RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

Generation, Characterization, and Count of Fluorescent Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous particles released by all cells in the external milieu. Depending on their origin, they are given different names: exosomes are nanovesicles that originate from the endosomal compartment, whereas microvesicles bud from plasma membrane. Both contain molecules that are crucial for the onset and spreading of different pathologies, from neurodegenerative diseases to cancer, and are considered promising disease markers. On the other hand, EVs are often used as therapeutic tools, and can be engineered to carry drugs and chemicals. This chapter describes a method to produce EVs, mainly exosomes, containing the green fluorescent protein (GFP) linked to an exosome anchoring protein (Nefmut). This enables counting and tracing of fluorescent EVs by different methods, including conventional flow cytometry.

Different Susceptibilities of Human Melanoma Cell Lines to G2/M Blockage and Cell Death Activation in Response to the Estrogen Receptor ß agonist LY500307.

Background: Gender differences in melanoma incidence, metastasis formation and disease progression are increasingly evident in epidemiological studies, with women showing significantly better survival than men. Among factors possibly underlying the disparities, sex hormones seem to play a key role. Nonetheless, functional mechanisms are still unclear, except for the antitumor ability of Estrogen Receptor (ER) ß, whose expression determination has often been suggested for melanoma prognosis. In this study, we aimed at evaluating the molecular mechanisms and functional effects associated with ERß signaling by using its agonist LY500307. Methods: We evaluated the antitumor effect of the specific synthetic ERß agonist LY500307 on some human melanoma cell lines, selected for different genetic background, expression levels of ERs and tumor progression. The expression of α and ß estrogen receptors was investigated taking advantage of The Cancer Genome Atlas database and confirmed on some selected melanoma cell lines. The biological effects of LY500307 were determined in vitro looking at melanoma cell proliferation, cell cycle profiles and migration demonstrating by western blot the involvement of some pathway specific markers. The LY500307-dependent induction of cell death was also analyzed by flow cytometry and western blot analysis of caspase 3 and poly adenosine diphosphate-ribose polymerase (PARP). Results: A significant decrease in the expression of both ERs, even more pronounced for ERα, has been found in patients with metastatic NRAS mutation. Treatment with LY500307 significantly reduced the proliferation of melanoma cells showing a cell cycle arrest at the G2/M boundary phase and promoting apoptosis with different sensitivities associated with disease stage and mutation. Indeed, the ERß agonist affects melanoma migration, inducing a reversion of the epithelial-mesenchymal transition, more evident in a low aggressive primary melanoma cell line. Conclusion: These results demonstrate the capability of LY500307 to reduce melanoma malignancy, counteracting cell viability and dissemination, overall suggesting a possible future use of LY500307 in personalized combined therapy.

Hyperactive HRAS dysregulates energetic metabolism in fibroblasts from patients with Costello syndrome via enhanced production of reactive oxidizing species.

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.

pH-responsive oleic acid based nanocarriers: Melanoma treatment strategies.

Numerous clinical observations indicate that, despite novel therapeutic approaches, a high percentage of melanoma patients is non-responder or suffers of severe drug-related toxicity. To overcome these problems, we considered the option of designing, preparing and characterizing nanoemulsions and niosomes containing oleic acid, a pH-sensitive monounsaturated fatty acid holding per se an antimetastatic and anti-inflammatory role in melanoma. These new nanostructures will allow in vivo administration of oleic acid, otherwise toxic in its free form. For pulmonary route chitosan, a mucoadhesive agent, was enclosed in these nanocarriers to improve residence time at the lung site. A deep physical and chemical characterization was carried out evaluating size, ζ -potential, microviscosity, polarity as well as stability over time and in culture media. Moreover, their pH-sensitivity was evaluated by fluorometric assay. Cytotoxicity and cellular uptake were assessed in cultured normal fibroblasts and human melanoma cell lines. Interestingly, results obtained confirm nanocarrier stability and pH-sensitivity, associated to absence of cell toxicity, efficient cellular uptake and retention. Therefore, these new pH-sensitive oleic acid-based nanostructures could represent, by combining drug delivery in a pH-dependent manner with the antimetastatic potential of this fatty acid, a powerful strategy for more specific medicine against metastatic melanoma.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Renal cancer: new models and approach for personalizing therapy.

BACKGROUND: Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making. METHODS: Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts. RESULTS: In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways. CONCLUSIONS: In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response.

HIV-1 Nef impairs key functional activities in human macrophages through CD36 downregulation.

Monocytes and macrophages utilize the class A and B scavenger receptors to recognize and perform phagocytosis of invading microbes before a pathogen-specific immune response is generated. HIV-1 Nef protein affects the innate immune system impairing oxidative burst response and phagocytic capacity of macrophages. Our data show that exogenous recombinant myristoylated Nef protein induces a marked CD36 downregulation in monocytes from Peripheral Blood Mononuclear Cells, in Monocyte-Derived Macrophages (MDMs) differentiated by cytokines and in MDMs contained in a mixed culture obtained expanding PBMCs under Human Erythroid Massive Amplification condition. Under the latter culture condition we identify three main populations after 6 days of expansion: lymphocytes (37.8 ± 14.7%), erythroblasts (46.7±6.1%) and MDMs (15.7 ± 7.5%). The Nef addition to the cell culture significantly downregulates CD36 expression in MDMs, but not in erythroid cells. Furthermore, CD36 inhibition is highly specific since it does not modify the expression levels of other MDM markers such as CD14, CD11c, CD86, CD68, CD206, Toll-like Receptor 2 and Toll-like Receptor 4. Similar results were obtained in MDMs infected with VSV-G pseudotyped HIV-1-expressing Nef. The reduced CD36 membrane expression is associated with decrease of correspondent CD36 mRNA transcript. Furthermore, Nef-induced CD36 downregulation is linked to both impaired scavenger activity with reduced capability to take up oxidized lipoproteins and to significant decreased phagocytosis of fluorescent beads and GFP-expressing Salmonella tiphymurium. In addition we observed that Nef induces TNF-α release in MDMs. Although these data suggest a possible involvement of TNF-α in mediating Nef activity, our results exclude a possible relationship between Nef-induced TNF-α release and Nef-mediated CD36 downregulation. The present work shows that HIV-1 Nef protein may have a role in the strategies elaborated by HIV-1 to alter pathogen disease outcomes, by modulating CD36 expression in macrophages, favoring the onset of opportunistic infections in HIV-1 infected people.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRß, TGFß, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

The expression of the glucocorticoid receptor in human erythroblasts is uniquely regulated by KIT ligand: implications for stress erythropoiesis.

Studies in mice indicated that activation of the erythroid stress pathway requires the presence of both soluble KIT ligand (KITL) and the glucocorticoid receptor (GR). To clarify the relative role of KITL and GR in stress erythropoiesis in humans, the biological activities of soluble full length- (fl-, 26-190 aa), carboxy-terminus truncated (tr-, 26-162 aa) human (hKITL) and murine (mKITL) KITL in cultures of cord blood (CB) mononuclear cells (MNCs) and CD34(pos) cells that mimic either steady state (growth factors alone) or stress (growth factors plus dexamethasone [DXM]) erythropoeisis were investigated. In steady state cultures, the KITLs investigated were equally potent in sustaining growth of hematopoietic colonies and expansion of megakaryocytes (MK) and erythroid precursors (EBs). By contrast, under stress erythropoiesis conditions, fl-hKITL generated greater numbers of EBs (fold increase [FI]=140) than tr-hKITL or mKITL (FI=20-40). Flow cytometric analyses indicated that only EBs generated with fl-hKITL remained immature (>70% CD36(pos)/CD235a(neg/low)), and therefore capable to proliferate, until day 8-12 in response to DXM. Signaling studies indicated that all KITLs investigated induced EBs to phosphorylate signal transducer and activator of transcription 5 (STAT5) but that extracellular-signaling-regulated-kinases (ERK) activation was observed mainly in the presence of fl-hKITL. EBs exposed to fl-hKITL also expressed higher levels of GRα than those exposed to mKITL (and tr-hKITL) which were reduced upon exposure to the ERK inhibitor U0126. These data reveal a unique requirement for fl-hKITL in the upregulation of GRα and optimal EB expansion in cultures that mimic stress erythropoiesis.

Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death.

The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.

Humanized culture medium for clinical expansion of human erythroblasts.

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMA(ser)). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6-12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10(-6) M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMA(ser) were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMA(def)). HEMA(def) sustained erythroid amplification as efficiently as HEMA(ser) for cord blood MNC and 10-fold higher than HEMA(ser) for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMA(def) by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.