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Abstract Gaucher Disease (GD), the most common lysosomal disorder, arises from mutations in the GBA1 gene and is characterized by a wide spectrum of phenotypes, ranging from mild hematological and visceral involvement to severe neurological disease. Neuronopathic patients display dramatic neuronal loss and increased neuroinflammation, whose molecular basis are still unclear. Using a combination of Drosophila dGBA1b loss-of-function models and GD patient-derived iPSCs differentiated towards neuronal precursors and mature neurons we showed that different GD- tissues and neuronal cells display an impairment of growth mechanisms with an increased cell death and reduced proliferation. These phenotypes are coupled with the downregulation of several Hippo transcriptional targets, mainly involved in cells and tissue growth, and YAP exclusion from nuclei. Interestingly, Hippo knock-down in the GBA-KO flies rescues the proliferative defect, suggesting that targeting the Hippo pathway can be a promising therapeutic approach to neuronopathic GD.

Abstract Background CBL syndrome is a RASopathy caused by heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. It is characterized by heterogeneous clinical phenotype, including developmental delay, facial dysmorphisms, cardiovascular malformations and an increased risk of cancer development, particularly juvenile myelomonocytic leukemia (JMML). Although the clinical phenotype has been progressively defined in recent years, immunological manifestations have not been well elucidated to date. Methods We studied the genetic, immunological, coagulative, and clinical profile of a family with CBL syndrome that came to our observation after the diagnosis of JMML, with homozygous CBL mutation, in one of the members. Results Variant analysis revealed the co-occurrence of CBL heterozygous mutation (c.1141 T > C) and SH2B3 mutation (c.1697G > A) in two other members. Patients carrying both mutations showed an ALPS-like phenotype characterized by lymphoproliferation, cytopenia, increased double-negative T-cells, impaired Fas-mediated lymphocyte apoptosis, altered cell death in PBMC and low TRECs expression. A coagulative work-up was also performed and showed the presence of subclinical coagulative alterations in patients carrying both mutations. Conclusion In the reported family, we described immune dysregulation, as part of the clinical spectrum of CBL mutation with the co-occurrence of SH2B3.

BackgroundInfusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS.MethodsThis is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation.ResultsMultivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients.DiscussionOur data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

BackgroundT cells engineered to target CD19 antigen on neoplastic B cells represent the most striking example of CAR-T cell therapy. The success rate of this therapy is affected by several limitations: target antigen loss, and/or acquisition of a senescent/exhausted phenotype by CAR and non-CAR T cells.Case presentationWe report on a patient affected by refractory Diffuse Large B-cell Lymphoma who was resistant to CAR T-cell therapy and to two cycles post CAR-T of pembrolizumab (PBZ) due to the evolution into a B-cell Hodgkin-like lymphoma. Owing to the CD30 expression and the Hodgkin-like phenotype, the patient was ultimately treated with Brentuximab-Vedotin and finally underwent remission. Upon PBZ treatment, 100% of circulating CAR-T+ cells showed a persistent CD8+ senescent/exhausted phenotype, while an increase in the percentage of senescent cells was found in the non-CAR CD8+ T cells compartment.ConclusionsPBZ is not able to reinvigorate exhausted CAR+ T cells and to confer durable clinical response. We hypothesize that the phenomenon is due to the senescent phenotype of CAR+ T cells, which did not allow PBZ-induced reactivation and proliferative rescue. The phenomenon, together with the loss of CAR-T target CD19 and the shift of non-CAR CD8+ T cells towards a senescent phenotype likely contributed to set up an immune landscape with poor antitumor capacity.

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms’ tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases (p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

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.