Mechanistic insights into the developmental origin of pediatric hematological disorders.

Embryonic and fetal hematopoietic stem and progenitor cells differ in some key properties from cells that are part of the adult hematopoietic system. These include higher proliferation and self-renewal capacity, different globin gene usage and differing lineage biases. While these evolved to cover specific requirements of embryonic development, they can have serious consequences for the pathogenesis of hematological malignancies that initiate pre-birth in fetal blood cells and may result in a particularly aggressive disease that does not respond well to treatments that have been designed for adult leukemias. This indicates that these infant/pediatric leukemias should be considered as developmental diseases, where a thorough understanding of their unique biology is essential for designing more effective therapies. In this review, we will highlight some of these unique fetal properties and detail the underlying molecular drivers of these phenotypes. We specifically focus on those that are pertinent to disease pathogenesis and that may therefore reveal disease vulnerabilities. We have also included an extensive description of the origins, phenotype and key molecular drivers of the main infant and pediatric leukemias that have a known prenatal origin. Importantly, successes in recent years in generating faithful models of these malignancies in which cellular origins, key drivers and potential vulnerabilities can be investigated have resulted in uncovering potential, new therapeutic avenues.

Iron regulates myeloma cell/macrophage interaction and drives resistance to bortezomib.

Iron plays a major role in multiple processes involved in cell homeostasis such as metabolism, respiration and DNA synthesis. Cancer cells exhibit pronounced iron retention as compared to healthy counterpart. This phenomenon also occurs in multiple myeloma (MM), a hematological malignancy characterized by terminally differentiated plasma cells (PCs), in which serum ferritin levels have been reported as a negative prognostic marker. The aim of current study is to evaluate the potential role of iron metabolism in promoting drug resistance in myeloma cancer cells with particular regard to the interactions between PCs and tumor-associated macrophages (TAMs) as a source of iron. Our data showed that myeloma cell lines are able to intake and accumulate iron and thus, increasing their scavenger antioxidant-related genes and mitochondrial mass. We further demonstrated that PCs pre-treated with ferric ammonium citrate (FAC) decreased bortezomib (BTZ)-induced apoptosis in vitro and successfully engrafted in zebrafish larvae treated with BTZ. Treating human macrophages with FAC, we observed a switch toward a M2-like phenotype associated with an increased expression of anti-inflammatory markers such as ARG1, suggesting the establishment of an iron-mediated immune suppressive tumor microenvironment favouring myeloma growth. Using mfap4:tomato mutant zebrafish larvae, we further confirmed the increase of PCs-monocytes interactions after FAC treatment which favour BTZ-resistance. Taken together our data support the hypothesis that targeting iron trafficking in myeloma microenvironment may represent a promising strategy to counteract a tumor-supporting milieu and drug resistance.

Correction: TLR4 signaling drives mesenchymal stromal cells commitment to promote tumor microenvironment transformation in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TLR4 signaling drives mesenchymal stromal cells commitment to promote tumor microenvironment transformation in multiple myeloma.

Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM-MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM-MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron Overload.

Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma‒optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.

Immune off-target effects of Brentuximab Vedotin in relapsed/refractory Hodgkin Lymphoma.

Hodgkin Lymphoma (HL) is associated with deep microenvironment re-shaping and myeloid dysfunction. Given that only limited data are available regarding the role of Brentuximab Vedotin (BV) as single agent in transplant-naive relapsed/refractory (R/R) patients and its off-target effects on immune system, we evaluated the amount of regulatory T-cells (T-regs), myeloid-derived suppressor cells (MDSC) subpopulations, and their functional marker, serum arginase-1 (s-Arg-1), in peripheral blood of 15 consecutive R/R HL patients. After a median of four BV cycles, the overall response rate (complete response + partial response) was 47%, with 4 (27%) complete metabolic remissions. BV reduced the absolute number of three MDSC subtypes and s-Arg-1 levels. Patients with baseline s-Arg-1 ≥200 ng/ml had inferior progression-free survival at 36 months compared to those with low s-Arg-1. T-regs dysfunction was recovered by BV: absolute T-regs count was increased after treatment with BV, independently of metabolic response achieved, with a significant reduction of CD30+ T-regs. Our data disclose off-target effects of BV in the microenvironment that could explain its deep and durable clinical efficacy.

Plasticity of High-Density Neutrophils in Multiple Myeloma is Associated with Increased Autophagy Via STAT3.

In both monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM) patients, immune functions are variably impaired, and there is a high risk of bacterial infections. Neutrophils are the most abundant circulating leukocytes and constitute the first line of host defense. Since little is known about the contribution of autophagy in the neutrophil function of MGUS and MM patients, we investigated the basal autophagy flux in freshly sorted neutrophils of patients and tested the plastic response of healthy neutrophils to soluble factors of MM. In freshly sorted high-density neutrophils obtained from patients with MGUS and MM or healthy subjects, we found a progressive autophagy trigger associated with soluble factors circulating in both peripheral blood and bone marrow, associated with increased IFNγ and pSTAT3S727. In normal high-density neutrophils, the formation of acidic vesicular organelles, a morphological characteristic of autophagy, could be induced after exposure for three hours with myeloma conditioned media or MM sera, an effect associated with increased phosphorylation of STAT3-pS727 and reverted by treatment with pan-JAK2 inhibitor ruxolitinib. Taken together, our data suggest that soluble factors in MM can trigger contemporary JAK2 signaling and autophagy in neutrophils, targetable with ruxolitinib.

Heme Oxygenase Inhibition Sensitizes Neuroblastoma Cells to Carfilzomib.

Neuroblastoma (NB) is an embryonic malignancy affecting the physiological development of adrenal medulla and paravertebral sympathetic ganglia in early infancy. Proteasome inhibitors (PIs) (i.e., carfilzomib (CFZ)) may represent a possible pharmacological treatment for solid tumors including NB. In the present study, we tested the effect of a novel non-competitive inhibitor of heme oxygenase-1 (HO-1), LS1/71, as a possible adjuvant therapy for the efficacy of CFZ in neuroblastoma cells. Our results showed that CFZ increased both HO-1 gene expression (about 18-fold) and HO activity (about 8-fold), following activation of the ER stress pathway. The involvement of HO-1 in CFZ-mediated cytotoxicity was further confirmed by the protective effect of pharmacological induction of HO-1, significantly attenuating cytotoxicity. In addition, HO-1 selective inhibition by a specific siRNA increased the cytotoxic effect following CFZ treatment in NB whereas SnMP, a competitive pharmacological inhibitor of HO, showed no changes in cytotoxicity. Our data suggest that treatment with CFZ produces ER stress in NB without activation of CHOP-mediated apoptosis, whereas co-treatment with CFZ and LS1/71 led to apoptosis activation and CHOP expression induction. In conclusion, our study showed that treatment with the non-competitive inhibitor of HO-1, LS1 / 71, increased cytotoxicity mediated by CFZ, triggering apoptosis following ER stress activation. These results suggest that PIs may represent a possible pharmacological treatment for solid tumors and that HO-1 inhibition may represent a possible strategy to overcome chemoresistance and increase the efficacy of chemotherapic regimens.

PMN-MDSC and arginase are increased in myeloma and may contribute to resistance to therapy.

OBJECTIVES: Despite improvement in overall response due to the introduction of the first-in-class proteasome inhibitor bortezomib (btz), multiple myeloma (MM) is still an incurable disease due to the immune-suppressive bone marrow (BM) environment. Thus, the authors aimed to identify the role of CD11b+CD15+CD14-HLA-DR- granulocytic-like myeloid-derived suppressor cells (PMN-MDSC) in MM patients treated up-front with novel agents. METHODS: In MM cell lines and primary cells derived by patients affected by MGUS and MM, we investigated sensitivity to bortezomib and lenalidomide in presence of Arg-1 and PMN-MDSC. RESULTS: The authors found that PMN-MDSC and their function through increased arginase-1 (Arg-1) are associated with MM progression. When the authors assessed cell viability of the human myeloma cell lines MM1.s, OPM2 and U266 treated with 5-20 nM btz for 24 h in PMN-MDSC conditioned media, they disclosed that amount of Arg-1 and Arg-1 inhibition could affect btz sensitivity in-vitro. PMN-MDSC and Arg-1 were increased in peripheral blood of newly diagnosed MM patients compared to healthy subjects. PMN-MDSC and arginase were reduced after exposure to lenalidomide-based regimen but increased after btz-based treatment. CONCLUSION: In MM, Arg-1 is mainly expressed by PMN-MDSC. PMN-MDSC and Arg-1 are reduced in vivo after lenalidomide but not bortezomib treatment.

Monocytic myeloid-derived suppressor cells as prognostic factor in chronic myeloid leukaemia patients treated with dasatinib.

Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in patients with chronic myeloid leukaemia (CML) inducing T cell tolerance. In this study, we found that therapy with tyrosine kinase inhibitors (TKI) decreased the percentage of granulocytic MDSC, but only patients treated with dasatinib showed a significant reduction in the monocytic subset (M-MDSC). Moreover, a positive correlation was observed between number of persistent M-MDSC and the value of major molecular response in dasatinib-treated patients. Serum and exosomes from patients with CML induced conversion of monocytes from healthy volunteers into immunosuppressive M-MDSC, suggesting a bidirectional crosstalk between CML cells and MDSC. Overall, we identified M-MDSC as prognostic factors in patients treated with dasatinib. It might be of interest to understand whether MDSC may be a candidate predictive markers of relapse risk following TKI discontinuation, suggesting their potential significance as practice of precision medicine.

Effect of Lipoic Acid on the Biochemical Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells.

Neuroblastoma (NB) is an extracranial solid cancer and the most common cancer in infancy. Despite the standard treatment for NB is based on the combination of chemotherapeutic drugs such as doxorubicin, vincristine, cyclophosphamide, and cisplatin, chemoresistance occurs over the time. The aim of the present research was to evaluate the effect of bortezomib (BTZ) (50 nM) on NB cell viability and how lipoic acid (ALA) (100 µM) modifies pharmacological response to this chemotherapeutic agent. Cell viability was assessed by ATP luminescence assay whereas expression of oxidative stress marker (i.e., heme oxygenase-1) and endoplasmic reticulum stress proteins was performed by real-time PCR, western blot, and immunofluorescence. Our data showed that BTZ treatment significantly reduced cell viability when compared to untreated cultures (about 40%). Interestingly, ALA significantly reduced the efficacy of BTZ (about 30%). Furthermore, BTZ significantly induced heme oxygenase-1 as a result of increased oxidative stress and such overexpression was prevented by concomitant treatment with ALA. Similarly, ALA significantly reduced BTZ-mediated endoplasmic reticulum stress as measured by reduction in BiP1 and IRE1α, ERO1α, and PDI expression. In conclusion, our data suggest that BTZ efficacy is dependent on cellular redox status and such mechanisms may be responsible of chemoresistance to this chemotherapeutic agent.

Antimicrobial and Anti-Proliferative Effects of Skin Mucus Derived from Dasyatis pastinaca (Linnaeus, 1758).

Resistance to chemotherapy occurs in various diseases (i.e., cancer and infection), and for this reason, both are very difficult to treat. Therefore, novel antimicrobial and chemotherapic drugs are needed for effective antibiotic therapy. The aim of the present study was to assess the antimicrobial and anti-proliferative effects of skin mucus derived from Dasyatis pastinaca (Linnaeus, 1758). Our results showed that skin mucus exhibited a significant and specific antibacterial activity against Gram-negative bacteria but not against Gram-positive bacteria. Furthermore, we also observed a significant antifungal activity against some strains of Candida spp. Concerning anti-proliferative activity, we showed that fish mucus was specifically toxic for acute leukemia cells (HL60) with an inhibition of proliferation in a dose dependent manner (about 52% at 1000 µg/mL of fish skin mucous, FSM). Moreover, we did not observe effects in healthy cells, in neuroblastoma cells (SH-SY5Y), and multiple myeloma cell lines (MM1, U266). Finally, it exhibited strong expression and activity of chitinase which may be responsible, at least in part, for the aforementioned results.

Toxic Effects of Zinc Chloride on the Bone Development in Danio rerio (Hamilton, 1822).

The increase of heavy metals in the environment involves a high exposure of aquatic organisms to these pollutants. The present study is planned to investigate the effects of zinc chloride (ZnCl2) on the bone embryonic development of Danio rerio and confirm the use of zebrafish as a model organism to study the teratogenic potential of this pollutant. Zebrafish embryos were exposed to different ZnCl2 concentrations and analyzed by ICP-MS. The skeletal anomalies were evaluated to confocal microscope after staining with calcein solution and RhodZin(TM)-3,AM. The data show a delay in hatching compared with the controls, malformations in the process of calcification and significant defects in growth. In conclusion, the current work demonstrates for the first time the Zn toxic effects on calcification process and confirm zebrafish (Danio rerio) as suitable alternative vertebrate model to study the causes and the mechanisms of the skeletal malformations.

Light and electron microscopy study of the spermatheca of Eupholidoptera chabrieri bimucronata (Ramme, 1927) and Uromenus brevicollis trinacriae La Greca 1964 (Orthoptera: Tettigoniidae).

A study by both optical and electron microscopy has been carried out on the spermatheca of Eupholidoptera chabrieri bimucronata and Uromenus brevicollis trinacriae (Orthoptera: Tettigoniidae). In both the examined species, the spermatheca consists of a sac/kidney-shaped seminal receptacle and a more or less tortuous spermathecal duct that opens into the common oviduct. The wall of both the organs consists of a pseudostratified epithelium surmounted by a cuticular intima; the latter is made up of a thicker endocuticle and an epicuticle. The epithelium shows two different cell types, irregularly arranged and with well differentiated functions: cuticle-forming and gland cells. In both the species, the cuticle-forming cells perform other functions, in addition to producing the cuticular intima. The gland cells never come in contact with the cuticular intima, have inside the reservoir a secretion whose appearance can diversify also in contiguous zones of the seminal receptacle. Based on our findings in both the species, the functions of the seminal receptacle would differ from those of the spermathecal duct. In the latter, some areas of the wall of the connecting tract show an activity of lysis, by contiguous epithelial cells, that could play a role in control and selection of spermatozoa. As for the feather-shaped spermatodesms, similar in both the species, freeze-fracture observations have shown that the acrosome of each spermatozoon regularly covers three-quarters of the extension of the acrosome of the following spermatozoon. Finally, the significance of our findings, compared with what is known in literature, is discussed.