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Background The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. Methods Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O 2 ) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. Results We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. Conclusions Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Rhabdomyosarcoma (RMS) is marked by a myogenesis differentiation blockade, and while the AKT/mTOR pathway is universally activated, its pharmacological inhibition has shown limited success. Here, we evaluate the activity of pan-AKT inhibitors Ipatasertib, ATP-competitive, and Miransertib, allosteric inhibitor, in RMS cell lines and fusion-positive/negative patient-derived xenografts (PDX). Unlike Miransertib, Ipatasertib show significant antitumor activity against a subset of RMS. Besides AKT, the other target of Ipatasertib, but not of Miransertib, is PRKG1, a cGMP-dependent protein kinase that shares the ATP binding pocket with AKT. We investigate the role of PRKG1 in PRKG1-depleted RMS cells and in xenograft models by transcriptomic approaches. PRKG1 silencing in RMS cells reduces tumor formation in xenograft models and induces a differentiated myogenic transcriptome. RMS show higher PRKG1 expression compared to any other developmental cancer, akin to fetal skeletal muscle. Importantly, PRKG1 expression in RMS correlates with mesodermal transcriptional signature and enhanced sensitivity to Ipatasertib, regardless of the fusion oncogene status. The antitumor activity of Ipatasertib is dose-dependent, reaching an effective intra-tumor concentration when administered at 25 mg/kg daily. This study unveils the role of PRKG1 in myogenesis and highlights the potential of PRKG1 as a clinical biomarker for Ipatasertib therapy in RMS. Despite frequent AKT/mTOR pathway activation in patient’s rhabdomyosarcoma (RMS), success of AKT inhibitors in the clinical has been limited. Here, using RMS patient-derived models, the authors demonstrate that the efficacy of the AKT inhibitor, ipatasertib, is in part due to its off-target effects on PRKG1, identifying PRKG1 as a potential biomarker for ipatasertib response.

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized by EWSR::ETS rearrangements whose cellular origin remains unclear. EWS::FLI1 expression in human pediatric mesenchymal stem cells (MSCs) induces a transcriptional response distinct from that of human adult MSCs, but fails to form tumors. Here we show that EWS::FLI1 expression in human embryonic mesenchymal stem cells (heMSCs) results in the acquisition of an ES transcriptome, with the oncogene not preferentially binding to gene promoters, but to intronic and intergenic microsatellites. In heMSCs, EWS::FLI1 directly regulates the expression of the DNA repair protein BRCA1, although cells expressing EWS::FLI1 show DNA damage. Xenografting of EWS::FLI1-transduced heMSCs results in the formation of tumors expressing characteristic ES markers. In summary, we show that EWS::FLI1 enforces an aberrant transcriptome and solely is able to endow transforming capacity when expressed in undifferentiated, early heMSCs. Ewing sarcoma (ES) is characterized by EWSR1/ETS gene rearrangements with unknown cellular origin. Here, authors show that expressing the EWS::FLI1 oncogene in human embryonic mesenchymal stem cells induces an Ewing sarcoma-like transcriptome and forms tumors in mice.

Neuroblastoma is a pediatric cancer that can present as low- or high-risk tumors (LR-NBs and HR-NBs), the latter group showing poor prognosis due to metastasis and strong resistance to current therapy. Whether LR-NBs and HR-NBs differ in the way they exploit the transcriptional program underlying their neural crest, sympatho-adrenal origin remains unclear. Here, we identified the transcriptional signature distinguishing LR-NBs from HR-NBs, which consists mainly of genes that belong to the core sympatho-adrenal developmental program and are associated with favorable patient prognosis and with diminished disease progression. Gain- and loss-of-function experiments revealed that the top candidate gene of this signature, Neurexophilin-1 (NXPH1) , has a dual impact on NB cell behavior in vivo: whereas NXPH1 and its receptor α-NRXN1 promote NB tumor growth by stimulating cell proliferation, they conversely inhibit organotropic colonization and metastasis. As suggested by RNA-seq analyses, these effects might result from the ability of NXPH1/α-NRXN signalling to restrain the conversion of NB cells from an adrenergic state to a mesenchymal one. Our findings thus uncover a transcriptional module of the sympatho-adrenal program that opposes neuroblastoma malignancy by impeding metastasis, and pinpoint NXPH1/α-NRXN signaling as a promising target to treat HR-NBs.

Neuroblastoma is the most common extracranial solid tumor of childhood, with heterogeneous clinical manifestations ranging from spontaneous regression to aggressive metastatic disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that senses plasmatic fluctuation in the extracellular concentration of calcium and plays a key role in maintaining calcium homeostasis. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma. The activation of CaSR with cinacalcet, a positive allosteric modulator of CaSR, reduces neuroblastoma tumor growth by promoting differentiation, endoplasmic reticulum (ER) stress and apoptosis. However, cinacalcet treatment results in unmanageable hypocalcemia in patients. Based on the bias signaling shown by calcimimetics, we aimed to identify a new drug that might exert tumor-growth inhibition similar to cinacalcet, without affecting plasma calcium levels. We identified a structurally different calcimimetic, AC-265347, as a promising therapeutic agent for neuroblastoma, since it reduced tumor growth by induction of differentiation, without affecting plasma calcium levels. Microarray analysis suggested biased allosteric modulation of the CaSR signaling by AC-265347 and cinacalcet towards distinct intracellular pathways. No upregulation of genes involved in calcium signaling and ER stress were observed in patient-derived xenografts (PDX) models exposed to AC-265347. Moreover, the most significant upregulated biological pathways promoted by AC-265347 were linked to RHO GTPases signaling. AC-265347 upregulated cancer testis antigens (CTAs), providing new opportunities for CTA-based immunotherapies. Taken together, this study highlights the importance of the biased allosteric modulation when targeting GPCRs in cancer. More importantly, the capacity of AC-265347 to promote differentiation of malignant neuroblastoma cells provides new opportunities, alone or in combination with other drugs, to treat high-risk neuroblastoma patients.

Ewing sarcoma (ES) is an aggressive bone and soft tissue neoplasm characterized by EWSR1/ETS rearrangements and whose cellular origin remains unclear. EWS-FLI1 expression in human pediatric mesenchymal stem cells (hpMSCs) induces a quantitatively and qualitatively different transcriptional response than its expression in human adult MSCs (haMSCs), but fails to form tumors in vivo. ES cells have early developmental lineage signatures distinct from postnatal MSCs. Here, we have generated MSCs from experimental teratomas out of human embryonic stem cells (heSCs). Transduction of these human embryonic mesenchymal stem cells (heMSCs) with EWS-FLI1 results in the acquisition of an ES transcriptome, although the oncogene does not preferentially bind to promoters, but to intronic and intergenic microsatellites with >10 CA dinucleotides and GGAA repeats, respectively. In heMSCs, EWS-FLI1 directly regulates BRCA1 expression, although EWS-FLI1-expressing cells show defects in DNA damage repair. Xenografting of EWS-FLI1-transduced heMSCs resulted in the formation of tumors expressing characteristic ES markers. In summary, EWS-FLI1 enforces an aberrant transcriptome and endows in vivo transforming capacity when expressed in an undifferentiated early heMSC. Our approach represents an innovative experimental method for understanding critical aspects of the biology of developmental tumors, from leukemia to sarcomas, in which few (even single) genetic alterations are able to transform a fetal stem cell.Competing Interest StatementThe authors have declared no competing interest.

Fundoscopy is the standard method for diagnosis and follow‐up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell‐free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow‐up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus‐encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT‐ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non‐cancer patients). According to clinical criteria, we grouped patients as having progression‐free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus‐derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression‐free patients and control non‐cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut‐off point for discriminating between progressive and progression‐free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut‐off point in those patients responding to chemotherapy. In contrast, one non‐responder patient remained with values above the cut‐off during follow‐up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow‐up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.

Background: Few studies have been undertaken to detect the presence of cardiovascular risk factors (CRFs) in healthy populations (individuals auto-reported as healthy). These risk factors include high body mass index (BMI), high waist-to-hip ratio (WHR), high systolic and diastolic blood pressure (SBP, DBP), high uric acid and high Castelli’s risk index (CRI); this last is the ratio of total cholesterol to HDL cholesterol (TC/HDL-c). In addition, the correlations between CRFs and the biomarker C-reactive protein (CRP) has not been explored in each sex. Aim: Therefore, this study aimed to determine sex differences in the abnormalities in blood and urine analyses, including CRFs and their correlation with CPR in a non-representative sample of healthy Mexican individuals. Results: A total of 238 subjects were included, 123 (51.7%) of whom were women. The main blood alterations detected were high serum lipids, including high total cholesterol, LDL-cholesterol, triglycerides, and the CRI, which were higher in men than in women. The men’s samples had a higher frequency of hypertensives and pre-hypertensives than the women’s sample. The CRP showed positive significant correlations with the CRFs: BMI, WHR, SBP, DBP, uric acid, and the CRI, with a higher correlation for BMI and WHR, and most of these correlations were higher in women than in men. Additionally, all these factors showed a positive correlation among them. Conclusion: In conclusion, the main alterations observed in blood are related to cardiovascular risk and were reported with a higher frequency in men when compared with women. This finding can be related to the higher values of WHR in this sex; additionally, the inflammatory marker CRP was more correlated with the cardiometabolic risk factors in women than in men, which suggests a different relationship between inflammation and cardiometabolic risk factors in each sex.

Neuroblastoma is a pediatric cancer that can present as low- or high-risk tumors (LR-NBs and HR-NBs), the latter group showing poor prognosis due to metastasis and strong resistance to current therapy. NBs are known to originate from alterations to cells in the sympatho-adrenal lineage derived from the neural crest, but whether LR-NBs and HR-NBs differ in the way they exploit the transcriptional program underlying their developmental origin remains unclear. Here, we compared the transcriptional landscapes of primary samples of LR-NBs, HR-NBs and human fetal adrenal gland, and thereby identified the transcriptional signature associated to NB formation that further distinguishes LR-NBs from HR-NBs. The majority of the genes comprising this signature belong to the core sympatho-adrenal developmental program, are associated with favorable patient prognosis and with diminished disease progression. The top candidate gene of this list, Neurexophilin-1 (NXPH1), encodes a ligand of the transmembrane receptors α-Neurexins (α-NRXNs). Our functional in vivo and in vitro assays reveal that NXPH1/α-NRXN signaling has a dual impact on NB behavior: whereas NXPH1 and α-NRXN1 promote NB tumor growth by stimulating cell proliferation, they conversely inhibit the ability of NB cells to form metastases. Our findings uncover a module of the neural crest-derived sympatho-adrenal developmental program that opposes neuroblastoma malignancy by impeding metastasis, and pinpoint NXPH1/α-NRXN signaling as a promising target to treat HR-NBs.

Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management.