifty-four PDOs were generated from 87 pretreated patients (take-on rate, 62%). The main PDO mutations were KRAS (96%), TP53 (88%), and CDKN2A/B (22%), with a 91% concordance rate with their tumor of origin. The mean turnaround time to chemogram was 6.8 weeks. In 91% of cases, ≥1 hit was identified (gemcitabine (n = 20 of 54), docetaxel (n = 18 of 54), and vinorelbine (n = 17 of 54), with a median of 3 hits/patient (range, 0-12). Our cohort included 34 evaluable patients with full clinical follow-up. We report a chemogram sensitivity of 83.3% and specificity of 92.9%. The overall response rate and progression-free survival were higher when patients received a hit treatment as compared to patients who received a nonhit drug (as part of routine management). Finally, we leveraged our PDO collection as a platform for drug validation and combo identification. We tested anti-KRASG12D (MRTX1133), alone or combined, and identified a specific synergy with anti-EGFR therapies in KRASG12D variants. CONCLUSIONS: We report the largest prospective study aiming at implementing PDO-based functional precision oncology and identify very robust predictive values in this clinical setting. In a clinically relevant turnaround time, we identify putative hits for 91% of patients, providing unexpected potential survival benefits in this very aggressive indication. Although this remains to be confirmed in interventional precision oncology trials, PDO collection already provides powerful opportunities for drugs and combinatorial treatment development.

Patient-derived organoids identify an apico-basolateral polarity switch associated with survival in colorectal cancer.

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFß and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

Iron Regulator Hepcidin Impairs Macrophage-Dependent Cardiac Repair After Injury.

BACKGROUND: Defective systemic and local iron metabolism correlates with cardiac disorders. Hepcidin, a master iron sensor, actively tunes iron trafficking. We hypothesized that hepcidin could play a key role to locally regulate cardiac homeostasis after acute myocardial infarction. METHODS: Cardiac repair was analyzed in mice harboring specific cardiomyocyte or myeloid cell deficiency of hepcidin and challenged with acute myocardial infarction. RESULTS: We found that the expression of hepcidin was elevated after acute myocardial infarction and the specific deletion of hepcidin in cardiomyocytes failed to improve cardiac repair and function. However, transplantation of bone marrow-derived cells from hepcidin-deficient mice ( Hamp-/-) or from mice with specific deletion of hepcidin in myeloid cells (LysMCRE/+/ Hampf/f) improved cardiac function. This effect was associated with a robust reduction in the infarct size and tissue fibrosis in addition to favoring cardiomyocyte renewal. Macrophages lacking hepcidin promoted cardiomyocyte proliferation in a prototypic model of apical resection-induced cardiac regeneration in neonatal mice. Interleukin (IL)-6 increased hepcidin levels in inflammatory macrophages. Hepcidin deficiency enhanced the number of CD45+/CD11b+/F4/80+/CD64+/MHCIILow/chemokine (C-C motif) receptor 2 (CCR2)+ inflammatory macrophages and fostered signal transducer and activator of transcription factor-3 (STAT3) phosphorylation, an instrumental step in the release of IL-4 and IL-13. The combined genetic suppression of hepcidin and IL-4/IL-13 in macrophages failed to improve cardiac function in both adult and neonatal injured hearts. CONCLUSIONS: Hepcidin refrains macrophage-induced cardiac repair and regeneration through modulation of IL-4/IL-13 pathways.

A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.

Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.

Serum Iron Protects from Renal Postischemic Injury.

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients (n=169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF-κB and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants.

Myeloid HIFs are dispensable for resolution of inflammation during skeletal muscle regeneration.

Besides their role in cellular responses to hypoxia, hypoxia-inducible factors (HIFs) are involved in innate immunity and also have anti-inflammatory (M2) functions, such as resolution of inflammation preceding healing. Whereas the first steps of the inflammatory response are associated with proinflammatory (M1) macrophages (MPs), resolution of inflammation is associated with anti-inflammatory MPs exhibiting an M2 phenotype. This M1 to M2 sequence is observed during postinjury muscle regeneration, which provides an excellent paradigm to study the resolution of sterile inflammation. In this study, using in vitro and in vivo approaches in murine models, we demonstrated that deletion of hif1a or hif2a in MPs has no impact on the acquisition of an M2 phenotype. Furthermore, using a multiscale methodological approach, we showed that muscles did not require macrophagic hif1a or hif2a to regenerate. These results indicate that macrophagic HIFs do not play a crucial role during skeletal muscle regeneration induced by sterile tissue damage.

Myeloid HIF-1 is protective in Helicobacter pylori-mediated gastritis.

Helicobacter pylori infection triggers chronic inflammation of the gastric mucosa that may progress to gastric cancer. The hypoxia-inducible factors (HIFs) are the central mediators of cellular adaptation to low oxygen levels (hypoxia), but they have emerged recently as major transcriptional regulators of immunity and inflammation. No studies have investigated whether H. pylori affects HIF signaling in immune cells and a potential role for HIF in H. pylori-mediated gastritis. HIF-1 and HIF-2 expression was examined in human H. pylori-positive gastritis biopsies. Subsequent experiments were performed in naive and polarized bone marrow-derived macrophages from wild-type (WT) and myeloid HIF-1α-null mice (HIF-1(Δmyel)). WT and HIF-1(Δmyel) mice were inoculated with H. pylori by oral gavage and sacrificed 6 mo postinfection. HIF-1 was specifically expressed in macrophages of human H. pylori-positive gastritis biopsies. Macrophage HIF-1 strongly contributed to the induction of proinflammatory genes (IL-6, IL-1ß) and inducible NO synthase in response to H. pylori. HIF-2 expression and markers of M2 macrophage differentiation were decreased in response to H. pylori. HIF-1(Δmyel) mice inoculated with H. pylori for 6 mo presented with a similar bacterial colonization than WT mice but, surprisingly, a global increase of inflammation, leading to a worsening of the gastritis, measured by an increased epithelial cell proliferation. In conclusion, myeloid HIF-1 is protective in H. pylori-mediated gastritis, pointing to the complex counterbalancing roles of innate immune and inflammatory phenotypes in driving this pathology.

Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype.

Hepcidin is a 25-amino-acid peptide demonstrated to be the iron regulatory hormone capable of blocking iron absorption from the duodenum and iron release from macrophages. Mutations affecting hepcidin regulators or the hepcidin gene itself cause hemochromatosis, a common genetic disorder. Hepcidin is produced mainly by the liver, but many cells and tissues express low levels of the hormone. To determine the contribution of these hepcidin-producing tissues in body iron homeostasis, we have developed a new mouse model in which the hepcidin gene can be conditionally inactivated. Here we compare a liver-specific knockout (KO) mouse model with total KO mice. We show that the liver-specific KO mice fully recapitulate the severe iron overload phenotype observed in the total KO mice, with increased plasma iron and massive parenchymal iron accumulation. This result demonstrates that the hepatocyte constitutes the predominant reservoir for systemic hepcidin and that the other tissues are unable to compensate.

CXCR4 is highly expressed at the tumor front but not in the center of prostate cancers.

OBJECTIVE: To evaluate the expression of CXCR4, its ligand SDF-1, ß-catenin and E-cadherin throughout the local tumor microenvironment of prostate cancer. PATIENTS AND METHODS: A total of 64 prostate cancer specimens, 24 frozen and 40 paraffin-embedded sections, were obtained from patients treated with radical prostatectomy for clinically localized cancer. Real-time RT-PCR was used for mRNA quantification of CXCR4 and SDF-1 in the tumor center (T), tumor front (F) and distant peritumoral tissue (D). Immunohistochemical analysis was used to investigate the expression patterns of CXCR4, E-cadherin and ß-catenin. Clinical records of these patients were studied for follow-up data, and the prognostic value of these molecules' expression was statistically assessed. RESULTS: CXCR4 mRNA and protein were significantly increased at the tumor front as compared to distant tissue or tumor center. In comparison, SDF-1 mRNA level gradually increased from the tumor center to the distant peritumoral tissue. High CXCR4 at the tumor front was associated with high Gleason score. Low SDF-1 at the tumor front was associated with locally advanced cancer and disease recurrence. Moreover, high CXCR4 staining at the tumor front and increased cytosolic E-cadherin expression in the same location was associated with locally advanced disease. CONCLUSIONS: CXCR4 seems overexpressed at the tumor front of prostate tumors, where it potentially promotes cell migration toward the SDF-1 centrifugal attracting gradient, as well as epithelial-mesenchymal transition. High CXCR4 and low SDF-1 levels at tumor front were both associated with adverse histological features.

KRAS, a New Target for Precision Medicine in Colorectal Cancer?

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, with significant public health concerns. This review examines the landscape of KRAS inhibition in colorectal cancer (CRC), focusing on recent advances in therapeutic strategies targeting this oncogene. Historically deemed undruggable due to its complex structure and essential role in tumorigenesis, KRAS mutations are prevalent in CRC and are associated with poor prognosis. However, breakthroughs in drug development have led to the emergence of KRAS inhibitors as promising treatment options. This review discusses various classes of KRAS inhibitors, including covalent and non-covalent inhibitors, as well as combination therapies aimed at enhancing efficacy and overcoming resistance mechanisms. It highlights recent clinical trials evaluating the efficacy of KRAS inhibitors either as monotherapy or in combination with other agents, such as anti-EGFR antibodies. Despite challenges such as resistance mechanisms and tumor heterogeneity, the development of KRAS inhibitors represents a significant advance in CRC treatment and holds promise for improving patient outcomes in the future.

Precision medicine for KRAS wild-type pancreatic adenocarcinomas.

BACKGROUND: KRAS mutation is the most common molecular alteration in pancreatic adenocarcinoma (PDAC), and around 10% of patients harbor KRAS wild-type tumors (KRASWT). METHODS: A retrospective chart review of clinical/molecular data was performed including all PDAC patients with a determined KRAS status (tumor molecular profiling on tissue or liquid biopsy). RESULTS: 342 patients were included with 54 KRASWT PDAC (16%) compared to 288 patients with KRASm PDAC. Median age was 61 years [IQR:54.0;67.0] and 164 pts (48%) were female. At diagnosis, KRASWT patients (63%) were more frequently diagnosed at a non-metastatic stage compared to KRASm patients (41%) (p = 0.003). Regarding metastatic sites, liver was less frequent in KRASWT (39%, p < 0.0001). Median overall survival (mOS) from initial diagnosis was significantly higher in the KRASWT group compared to KRASm (50.8 months, CI95% [32.0-NR] vs 21.1 months, CI95% [18.9-23.4] (p < 0.004 after adjustment on age, ECOG and stage at diagnosis). In first-line systemic treatment, (mostly FOLFIRINOX) progression-free survival (PFS) was also higher in KRASWT. Based on ESCAT classification, a putative actionable alteration (ESCAT I-III) was identified in 19 (36%) KRASWT pts and 46 (16%) KRASm patients (p < 0.0001) with more alterations in FGFR2, BRAF(V600E), NRTK and more MSI tumors. KRASWT harbored also fewer alterations in TP53, CDKN2A, and SMAD4. 12 KRASWT patients received a molecularly-matched treatment with clinical benefit and improved outcomes compared to KRASm patients. CONCLUSIONS: KRASWT patients display distinct disease characteristics and outcomes with prolonged overall survival. KRASWT patients also harbor more actionable molecular alterations, leading to higher survival rates after receiving molecularly matched treatments.

Identification of CAD as an androgen receptor interactant and an early marker of prostate tumor recurrence.

Markers of prostate tumor recurrence after radical prostatectomy are lacking and highly demanded. The androgen receptor (AR) is a nuclear receptor that plays a pivotal role in normal and cancerous prostate tissue. AR interacts with a number of proteins modulating its stability, localization, and activity. To test the hypothesis that an increased expression of AR partners might foster tumor development, we immunopurified AR partners in human tumors xenografted into mice. One of the identified AR partners was the multifunctional enzyme carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the 3 initial steps of pyrimidine biosynthesis. We combined experiments in C4-2, LNCaP, 22RV1, and PC3 human prostate cell lines and analysis of frozen radical prostatectomy samples to study the CAD-AR interaction. We show here that in prostate tumor cells, CAD fosters AR translocation into the nucleus and stimulates its transcriptional activity. Notably, in radical prostatectomy specimens, CAD expression was not correlated with proliferation markers, but a higher CAD mRNA level was associated with local tumor extension (P=0.049) and cancer relapse (P=0.017). These results demonstrate an unsuspected function for a key metabolic enzyme and identify CAD as a potential predictive marker of cancer relapse.

Primary Colorectal Tumor Displays Differential Genomic Expression Profiles Associated with Hepatic and Peritoneal Metastases.

BACKGROUND: Despite improvements in characterization of CRC heterogeneity, appropriate risk stratification tools are still lacking in clinical practice. This study aimed to elucidate the primary tumor transcriptomic signatures associated with distinct metastatic routes. METHODS: Primary tumor specimens obtained from CRC patients with either isolated LM (CRC-Liver) or PM (CRC-Peritoneum) were analyzed by transcriptomic mRNA sequencing, gene set enrichment analyses (GSEA) and immunohistochemistry. We further assessed the clinico-pathological associations and prognostic value of our signature in the COAD-TCGA independent cohort. RESULTS: We identified a significantly different distribution of Consensus Molecular Subtypes between CRC-Liver and CRC-peritoneum groups. A transcriptomic signature based on 61 genes discriminated between liver and peritoneal metastatic routes. GSEA showed a higher expression of immune response and epithelial invasion pathways in CRC-Peritoneum samples and activation of proliferation and metabolic pathways in CRC-Liver samples. The biological relevance of RNA-Seq results was validated by the immunohistochemical expression of three significantly differentially expressed genes (ACE2, CLDN18 and DUSP4) in our signature. In silico analysis of the COAD-TCGA showed that the CRC-Peritoneum signature was associated with negative prognostic factors and poor overall and disease-free survivals. CONCLUSIONS: CRC primary tumors spreading to the liver and peritoneum display significantly different transcriptomic profiles. The implementation of this signature in clinical practice could contribute to identify new therapeutic targets for stage IV CRC and to define individualized follow-up programs in stage II-III CRC.

Implementing patient derived organoids in functional precision medicine for patients with advanced colorectal cancer.

BACKGROUND: Patient Derived Organoids (PDOs) emerged as the best technology to develop ex vivo tumor avatars. Whether drug testing on PDOs to identify efficient therapies will bring clinical utility by improving patient survival remains unclear. To test this hypothesis in the frame of clinical trials, PDO technology faces three main challenges to be implemented in routine clinical practices: i) generating PDOs with a limited amount of tumor material; ii) testing a wide panel of anti-cancer drugs; and iii) obtaining results within a time frame compatible with patient disease management. We aimed to address these challenges in a prospective study in patients with colorectal cancer (CRC). METHODS: Fresh surgical or core needle biopsies were obtained from patients with CRC. PDOs were established and challenged with a panel of 25 FDA-approved anti-cancer drugs (chemotherapies and targeted therapies) to establish a scoring method ('chemogram') identifying in vitro responders. The results were analyzed at the scale of the cohort and individual patients when the follow-up data were available. RESULTS: A total of 25 PDOs were successfully established, harboring 94% concordance with the genomic profile of the tumor they were derived from. The take-on rate for PDOs derived from core needle biopsies was 61.5%. A chemogram was obtained with a 6-week median turnaround time (range, 4-10 weeks). At least one hit (mean 6.16) was identified for 92% of the PDOs. The number of hits was inversely correlated to disease metastatic dissemination and the number of lines of treatment the patient received. The chemograms were compared to clinical data obtained from 8 patients and proved to be predictive of their response with 75% sensitivity and specificity. CONCLUSIONS: We show that PDO-based drug tests can be achieved in the frame of routine clinical practice. The chemogram could provide clinicians with a decision-making tool to tailor patient treatment. Thus, PDO-based functional precision oncology should now be tested in interventional trials assessing its clinical utility for patients who do not harbor activable genomic alterations or have developed resistance to standard of care treatments.

Hepatic hypoxia-inducible factor-2 down-regulates hepcidin expression in mice through an erythropoietin-mediated increase in erythropoiesis.

BACKGROUND: Iron metabolism, regulated by the iron hormone hepcidin, and oxygen homeostasis, dependent on hypoxia-inducible factors, are strongly interconnected. We previously reported that in mice in which both liver hypoxia-inducible factors-1 and -2 are stabilized (the hepatocyte von Hippel-Lindau knockout mouse model), hepcidin expression was strongly repressed and we hypothesized that hypoxia-inducible factor-2 could be the major regulatory component contributing to the hepcidin down-regulation. DESIGN AND METHODS: We generated and analyzed hepatocyte-specific knockout mice harboring either hypoxia-inducible factor-2α deficiency (Hif2a knockout) or constitutive hypoxia-inducible factor-2α stabilization (Vhlh/Hif1a knockout) and ex vivo systems (primary hepatocyte cultures). Hif2a knockout mice were fed an iron-deficient diet for 2 months and Vhlh/Hif1a knockout mice were treated with neutralizing erythropoietin antibody. RESULTS: We demonstrated that hypoxia-inducible factor-2 is dispensable in hepcidin gene regulation in the context of an adaptive response to iron-deficiency anemia. However, its overexpression in the double Vhlh/Hif1a hepatocyte-specific knockout mice indirectly down-regulates hepcidin expression through increased erythropoiesis and erythropoietin production. Experiments in primary hepatocytes confirmed the non-autonomous role of hypoxia-inducible factor-2 in hepcidin regulation. CONCLUSIONS: While our results indicate that hypoxia-inducible factor-2 is not directly involved in hepcidin repression, they highlight the contribution of hepatic hypoxia-inducible factor-2 to the repression of hepcidin through erythropoietin-mediated increased erythropoiesis, a result of potential clinical interest.

Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing.

Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the importance and regulation of these changes remain unclear. We found that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the mouse intestine after experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified conventional dendritic cells (cDCs) as a source of hepcidin that is induced by microbial stimulation in mice, prominent in the inflamed intestine of humans, and essential for tissue repair. cDC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and consequently facilitated intestinal repair. Collectively, these results identify a pathway whereby cDC-derived hepcidin promotes mucosal healing in the intestine through means of nutritional immunity.

Pulmonary Iron Homeostasis in Hepcidin Knockout Mice.

Pulmonary iron excess is deleterious and contributes to a range of chronic and acute inflammatory diseases. Optimal lung iron concentration is maintained through dynamic regulation of iron transport and storage proteins. The iron-regulatory hormone hepcidin is also expressed in the lung. In order to better understand the interactions between iron-associated molecules and the hepcidin-ferroportin axis in lung iron balance, we examined lung physiology and inflammatory responses in two murine models of systemic iron-loading, either hepcidin knock-out (Hepc KO) or liver-specific hepcidin KO mice (Hepc KOliv), which do (Hepc KOliv) or do not (Hepc KO) express lung hepcidin. We have found that increased plasma iron in Hepc KO mice is associated with increased pulmonary iron levels, consistent with increased cellular iron uptake by pulmonary epithelial cells, together with an increase at the apical membrane of the cells of the iron exporter ferroportin, consistent with increased iron export in the alveoli. Subsequently, alveolar macrophages (AM) accumulate iron in a non-toxic form and this is associated with elevated production of ferritin. The accumulation of iron in the lung macrophages of hepcidin KO mice contrasts with splenic and hepatic macrophages which contain low iron levels as we have previously reported. Hepc KOliv mice with liver-specific hepcidin deficiency demonstrated same pulmonary iron overload profile as the Hepc KO mice, suggesting that pulmonary hepcidin is not critical in maintaining local iron homeostasis. In addition, the high iron load in the lung of Hepc KO mice does not appear to enhance acute lung inflammation or injury. Lastly, we have shown that intraperitoneal LPS injection is not associated with pulmonary hepcidin induction, despite high levels of inflammatory cytokines. However, intranasal LPS injection stimulates a hepcidin response, likely derived from AM, and alters pulmonary iron content in Hepc KO mice.

Iron regulatory protein 1 sustains mitochondrial iron loading and function in frataxin deficiency.

Mitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron regulatory protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool. Surprisingly, our data indicate that IRP1 activation sustains mitochondrial iron supply and function rather than driving detrimental iron overload. Mitochondrial iron accumulation is shown to depend on mitochondrial dysfunction and heme-dependent upregulation of the mitochondrial iron importer mitoferrin-2. Our results uncover an unexpected protective role of IRP1 in pathological conditions associated with altered Fe-S metabolism.

Copper deficiency leads to anemia, duodenal hypoxia, upregulation of HIF-2α and altered expression of iron absorption genes in mice.

Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter - Dmt1) and ferric reductase - Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.