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Background: Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. Methods: We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages—onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. Results: We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. Conclusions: Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL. [ABSTRACT FROM AUTHOR]

Background: Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long "vein-to-vein" time. Methods: We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). Results: CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38− stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. Conclusions: Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells. [ABSTRACT FROM AUTHOR]

Background: Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long "vein-to-vein" time. Methods: We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). Results: CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38− stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. Conclusions: Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells. [ABSTRACT FROM AUTHOR]

The article looks at a study which evaluates impact of azacytidine treatment on aberrant DNA methylation in Juvenile Myelomonocytic Leukaemia (JMML) patients. Topics discussed include membrane elements such as adhesion molecules, developmental processes and cell cycle-related genes, assessment and genomic DNA extraction and sequence analysis and scenario of pathogenetic epigenetic regulation and specific levels of hypermethylation.

Despite progress in treating B-cell precursor acute lymphoblastic leukemia (BCP-ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high-risk relapsed patients. Che-1/AATF (Che-1) is an RNA polymerase II-binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che-1 is overexpressed in pediatric BCP-ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP-ALL cells. Furthermore, we report that c-Myc regulates Che-1 expression by direct binding to its promoter and describe a strict correlation between Che-1 expression and c-Myc expression. RNAseq analyses upon Che-1 or c-Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP-seq experiments suggest that Che-1 acts as a downstream effector of c-Myc. These results identify the pivotal role of Che-1 in the control of BCP-ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP-ALL. [ABSTRACT FROM AUTHOR]

Purpose of Review: Hematopoietic stem cell transplantation (HSCT) is a treatment option for children with malignant and non-malignant disorders as well as an expanding number of inherited disorders. However, only a limited portion of patients in the need of an allograft have an HLA-compatible, either related or unrelated, donor. Haploidentical HSCT is now considered a valid treatment option, especially in view of the recent insights in terms of graft manipulation. This review will offer an overview of clinical results obtained through the use of haploidentical HSCT in non-malignant diseases. We will analyze major advantages and drawbacks of both T cell depleted and unmanipulated HSCT, discussing future challenges for further improving patients' outcome. Recent Findings: T cell depletion (TCD) aims to reduce the morbidity and mortality associated with graft-versus-host disease (GvHD). However, the delayed immune recovery and the risk of graft failure still remain potential problems. In the last years, the use of post-transplant cyclophosphamide has been shown to be an alternative effective strategy to prevent GvHD in recipients of haploidentical HSCT. Summary: Recent data suggest that both T cell depleted and T cell-replete haplo-HSCT are suitable options to treat children with several types of non-malignant disorders lacking an HLA-identical donor. [ABSTRACT FROM AUTHOR]

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high-risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft-versus-host disease and could also contribute to anti-microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34+ cells are enriched in lymphoid-committed precursors, while PB CD34+ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56+CD161+ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56+CD161+ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34+ cultures was lower for G-CSF-mobilized HSCs (good mobilizers) than for G-CSF+plerixafor-mobilized HSC (poor mobilizers). Moreover, G-CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G-CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT. [ABSTRACT FROM AUTHOR]

Background: Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis. Methods: Morphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs). Results: Despite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs. Conclusions: Our results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment. [ABSTRACT FROM AUTHOR]

Vitiligo is characterized by the progressive disappearance of pigment cells from skin and hair follicle. Several in vitro and in vivo studies show evidence of an altered redox status, suggesting that loss of cellular redox equilibrium might be the pathogenic mechanism in vitiligo. However, despite the numerous data supporting a pathogenic role of oxidative stress, there is still no consensus explanation underlying the oxidative stress-driven disappear of melanocytes from the epidermis. In this study, in vitro characterization of melanocytes cultures from non-lesional vitiligo skin revealed at the cellular level aberrant function of signal transduction pathways common with neurodegenerative diseases including modification of lipid metabolism, hyperactivation of mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB), constitutive p53-dependent stress signal transduction cascades, and enhanced sensibility to pro-apoptotic stimuli. Notably, these long-term effects of subcytotoxic oxidative stress are also biomarkers of pre-senescent cellular phenotype. Consistent with this, vitiligo cells showed a significant increase in p16 that did not correlate with the chronological age of the donor. Moreover, vitiligo melanocytes produced many biologically active proteins among the senescence-associated secretory phenotype (SAPS), such as interleukin-6 (IL-6), matrix metallo proteinase-3 (MMP3), cyclooxygenase-2 (Cox-2), insulin-like growth factor-binding protein-3 and 7 (IGFBP3, IGFBP7). Together, these data argue for a complicated pathophysiologic puzzle underlying melanocytes degeneration resembling, from the biological point of view, neurodegenerative diseases. Our results suggest new possible targets for intervention that in combination with current therapies could correct melanocytes intrinsic defects. [ABSTRACT FROM AUTHOR]

While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both β-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector bcatenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/ β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibitiondependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling plays important roles in many developmental processes including neural crest-derived melanocyte development and migration. However, the effective contribution of Wnt/β-catenin pathway in melanogenesis in adult human melanocytes has not been fully elucidated. Here, we report that in melanoma cells and in normal human melanocytes, melanogenesis stimulation by α-melanocyte-stimulating hormone (α-MSH) induces phosphorylation of β-catenin-Ser675 and stabilization of β-catenin protein. Activation of protein kinase A by α-MSH attenuates glycogen synthase kinase-3β, which regulates ubiquitin-dependent degradation of β-catenin, suggesting a coordinated mechanism of β-catenin activity stimulation. Consistent with increased nuclear β-catenin, cyclic adenosine monophosphate (cAMP) elevation facilitates β-catenin-dependent transactivation of many Wnt target genes. Moreover, chromatin immunoprecipitation assays demonstrated an increased association of β-catenin with the proximal promoter of microphthalmia-associated transcription factor, the master regulator of pigmentation. These results demonstrate the existence of cross talk between the cAMP and Wnt pathways in melanocytes, suggesting that β-catenin could play a key role in the physiological regulation of epidermal melanogenesis. [ABSTRACT FROM AUTHOR]

The article describes the case of a nine-year-old patient with erythropoietic protoporphyria. It discusses the patient's medical history, the symptoms shown by the patient, the results of the patient's medical examinations, and the importance for pediatricians to obtain knowledge of the patient's history in the diagnosis of the disease.

The article presents a case study of a nine-year-old boy who had a two-year history of swelling and burning sensations on the dorsal surface of his hands, face and nose and was diagnosed with Erythropoietic protoporphyria. Topics include development of the symptoms such as blisters and purpuric rash with exposure to sun or warm temperature from the age of two, detection of higher levels of protoporphyrin IX in plasma, and role of protoporphyrin in causing itching and burning sensation.