Search

ERCC excision repair 6 like 2 (ERCC6L2) gene encodes for different helicase-like protein members of the Snf2 family involved in transcription-coupled nucleotide excision repair and in cell proliferation. Germline homozygous mutations in children and adults predispose to a peculiar bone marrow failure phenotype characterized by mild hematological alterations with a high risk of developing acute myeloid leukemia. The outcome for patients with leukemia progression is dismal while patients undergoing hematopoietic stem cell transplantation in the early stage have better outcomes. The ERCC6L2-related hematological disease presents a high penetrance, posing important questions regarding the treatment strategies and possible preemptive approaches. This review describes the biological function of ERCC6L2 and the clinical manifestations of the associated disease, trying to focus on the unsolved clinical questions.

Astolf et al provide the first report of acute promyelocytic leukemia driven by viral insertion into the RARA locus. This represents a clear demonstration of a pathology driven by the member of the anelloviruses, a group of viruses otherwise thought to have minimal or no pathogenic potential.

Pediatric acute myeloid leukemia (AML) represents an aggressive disease and is the leading cause of childhood leukemic mortality. The genomic landscape of pediatric AML has been recently mapped and redefined thanks to large-scale sequencing efforts. Today, understanding how to incorporate the growing list of genetic lesions into a risk stratification algorithm for pediatric AML is increasingly challenging given the uncertainty regarding the prognostic impact of rare lesions. Here we review some uncommon cytogenetic lesions to be considered for inclusion in the high-risk groups of the next pediatric AML treatment protocols. We describe their main clinical characteristics, biological background and outcome. We also provide some suggestions for the management of these rare but challenging patients and some novel targeted therapeutic options.

Gaucher disease is a lysosomal storage disorder characterized by β-glucosidase enzyme deficiency and substrate accumulation, especially in cells of the reticuloendothelial system. Typical features of the disease are the unrestrained activation of inflammatory mechanisms, whose molecular pathways are still unclear. To investigate biological mechanisms underlying the macrophage activation in GD, we derived iPSCs from a healthy donor and a GD patient line and differentiated them into hematopoietic progenitors. While GD iPSCs are able to efficiently give rise to CD33+/CD45+ myeloid progenitors, the maturation towards the CD14+/CD163+ monocyte/macrophages fate resulted enhanced in the GD lines, that in addition displayed a decreased growth potential compared to control cells either in semisolid or in liquid culture. The GD lines growth impairment was associated with a significant upregulation of RIPK3 and MLKL, two key effectors of necroptosis, the inflammation related cell death pathway. The activation of necroptosis, which has already been linked to neuronopathic GD, may play a role in the disease proinflammatory condition and in the identified cell growth defects. Understanding the GD macrophage role in the alteration of mechanisms linked to cellular metabolism imbalance, cell death and inflammation are crucial in identifying new ways to approach the disease.

Gaucher disease (GD) is a monogenic disorder characterized byβ-glucocerebrosidase enzyme deficiency. Typical features of the diseaseare the unrestrained activation of inflammatory mechanisms andneuronal cell death in neuronopathic conditions, whose molecularpathways are still not fully understood. Recent evidences show thatupregulation of the Hippo pathway is associated with neuroinflamma-tion and neuronal cell death. We analyzed the Hippo pathway activitytaking advantage of a Drosophila Gaucher-like model characterized bydGBA1b knock-out (GBA1bKO).WefoundderegulationofCycE,dIAPandMYC, direct targets of the pathway both in Drosophila and mammals,which were severely reduced at transcript and protein level,suggestingacell growth impairment. Analyzing upstream components we foundthat Fat, an atypical cadherin upstream of the kinase complex, wasupregulated. Moreover the glypicans Dally and Dally-like, known to benegatively regulated by Fat and involved in glial and synapsedevelopment, were found downregulated in the GBA1bKObackground,supporting a role in neurological damage. On the other hand, theaberrant inflammatory condition of the hematological compartmentwas investigated in the human model of induced Pluripotent Stem Cells(iPSC), differentiated into hematopoietic progenitors. While GD iPSCswere able to differentiate into CD34+/CD43+/CD45+ progenitors, theyshowed a decreased proliferative potential compared to healthy donoriPSC either in semisolid and liquid culture, therefore exhibiting a growthimpairment. This effect was coupled with the activation of necroptosispathway with a significant upregulation of RIPK3 and MLKL both in thepluripotent and differentiated state. Correction of the mutation to rescuethe phenotype is ongoing, taking advantage of AAVS1 editing approachin order to develop a fully comparable cell model useful in a cell therapyperspective. These data obtained with suitable GD models support therole of Hippo and necroptosis effectors in GD, amenable of furtherconsideration as potential therapeutic targets.

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

Nowadays, thanks to extensive studies and progress in precision medicine, pediatric leukemia has reached an extremely high overall survival rate. Nonetheless, a fraction of relapses and refractory cases is still present, which are frequently correlated with poor prognosis. Although several molecular features of these diseases are known, still the field of energy metabolism, which is widely studied in adult, has not been frequently explored in childhood leukemias. Metabolic reprogramming is a hallmark of cancer and is deeply connected with other genetic and signaling aberrations generally known to be key features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This review aims to clear the current knowledge on metabolic rewiring in pediatric ALL and AML, also highlighting the influence of the main signaling pathways and suggesting potential ideas to further exploit this field to discover new prognostic biomarkers and, above all, beneficial therapeutic options.

Supplemental Digital Content is available in the text.

La malattia di Gaucher è un disordine monogenico caratterizzato dal deficit dell’enzima β-glucocerebrosidasi. Dal momento che l’innesco aberrante di meccanismi infiammatori cellulari è un aspetto peculiare della malattia, ci siamo concentrati sullo studio del pathway della necroptosi, forma di morte cellulare programmata dettata dall’infiammazione. Dall’analisi dell’espressione genica e dalla valutazione dei livelli proteici in modelli cellulari Gaucher-like, ottenuti tramite inibizione enzimatica, abbiamo evidenziato come la chinasi RIP1, primo effettore della via di segnalazione sia iperattivato nello stato patologico. Per disporre di un sistema d’analisi più accurato stiamo mettendo a punto modelli che sfruttano cellule staminali pluripotenti indotte (iPSCs) derivate da paziente e donatore sano. Tramite gene editing la più comune mutazione puntiforme causante la malattia (N370S), è stata inserita in iPSCs derivate da donatore sano. Le cellule sono state trasfettate, tramite nucleofezione, con il plasmide pSpCas9(BB)-2A-Puro codificante per la Cas9 e per una guida a RNA in grado di indirizzarne il taglio sito-specifico. La sequenza stampo per l’inserimento della mutazione è stata introdotta sotto forma di DNA a singolo filamento. La presenza della mutazione è stata verificata tramite PCR effettuata con primer specifici su pool di cellule trasfettate. Parallelamente, da cellule mononucleate del sangue periferico di un paziente Gaucher con mutazione N370S/L444P sono state ottenute iPSCs grazie a trasduzione con i vettori virali Sendai codificanti per i fattori di riprogrammazione: Klf4, cMyc e KOS. Il monitoraggio del differenziamento verso destino ematopoietico sarà portato avanti concentrandosi nello specifico sul tipo cellulare macrofagico in quanto maggior target dell’accumulo sfingolipidico caratterizzante la malattia.

Necrotizing enterocolitis (NEC) is a gut inflammatory disorder which constitutes one of the leading causes of morbidity and mortality for preterm infants. The pathophysiology of NEC is yet to be fully understood; several observational studies have led to the identification of multiple factors involved in the pathophysiology of the disease, including gut immaturity and dysbiosis of the intestinal microbiome. Given the complex interactions between microbiota, enterocytes, and immune cells, and the limited access to fetal human tissues for experimental studies, animal models have long been essential to describe NEC mechanisms. However, at present there is no animal model perfectly mimicking human NEC; furthermore, the disease mechanisms appear too complex to be studied in single-cell cultures. Thus, researchers have developed new approaches in which intestinal epithelial cells are exposed to a combination of environmental and microbial factors which can potentially trigger NEC. In addition, organoids have gained increasing attention as promising models for studying NEC development. Currently, several in vitro models have been proposed and have contributed to describe the disease in deeper detail. In this paper, we will provide an updated review of available in vitro models of NEC and an overview of current knowledge regarding its molecular underpinnings.

BCOR is a gene that encodes for an epigenetic regulator involved in the specification of cell differentiation and body structure development and takes part in the noncanonical polycomb repressive complex 1. This review provides a comprehensive summary of BCOR’s involvement in oncology, illustrating that various BCOR aberrations, such as the internal tandem duplications of the PCGF Ub-like fold discriminator domain and different gene fusions (mainly BCOR–CCNB3, BCOR–MAML3 and ZC3H7B–BCOR), represent driver elements of various sarcomas such as clear cell sarcoma of the kidney, primitive mesenchymal myxoid tumor of infancy, small round blue cell sarcoma, endometrial stromal sarcoma and histologically heterogeneous CNS neoplasms group with similar genomic methylation patterns known as CNS-HGNET-BCOR. Furthermore, other BCOR alterations (often loss of function mutations) recur in a large variety of mesenchymal, epithelial, neural and hematological tumors, suggesting a central role in cancer evolution.

The Hedgehog (Hh) pathway is a key signaling system that controls proliferation and differentiation of embryonic cells, whereas in adult organisms it is involved in the control of tissue homeostasi...

Summary The scenario of paediatric acute myeloid leukaemia (AML), particularly non‐Down syndrome acute megakaryoblastic leukaemia (non‐DS‐AMKL), has been recently revolutionized by the advent of large‐scale, genomic sequencing technologies. In this changing landscape, a significantly relevant discovery has been represented by the identification of the CBFA2T3‐GLIS2 fusion gene, which is the result of a cryptic inversion of chromosome 16. It is the most frequent chimeric oncogene identified to date in non‐DS‐AMKL, although it seems not to be exclusively restricted to the French‐American‐British M7 subgroup. The CBFA2T3‐GLIS2 fusion gene characterizes a subtype of leukaemia that is specific to paediatrics, having never been identified in adults. It characterizes an extremely aggressive leukaemia, as the presence of this fusion is associated with a grim outcome in almost all of the case series reported, with overall survival rates ranging between 15% and 30%. Although the molecular basis that underlies this leukaemia subtype is still far from being completely elucidated, unique functional properties induced by CBFA2T3‐GLIS2 in the leukaemogenesis driving process have been recently identified. We here review the peculiarities of CBFA2T3‐GLIS2‐positive AML, describing its intriguing clinical and biological behaviour and providing some challenging targeting opportunities.

Simple Summary The use of innovative technologies has revolutionized cancer research in recent years, and in the field of pediatric oncohematology, great results have been achieved. However, within this context, acute myeloid leukemia still represents a considerable challenge for clinicians, as frequent cases of relapse or refractory disease, especially in specific subgroups, remain present. With this review, the authors aim to recapitulate the main features of this extremely heterogeneous malignancy, by highlighting the concept of clonal evolution and how, thanks also to the impact of new high throughput techniques, this could be exploited to deepen the current knowledge on molecular mechanisms driving this disease. Overall, this study will seek to pave the way for making new tools available to further improve diagnosis and treatment protocols in pediatric patients. Abstract Despite improvements in therapeutic protocols and in risk stratification, acute myeloid leukemia (AML) remains the leading cause of childhood leukemic mortality. Indeed, the overall survival accounts for ~70% but still ~30% of pediatric patients experience relapse, with poor response to conventional chemotherapy. Thus, there is an urgent need to improve diagnosis and treatment efficacy prediction in the context of this disease. Nowadays, in the era of high throughput techniques, AML has emerged as an extremely heterogeneous disease from a genetic point of view. Different subclones characterized by specific molecular profiles display different degrees of susceptibility to conventional treatments. In this review, we describe in detail this genetic heterogeneity of pediatric AML and how it is linked to relapse in terms of clonal evolution. We highlight some innovative tools to characterize minor subclones that could help to enhance diagnosis and a preclinical model suitable for drugs screening. The final ambition of research is represented by targeted therapy, which could improve the prognosis of pediatric AML patients, as well as to limit the side toxicity of current treatments.

Background CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. Methods We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. Results As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. Conclusions Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0396-0) contains supplementary material, which is available to authorized users.

L-Asparaginase (L-Asp) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid, and its depletion induces leukemic cell death. L-Asp is an important component of treatment regimens for Acute Lymphoblastic Leukemia (ALL). Sensitivity to L-Asp is due to the absence of L-Asparagine synthetase (ASNS), the enzyme that catalyzes the biosynthesis of L-asparagine. ASNS gene is located on 7q21.3, and its increased expression in ALLs correlates with L-Asp resistance. Chromosome 7 monosomy (-7) is a recurrent aberration in myeloid disorders, particularly in adverse-risk Acute Myeloid Leukemias (AMLs) and therapy-related myeloid neoplasms (t-MN), that leads to a significant downregulation of the deleted genes, including ASNS. Therefore, we hypothesized that -7 could affect L-Asp sensitivity in AMLs. By treating AML cell lines and primary cells from pediatric patients with L-Asp, we showed that -7 cells were more sensitive than AML cells without -7. Importantly, both ASNS gene and protein expression were significantly lower in -7 AML cell lines, suggesting that haploinsufficiency of ASNS might induce sensitivity to L-Asp in AMLs. To prove the role of ASNS haploinsufficiency in sensitizing AML cells to L-Asp treatment, we performed siRNA-knockdown of ASNS in AML cell lines lacking -7, and observed that ASNS knockdown significantly increased L-Asp cytotoxicity. In conclusion, -7 AMLs showed high sensitivity to L-Asp treatment due to low expression of ASNS. Thus, L-Asp may be considered for treatment of AML pediatric patients carrying -7, in order to improve the outcome of adverse-risk AMLs and t-MN patients.

// Riccardo Masetti 1, * , Ilaria Castelli 1, * , Annalisa Astolfi 2 , Salvatore Nicola Bertuccio 1 , Valentina Indio 2 , Marco Togni 1, 3 , Tamara Belotti 1 , Salvatore Serravalle 1 , Giuseppe Tarantino 2 , Marco Zecca 4 , Martina Pigazzi 5 , Giuseppe Basso 5 , Andrea Pession 1, # , Franco Locatelli 6, 7, # 1 Department of Pediatrics “Lalla Seragnoli”, Hematology-Oncology Unit, University of Bologna, Bologna, Italy 2 Interdepartmental Centre of Cancer Research “G. Prodi”, University of Bologna, Bologna, Italy 3 Current address: Stem Cell Group, University College London Cancer Institute, University College London, London, United Kingdom 4 Department of Pediatric Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 5 Department of Woman and Child Health, Laboratory of Hematology-Oncology, University of Padova, Padova, Italy 6 Department of Pediatric Hematology-Oncology, IRCCS Ospedale Bambino Gesu, Rome, Italy 7 University of Pavia, Pavia, Italy * These authors have contributed equally to this work # Both authors have shared co-senior authorship Correspondence to: Riccardo Masetti, email: riccardo.masetti@gmail.com Keywords: pediatric acute myeloid leukemia, acute myeloid leukemia relapse, whole-exome massively parallel sequencing, SETD2 mutation, FLT3-TKD mutation Received: April 19, 2016 Accepted: July 10, 2016 Published: July 22, 2016 ABSTRACT Despite significant improvement in treatment of childhood acute myeloid leukemia (AML), 30% of patients experience disease recurrence, which is still the major cause of treatment failure and death in these patients. To investigate molecular mechanisms underlying relapse, we performed whole-exome sequencing of diagnosis-relapse pairs and matched remission samples from 4 pediatric AML patients without recurrent cytogenetic alterations. Candidate driver mutations were selected for targeted deep sequencing at high coverage, suitable to detect small subclones (0.12%). BiCEBPα mutation was found to be stable and highly penetrant, representing a separate biological and clinical entity, unlike WT1 mutations, which were extremely unstable. Among the mutational patterns underlying relapse, we detected the acquisition of proliferative advantage by signaling activation (PTPN11 and FLT3-TKD mutations) and the increased resistance to apoptosis (hyperactivation of TYK2). We also found a previously undescribed feature of AML, consisting of a hypermutator phenotype caused by SETD2 inactivation. The consequent accumulation of new mutations promotes the adaptability of the leukemia, contributing to clonal selection. We report a novel ASXL3 mutation characterizing a very small subclone (

T-Acute Lymphoblastic Leukemia (T-ALL) remains a subgroup of pediatric ALL, with a lower response to standard chemotherapy. Some recent studies established the fundamental role of epigenetic aberrations such as DNA hypermethylation, to influence patients’ outcome and response to chemotherapy. Moreover, L-asparaginase is an important chemotherapeutic agent for treatment of ALL and resistance to this drug has been linked toASNSexpression, which can be silenced through methylation. Therefore, we tested whether the sensitivity of T-ALL cell lines towards L-asparaginase is correlated to the epigenetic status ofASNSgene and whether the sensitivity can be modified by concurrent demethylating treatment. Hence we treated different T-ALL cell lines with L-asparaginase and correlated different responses to the treatment withASNSexpression. Then we demonstrated that theASNSexpression was dependent on the methylation status of the promoter. Finally we showed that, despite the demethylating effect on theASNSgene expression, the combined treatment with the demethylating agent Decitabine could synergistically improve the L-asparaginase sensitivity in those T-ALL cell lines characterized by hypermethylation of theASNSgene. In conclusion, this preclinical study identified an unexpected synergistic activity of L-asparaginase and Decitabine in the subgroup of T-ALL with lowASNSexpression due to hypermethylation of theASNSpromoter, while it did not restore sensitivity in the resistant cell lines characterized by higherASNSexpression.

Acute pancreatitis is a pancreatic inflammation that recognises Salmonella typhi among its aetiological agents. In this article the authors describe two cases of acute pancreatitis secondary to typhoid fever, evolving towards complete recovery. These two cases, besides confirming that Salmonella typhi can be responsible for acute pancreatitis, remind us that during typhoid fever, amylase enzyme test should be always assessed. Moreover, salmonella infection must also be considered in cases of non-alcoholic or non-lithiasic pancreatitis.

The authors describe a clinical case in which they found the unusual combination of acute hepatitis caused by HCV and pericarditis in a young person, resulting in complete recovery from the pericarditis but in a deterioration of the chronic HCV. A close examination of the literature on this subject revealed that, although no similar case was recorded, an aetiological relationship between the hepatitis C virus and pericarditis cannot be excluded since an HCV infection often gives rise to extra-hepatic cardiac problems.