DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells.

We have recently identified the uncharacterized ZNF555 protein as a component of a productive complex involved in the morbid function of the 4qA locus in facioscapulohumeral dystrophy. Subsequently named DiPRO1 (Death, Differentiation, and PROliferation related PROtein 1), our study provides substantial evidence of its role in the differentiation and proliferation of human myoblasts. DiPRO1 operates through the regulatory binding regions of SIX1, a master regulator of myogenesis. Its relevance extends to mesenchymal tumors, such as rhabdomyosarcoma (RMS) and Ewing sarcoma, where DiPRO1 acts as a repressor via the epigenetic regulators TIF1B and UHRF1, maintaining methylation of cis-regulatory elements and gene promoters. Loss of DiPRO1 mimics the host defense response to virus, awakening retrotransposable repeats and the ZNF/KZFP gene family. This enables the eradication of cancer cells, reprogramming the cellular decision balance towards inflammation and/or apoptosis by controlling TNF-α via NF-kappaB signaling. Finally, our results highlight the vulnerability of mesenchymal cancer tumors to si/shDiPRO1-based nanomedicines, positioning DiPRO1 as a potential therapeutic target.

Immune system and intestinal microbiota determine efficacy of androgen deprivation therapy against prostate cancer.

BACKGROUND: Prostate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). We investigated a mouse model of PC as well as specimens from PC patients to unravel an unsuspected contribution of thymus-derived T lymphocytes and the intestinal microbiota in the efficacy of ADT. METHODS: Preclinical experiments were performed in PC-bearing mice, immunocompetent or immunodeficient. In parallel, we prospectively included 65 HSPC and CRPC patients (Oncobiotic trial) to analyze their feces and blood specimens. RESULTS: In PC-bearing mice, ADT increased thymic cellularity and output. PC implanted in T lymphocyte-depleted or athymic mice responded less efficiently to ADT than in immunocompetent mice. Moreover, depletion of the intestinal microbiota by oral antibiotics reduced the efficacy of ADT. PC reduced the relative abundance of Akkermansia muciniphila in the gut, and this effect was reversed by ADT. Moreover, cohousing of PC-bearing mice with tumor-free mice or oral gavage with Akkermansia improved the efficacy of ADT. This appears to be applicable to PC patients because long-term ADT resulted in an increase of thymic output, as demonstrated by an increase in circulating recent thymic emigrant cells (sjTRECs). Moreover, as compared with HSPC controls, CRPC patients demonstrated a shift in their intestinal microbiota that significantly correlated with sjTRECs. While feces from healthy volunteers restored ADT efficacy, feces from PC patients failed to do so. CONCLUSIONS: These findings suggest the potential clinical utility of reversing intestinal dysbiosis and repairing acquired immune defects in PC patients.

Cancer Induces a Stress Ileopathy Depending on ß-Adrenergic Receptors and Promoting Dysbiosis that Contributes to Carcinogenesis.

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of ß-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies. SIGNIFICANCE: Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a ß-adrenergic receptor-dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.-related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy. This article is highlighted in the In This Issue feature, p. 873.

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Ileal immune tonus is a prognosis marker of proximal colon cancer in mice and patients.

Ileal epithelial cell apoptosis and the local microbiota modulate the effects of oxaliplatin against proximal colon cancer by modulating tumor immunosurveillance. Here, we identified an ileal immune profile associated with the prognosis of colon cancer and responses to chemotherapy. The whole immune ileal transcriptome was upregulated in poor-prognosis patients with proximal colon cancer, while the colonic immunity of healthy and neoplastic areas was downregulated (except for the Th17 fingerprint) in such patients. Similar observations were made across experimental models of implanted and spontaneous murine colon cancer, showing a relationship between carcinogenesis and ileal inflammation. Conversely, oxaliplatin-based chemotherapy could restore a favorable, attenuated ileal immune fingerprint in responders. These results suggest that chemotherapy inversely shapes the immune profile of the ileum-tumor axis, influencing clinical outcome.

Autophagy-mediated metabolic effects of aspirin.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b-/- or Bcln1+/-) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.

Chemical activation of SAT1 corrects diet-induced metabolic syndrome.

The pharmacological targeting of polyamine metabolism is currently under the spotlight for its potential in the prevention and treatment of several age-associated disorders. Here, we report the finding that triethylenetetramine dihydrochloride (TETA), a copper-chelator agent that can be safely administered to patients for the long-term treatment of Wilson disease, exerts therapeutic benefits in animals challenged with hypercaloric dietary regimens. TETA reduced obesity induced by high-fat diet, excessive sucrose intake, or leptin deficiency, as it reduced glucose intolerance and hepatosteatosis, but induced autophagy. Mechanistically, these effects did not involve the depletion of copper from plasma or internal organs. Rather, the TETA effects relied on the activation of an energy-consuming polyamine catabolism, secondary to the stabilization of spermidine/spermine N1-acetyltransferase-1 (SAT1) by TETA, resulting in enhanced enzymatic activity of SAT. All the positive effects of TETA on high-fat diet-induced metabolic syndrome were lost in SAT1-deficient mice. Altogether, these results suggest novel health-promoting effects of TETA that might be taken advantage of for the prevention or treatment of obesity.

Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer.

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (TFH) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of TFH cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of TFH cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1+ TFH cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC.

Comedications influence immune infiltration and pathological response to neoadjuvant chemotherapy in breast cancer.

Immunosurveillance plays an important role in breast cancer (BC) prognosis and progression, and can be geared by immunogenic chemotherapy. In a cohort of 1023 BC patients treated with neoadjuvant chemotherapy (NAC), 40% of the individuals took comedications mostly linked to aging and comorbidities. We systematically analyzed the off-target effects of 1178 concurrent comedications (classified according to the Anatomical Therapeutic Chemical (ATC) Classification System) on the density of tumor-infiltrating lymphocytes (TILs) and pathological complete responses (pCR). At level 1 of the ATC system, the main anatomical classes of drugs were those targeting the nervous system (class N, 39.1%), cardiovascular disorders (class C, 26.6%), alimentary and metabolism (class A, 16.9%), or hormonal preparations (class H, 6.5%). At level 2, the most frequent therapeutic classes were psycholeptics (N05), analgesics (N02), and psychoanaleptics (N06). Pre-NAC TIL density in triple-negative BC (TNBC) was influenced by medications from class H, N, and A, while TIL density in HER2+ BC was associated with the use of class C. Psycholeptics (N05) and agents acting on the renin-angiotensin system (C09) were independently associated with pCR in the whole population of BC or TNBC, and in HER2-positive BC, respectively. Importantly, level 3 hypnotics (N05C) alone were able to reduce tumor growth in BC bearing mice and increased the anti-cancer activity of cyclophosphamide in a T cell-dependent manner. These findings prompt for further exploration of drugs interactions in cancer, and for prospective drug-repositioning strategies to improve the efficacy of NAC in BC.

Sustained Type I interferon signaling as a mechanism of resistance to PD-1 blockade.

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNß transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.

Transcriptional landscape of a RETC634Y-mutated iPSC and its CRISPR-corrected isogenic control reveals the putative role of EGR1 transcriptional program in the development of multiple endocrine neoplasia type 2A-associated cancers.

MEN2A is a hereditary cancer-predisposing syndrome that affects patients with germline RET mutations. The effects of this oncogenic tyrosine kinase in the context of primitive stem cells are not known. In order to study these events, we generated a MEN2A induced Pluripotent Stem Cell (iPSC) line from a patient with RET mutation and an isogenic counterpart by CRISPR-Cas9 correction of the mutation. Whole exome sequencing of iPSC before and after CRISPR-Cas9 genome edition revealed no major exonic off target effect of the CRISPR correction. However, an integrative differential gene expression analysis of iPSC with oncogenic RETC634Y and its gene-corrected iPSC with RETY634C as well as RETwt iPSCs revealed activation of the Early Growth Response 1 (EGR1) transcriptional program in RET-mutated iPSC, a pathway shown to be involved in RET-induced oncogenesis. These data constitute the first proof of concept of the feasibility of the use of an iPSC and its genome-corrected counterpart to unravel the molecular mechanisms underlying the development of the hereditary MEN2A cancer predisposing syndrome.

Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis induce sustained clinical responses in a sizable minority of cancer patients. We found that primary resistance to ICIs can be attributed to abnormal gut microbiome composition. Antibiotics inhibited the clinical benefit of ICIs in patients with advanced cancer. Fecal microbiota transplantation (FMT) from cancer patients who responded to ICIs into germ-free or antibiotic-treated mice ameliorated the antitumor effects of PD-1 blockade, whereas FMT from nonresponding patients failed to do so. Metagenomics of patient stool samples at diagnosis revealed correlations between clinical responses to ICIs and the relative abundance of Akkermansia muciniphila Oral supplementation with A. muciniphila after FMT with nonresponder feces restored the efficacy of PD-1 blockade in an interleukin-12-dependent manner by increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes into mouse tumor beds.

Combined therapy of colon carcinomas with an oncolytic adenovirus and valproic acid.

The anti-tumor potential of oncolytic adenoviruses (CRAds) has been demonstrated in preclinical and clinical studies. While these agents failed to eradicate tumors when used as a monotherapy, they may be more effective if combined with conventional treatments such as radiotherapy or chemotherapy. This study seeks to evaluate the combination of a CRAd bearing a ∆24 deletion in E1A with valproic acid (VPA), a histone deacetylase inhibitor, for the treatment of human colon carcinomas. This combination led to a strong inhibition of cell growth both in vitro and in vivo compared to treatment with CRAd or VPA alone. This effect did not stem from a better CRAd replication and production in the presence of VPA. Inhibition of cell proliferation and cell death were induced by the combined treatment. Moreover, whereas cells treated only with CRAd displayed a polyploidy (> 4N population), this phenotype was increased in cells treated with both CRAd and VPA. In addition, the increase in polyploidy triggered by combined treatment with CRAd and VPA was associated with the enhancement of H2AX phosphorylation (γH2AX), a hallmark of DNA damage, but also with a decrease of several DNA repair proteins. Finally, viral replication (or E1A expression) was shown to play a key role in the observed effects since no enhancement of polyploidy nor increase in γH2AX were found following cell treatment with a replication-deficient Ad and VPA. Taken together, our results suggest that CRAd and VPA could be used in combination for the treatment of colon carcinomas.

Molecular Imaging to Predict Response to Targeted Therapies in Renal Cell Carcinoma.

Molecular magnetic resonance imaging targeted to an endothelial integrin involved in neoangiogenesis was compared to DCE-US and immunochemistry to assess the early response of three different therapeutic agents in renal cell carcinoma. Human A498 renal cells carcinoma was subcutaneously inoculated into 24 nude mice. Mice received either phosphate-buffered saline solution, sunitinib, everolimus, or bevacizumab during 4 days. DCE-US and molecular MRI targeting αvß3 were performed at baseline and 4 days after treatment initiation. PI, AUC, relaxation rate variations ΔR2⁎, and percentage of vessels area quantified on CD31-stained microvessels were compared. Significant decreases were observed for PI and AUC parameters measured by DCE-US for bevacizumab group as early as 4 days, whereas molecular αvß3-targeted MRI was able to detect significant changes in both bevacizumab and everolimus groups. Percentage of CD31-stained microvessels was significantly correlated with DCE-US parameters, PI (R = 0.87, p = 0.0003) and AUC (R = 0.81, p = 0.0013). The percentage of vessel tissue area was significantly reduced (p < 0.01) in both sunitinib and bevacizumab groups. We report an early detection of neoangiogenesis modification after induction of targeted therapies, using DCE-US or αvß3-targeted MRI. We consider these outcomes should encourage clinical trial developments to further evaluate the potential of this molecular MRI technique.

Tetraspanin 8 (TSPAN 8) as a potential target for radio-immunotherapy of colorectal cancer.

Tetraspanin 8 (TSPAN8) overexpression is correlated with poor prognosis in human colorectal cancer (CRC). A murine mAb Ts29.2 specific for human TSPAN8 provided significant efficiency for immunotherapy in CRC pre-clinical models. We therefore evaluate the feasability of targeting TSPAN8 in CRC with radiolabeled Ts29.2. Staining of tissue micro-arrays with Ts29.2 revealed that TSPAN8 espression was restricted to a few human healthy tissues. DOTA-Ts29.2 was radiolabeled with 111In or 177Lu with radiochemical purities >95%, specific activity ranging from 300 to 600 MBq/mg, and radioimmunoreactive fractions >80%. The biodistribution of [111In]DOTA-Ts29.2 in nude mice bearing HT29 or SW480 CRC xenografts showed a high specificity of tumor localization with high tumor/blood ratios (HT29: 4.3; SW480-TSPAN8: 3.9 at 72h and 120h post injection respectively). Tumor-specific absorbed dose calculations for [177Lu]DOTA-Ts29.2 was 1.89 Gy/MBq, establishing the feasibility of using radioimmunotherapy of CRC with this radiolabeled antibody. A significant inhibition of tumor growth in HT29 tumor-bearing mice treated with [177Lu]DOTA-Ts29.2 was observed compared to control groups. Ex vivo experiments revealed specific DNA double strand breaks associated with cell apoptosis in [177Lu]DOTA-Ts29.2 treated tumors compared to controls. Overall, we provide a proof-of-concept for the use of [111In/177Lu]DOTA-Ts29.2 that specifically target in vivo aggressive TSPAN8-positive cells in CRC.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

Dual inhibition using cabozantinib overcomes HGF/MET signaling mediated resistance to pan-VEGFR inhibition in orthotopic and metastatic neuroblastoma tumors.

MET is expressed on neuroblastoma cells and may trigger tumor growth, neoangiogenesis and metastasis. MET upregulation further represents an escape mechanism to various anticancer treatments including VEGF signaling inhibitors. We developed in vitro a resistance model to pan-VEGFR inhibition and explored the simultaneous inhibition of VEGFR and MET in neuroblastoma models in vitro and in vivo using cabozantinib, an inhibitor of the tyrosine kinases including VEGFR2, MET, AXL and RET. Resistance in IGR-N91-Luc neuroblastoma cells under continuous in vitro exposure pressure to VEGFR1-3 inhibition using axitinib was associated with HGF and p-ERK overexpression. Cabozantinib exhibited anti-proliferative effects in neuroblastoma cells and reduced cell migration in vitro as measured by phase-contrast with IncuCyte system. In vivo, an enhanced number of animals with IGR-N91-Luc metastases was noted following axitinib treatment as compared to control animals. Orally administered cabozantinib per gavage at 30 and 60 mg/kg/day significantly inhibited tumor growth of orthotopic adrenal IGR-N91-Luc and metastatic IMR-32-Luc xenografts. Antitumor activity was associated with decreased vascularization, inhibition of p-SRC and induction of apoptotic cell death. Activation of the HGF-mediated MET pathway is involved in escape to selective VEGFR inhibition in neuroblastoma suggesting combined inhibition of MET and VEGFR signaling to reduce secondary resistance and enhanced invasiveness.

CXCR4/CXCL12 axis counteracts hematopoietic stem cell exhaustion through selective protection against oxidative stress.

Hematopoietic stem cells (HSCs) undergo self-renewal to maintain hematopoietic homeostasis for lifetime, which is regulated by the bone marrow (BM) microenvironment. The chemokine receptor CXCR4 and its ligand CXCL12 are critical factors supporting quiescence and BM retention of HSCs. Here, we report an unknown function of CXCR4/CXCL12 axis in the protection of HSCs against oxidative stress. Disruption of CXCR4 receptor in mice leads to increased endogenous production of reactive oxygen species (ROS), resulting in p38 MAPK activation, increased DNA double-strand breaks and apoptosis leading to marked reduction in HSC repopulating potential. Increased ROS levels are directly responsible for exhaustion of the HSC pool and are not linked to loss of quiescence of CXCR4-deficient HSCs. Furthermore, we report that CXCL12 has a direct rescue effect on oxidative stress-induced HSC damage at the mitochondrial level. These data highlight the importance of CXCR4/CXCL12 axis in the regulation of lifespan of HSCs by limiting ROS generation and genotoxic stress.

Iron Overload Exacerbates Busulfan-Melphalan Toxicity Through a Pharmacodynamic Interaction in Mice.

PURPOSE: Busulfan-melphalan high-dose chemotherapy followed by autologous stem cell transplantation is an essential consolidation treatment of high-risk neuroblastoma in children. Main treatment limitation is hepatic veno-occlusive disease, the most severe and frequent extra-hematological toxicity. This life threatening toxicity has been related to a drug interaction between busulfan and melphalan which might be increased by prior disturbance of iron homeostasis, i.e. an increased plasma ferritin level. METHODS: We performed an experimental study of busulfan and melphalan pharmacodynamic and pharmacokinetics in iron overloaded mice. RESULTS: Iron excess dramatically increased the toxicity of melphalan or busulfan melphalan combination in mice but it did not modify the clearance of either busulfan or melphalan. We show that prior busulfan treatment impairs the clearance of melphalan. This clearance alteration was exacerbated in iron overloaded mice demonstrating a pharmacokinetic interaction. Additionally, iron overload increased melphalan toxicity without altering its pharmacokinetics, suggesting a pharmacodynamic interaction between iron and melphalan. Based on iron homeostasis disturbance, we postulated that prior induction of ferritin, through Nrf2 activation after oxidative stress, may be associated with the alteration of melphalan metabolism. CONCLUSION: Iron overload increases melphalan and busulfan-melphalan toxicity through a pharmacodynamic interaction and reveals a pharmacokinetic drug interaction between busulfan and melphalan.