Search

Le traslocazioni di MLL, presenti in oltre il 20% delle AML pediatriche, si associano a prognosi sfavorevole. Pertanto, continui sforzi sono necessari per identificare nuove terapie efficaci per questi pazienti. EPZ- 5676 è un inibitore di DOT1L, metiltranferasi con attività leucemogenica in presenza di riarrangiamenti di MLL (MLL-r). Sorafenib è un inibitore di tirosino-chinasi, tra cui FLT3, frequentemente utilizzato nel trattamento dei pazienti MLL-r. Scopo dello studio è la valutazione degli effetti citotossici e biologici della combinazione EPZ-5676 e Sorafenib. Sono state utilizzate linee cellulari con o senza MLL-r, e cellule primarie ottenute da pazienti AML pediatrici con MLL-r. Sono stati valutati gli effetti citotossici e biologici dei singoli farmaci e della loro combinazione mediante analisi citofluorimetriche, Western Blotting e RQ-PCR. EPZ-5676 inibisce DOT1L inducendo demetilazione del target H3k79 in tutte le cellule, ma la proliferazione cellulare è significativamente inibita solo nelle cellule MOLM13 e nOMO1 (MLL-r) e OCI-AML3 (non MLL-r). Ciò si associa ad induzione dell’apoptosi o effetti citostatici. EPZ-5676 riduce l’espressione dei geni HOXA9, MEIS1 e FLT3 in tutte le cellule; STAT5 e c-Myc nelle cellule MLL-r. Inoltre, EPZ-5676 modula parzialmente le vie di sopravvivenza PI3k/Akt e MAPk. La combinazione EPZ-5676 e Sorafenib risulta sinergica nelle linee cellulari (anche in assenza di MLL-r) e nelle cellule primarie, in cui induce un significativo incremento dell’apoptosi. In conclusione, gli effetti di EPZ-5676 non sembrano dipendere esclusivamente dalla presenza di MLL-r. Inoltre, EPZ-5676 è sinergico con Sorafenib, suggerendo un nuovo approccio terapeutico per il trattamento dei pazienti pediatrici affetti da AML.

Background: About 20% of the childhood AML present MLL gene translocations, that are associated with a very poor prognosis. That is why there is a strong interest in developing novel therapeutic strategies for these patients. As DOT1L is responsible for H3K79 methylation and is associated with MLL-r leukemogenesis, DOT1L inhibitors entered in clinical trials as promising treatments. MLL-r AML also showed an overexpression of FLT3 receptor tyrosine kinase. Therefore, FLT3 inhibitors, such as the multi-tyrosine kinase inhibitor Sorafenib, demonstrated encouraging efficacy in AML. Aims: To investigate the efficacy of a combination treatment using DOT1L inhibitor EPZ-5676 and Sorafenib to treat MLL-r AML. Methods: MLL-r (MOLM13, NOMO1, THP1) and non MLL-r (OCI-AML3, HL60 and U937 harboring CALM-AF10 translocation) AML cell lines, and MLL-r primary samples from pediatric AML patients were used. Flow cytometry analyses were performed to assess absolute cell counting and apoptosis (AnnexinV FITC/PI staining). Protein expression and H3K79 methylation were quantified by Western blot. mRNA expression was studied by quantitative Real-Time PCR (Q-PCR). Results: Firstly, the specific effects of DOT1L inhibition were examined in AML cell lines treated with increasing concentrations of EPZ-5676, up to 18 days. Cell growth was significantly inhibited after at least 8 days of treatment in MOLM13 and NOMO1 cells, but time-dependent apoptosis occurred only in MOLM13, thus suggesting that DOT1L inhibition alone might not be able to induce cytotoxicity. Whereas no effects were observed in HL60, U937 (non MLL-r), or THP1 (MLL-r) cells, both cell growth impairment and apoptosis were detected in non MLL-r OCI-AML3 cells, so that the impact of DOT1L inhibition could not exclusively rely on MLL rearrangements. Repression of DOT1L occurred since the 4th day of treatment, as demonstrated by the complete loss of H3K79me2. To further explore the consequence of this phenomenon, both MLL targets and key component of signaling pathways involved in AML survival (i.e. PI3K, FLT3 and MAPK) were investigated in cells treated with 1 uM EPZ-5676, up to 28 days. Gene expression of HOXA9, MEIS1, FLT3 and CDK6 protein (MLL target) decreased in all cell lines, whereas STAT5 and c-Myc mRNAs, along with STAT5 protein expression, were downregulated only in MLL-r cells. Furthermore, in MOLM13 and NOMO1 cells p-Erk was strongly reduced. Finally, p-Akt slightly decreased in nearly every case, whereas a strong induction in p-S6RP was observed only in U937 cells after prolonged treatment, suggesting the possible involvement of PI3K pathway in drug resistance mechanisms. To increase the benefit of DOT1L inhibition, both AML cell lines and MLL-r primary samples were pretreated with increasing concentration of EPZ- 5676 for 4/8 days, following 24/48h treatment with Sorafenib. This combination resulted in a synergistic effect in nearly all cases. Importantly, EPZ-5676 pretreatment increased Sorafenib-induced cell growth inhibition in EPZ-5676 refractory cells HL60, U937 and THP1. Moreover, in primary AML samples both EPZ-5676 and Sorafenib showed a limited effect as single agent, whereas their combination induced a drastic increase of apoptosis. Summary/Conclusions: These results demonstrated that the single administration of EPZ-5676 has a limited antileukemic activity, which is not restricted to MLL-r AMLs. However, the combination of EPZ-5676 with Sorafenib revealed a synergistic effect in both MLL-r and non MLL-r AMLs, paving the way to innovative and more effective treatments for pediatric AML patients.

Extraskeletal myxoid chondrosarcoma (EMC) is a very rare sarcoma most often arising in the soft tissue. Rare EMC of the bone have been reported. EMC exhibits distinctive clinico-pathological and genetic features; however, despite the name, it lacks any feature of cartilaginous differentiation. EMC is characterized by the rearrangement of the NR4A3, which, in most cases (about 62-75%), is fused with EWSR1 and less frequently with other partners, including TAF15 (27%), TCF12 (4%), TFG, and FUS. We herein report the identification by whole-transcriptome sequencing of HSPA8 as a novel fusion partner of NR4A3 in a case of EMC. FISH analysis confirmed the presence of a genomic HSPA8-NR4A3 translocation in the vast majority of tumor cells. Our findings expand the spectrum of NR4A3 fusion partners involved in EMC pathobiology.

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.

Despite enormous improvements in pre-clinical and clinical research, acute leukemia still represents an open challenge for pediatric hematologists; both for a significant relapse rate and for long term therapy-related sequelae. In this context, the use of an innovative technology, such as induced pluripotent stem cells (iPSCs), allows to finely reproduce the primary features of the malignancy and can be exploited as a model to study the onset and development of leukemia in vitro. The aim of this review is to explore the recent literature describing iPSCs as a key tool to study different types of hematological malignancies, comprising acute myeloid leukemia, non-down syndrome acute megakaryoblastic leukemia, B cell acute lymphoblastic leukemia, and juvenile myelomonocytic leukemia. This model demonstrates a positive impact on pediatric hematological diseases, especially in those affecting infants whose onsets is found in fetal hematopoiesis. This evidence highlights the importance of achieving an in vitro representation of the human embryonic hematopoietic development and timing-specific modifications, either genetic or epigenetic. Moreover, further insights into clonal evolution studies shed light in the way of a new precision medicine era, where patient-oriented decisions and therapies could further improve the outcome of pediatric cases. Nonetheless, we will also discuss here the difficulties and limitations of this model.

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.

Supplemental Digital Content is available in the text.

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.

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.

The genomic landscape of children with acute myeloid leukemia (AML) who do not carry any cytogenetic abnormality (CN-AML) is particularly heterogeneous and challenging, being characterized by different clinical outcomes. To provide new genetic insights into this AML subset, we analyzed through RNA-seq 13 pediatric CN-AML cases, corroborating our findings in an independent cohort of 168 AML patients enrolled in the AIEOP AML 2002/01 study. We identified a chimeric transcript involving NUP98 and PHF23, resulting from a cryptic t(11;17)(p15;p13) translocation, demonstrating, for the first time, that NUP98-PHF23 is a novel recurrent (2.6 %) abnormality in pediatric CN-AML.

Pediatric early T-cell precursor leukemia with NF1 deletion and high-sensitivity in vitro to tipifarnib

NA