Active notch protects MAPK activated melanoma cell lines from MEK inhibitor cobimetinib

The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAFV600E metastatic melanoma (MM) and promotes migration in GNAQQ209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAFV600E and BRAF wt MM and GNAQQ209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAFV600E MM and GNAQQ209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAFV600E MM and GNAQQ209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQQ209L UM cells treated with the MEK inhibitor

Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma

Background: The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the identifcation and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1+ EV mediate resistance to anti-PD1, instead the role of PD1+ EV is not fully understood. Methods: We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1+ EVs and PD1+ EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identifed EV-based biomarkers. Additionally we assessed the involvement of PD1+ EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids. Results: The level of PD-L1+ EVs released from melanoma and CD8+ T cells and that of PD1+ EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were signifcantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Signifcant correlations were found for PD-L1+ EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1+ EVs, from T cells and B cells, and high level of PD-L1+ EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1+ EVs from melanoma and PD1+ EVs from T cells in predicting PFS was confrmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafcking and tumor spheroids killing. Conclusion: Our study identifed circulating PD1+ EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy

The Interaction between Reactive Peritoneal Mesothelial Cells and Tumor Cells via Extracellular Vesicles Facilitates Colorectal Cancer Dissemination

Simple SummaryEmerging evidence has suggested that cancer-derived extracellular vesicles (EVs) have a crucial role in mediating directional metastasis to the peritoneal surface in colorectal cancer (CRC). We investigated the EV-mediated crosstalk between tumor and mesothelial cells which may drive remodeling of the premetastatic niche to allow tumor spread to the peritoneal surface. Our findings demonstrated that cancer-derived EVs triggered apoptosis and reduced mesothelial cell invasiveness and mesothelial-to-mesenchymal transition. On the other hand, mesothelial cells actively supported tumor invasion by releasing EVs, which induced upregulation of the major pro-invasive system in tumor cells. For the first time, we provide evidence of EV-driven mechanisms of CRC progression in patient-derived models, highlighting the crucial role of EVs in the reprogramming of mesothelial and tumor cells to establish the metastatic process.Advanced colorectal cancer (CRC) is highly metastatic and often results in peritoneal dissemination. The extracellular vesicles (EVs) released by cancer cells in the microenvironment are important mediators of tumor metastasis. We investigated the contribution of EV-mediated interaction between peritoneal mesothelial cells (MCs) and CRC cells in generating a pro-metastatic environment in the peritoneal cavity. Peritoneal MCs isolated from peritoneal lavage fluids displayed high CD44 expression, substantial mesothelial-to-mesenchymal transition (MMT) and released EVs that both directed tumor invasion and caused reprogramming of secretory profiles by increasing TGF-beta 1 and uPA/uPAR expression and MMP-2/9 activation in tumor cells. Notably, the EVs released by tumor cells induced apoptosis by activating caspase-3, peritoneal MC senescence, and MMT, thereby augmenting the tumor-promoting potential of these cells in the peritoneal cavity. By using pantoprazole, we reduced the biogenesis of EVs and their pro-tumor functions. In conclusion, our findings provided evidence of underlying mechanisms of CRC dissemination driven by the interaction of peritoneal MCs and tumor cells via the EVs released in the peritoneal cavity, which may have important implications for the clinical management of patients

SMARCE1-related meningiomas: A clear example of cancer predisposing syndrome

We report the case of a 16-year-old girl presenting with spinal clear-cell multiple meningiomas (CCMs). In view of this presentation, we sequenced a bioinformatic panel of genes associated with susceptibility to meningioma, identifying a germline heterozygous variant in SMARCE1. Somatic DNA investigations in the CCM demonstrated the deletion of the wild-type allele (loss of heterozygosity, LOH), supporting the causative role of this variant. Family segregation study detected the SMARCE1 variant in the asymptomatic father and in the asymptomatic sister who, nevertheless, presents 2 spinal lesions. Germline heterozygous loss-of-function (LoF) variants in SMARCE1, encoding a protein of the chromatin-remodeling complex SWI/SNF, have been described in few fa-milial cases of susceptibility to meningioma, in particular the CCM subtype. Our case confirms the role of NGS in investigating predisposing genes for meningiomas (multiple or recurrent), with specific regard to SMARCE1 in case of pediatric CCM. In addition to the age of onset, the presence of familial clustering or the coexistence of multiple synchronous meningiomas also supports the role of a genetic predisposition that deserves a molecular assessment. Additionally, given the incomplete penetrance, it is of great importance to follow a specific screening or follow-up program for symptomatic and asymptomatic carriers of pathogenic variants in SMARCE1

Cervical cancer benefits from trabectedin combination with the β-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting β-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity.Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry.Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines.Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by β-adrenergic receptor activation in both ovarian and cervical cancer models

Combination of an Antigen-Specific Therapy and an Immunomodulatory Treatment to Simultaneous Block Recurrent Autoimmunity and Alloreactivity in Non-Obese Diabetic Mice

<div><p>Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity <i>via</i> antigen-specific immunization, and alloreactivity <i>via</i> granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB_9-23 peptide alone or in combination with two islet peptides (IGRP_206-214and GAD_524-543) in incomplete Freund’s adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB_9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB_9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4⁺CD25⁺ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB_9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB_9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.</p></div

G-CSF/RAPA treatment induces transplant tolerance to allogeneic islets in NOD mice that depends on IL-10 production.

<p>A, spontaneously diabetic female NOD mice were transplanted with allogeneic islets from BALB/c donors. Recipients were treated with G-CSF/RAPA and monitored for graft survival. Anti-IL-10 was administered in NOD mice transplanted and treated with G-CSF/RAPA. Arrow indicates the time when anti-IL-10 mAb treatments initiated. Overall graft survival is shown. Graph shows the percentage of islet graft survival after transplantation. B, correlation between blood glucose levels at the time of transplant and the number of days of syngeneic islet engraftment. C, graph shows the percentage of islet graft survival in G-CSF/RAPA vs. G-CSF/RAPA/insB_9–23/IFA-treated recipients. Survival curves were compared with the log-rank test. <i>P</i> values are indicated in the graphs.</p

InsB_9–23/IFA immunization temporarily controls recurrent autoimmunity in NOD mice.

<p>A, diabetic NOD mice were transplanted with islets from NOD donors (syngeneic) and treated with insB_9–23/IFA or PBS/IFA. Percentage of islet graft survival after transplantation is shown. B, correlation between blood glucose levels at the time of transplant and the number of days of syngeneic islet engraftment for all mice. Each symbol represents one mouse. Survival curves were compared with the log-rank test. Correlation was done with Pearson coefficient. <i>P</i> values and R² values are indicated in the graphs.</p

InsB_9–23/IFA treatment efficacy is dependent on CD4⁺CD25⁺ Treg cells.

<p>Spontaneously diabetic female NOD mice were transplanted with syngeneic islets and with insB_9–23/IFA. A, some mice were received anti-CD25 mAb administration injected at the same time of transplantation or B, 10 days after transplantation. Arrows indicate the time when anti-CD25 mAb treatments initiated. Overall graft survival is shown. Survival curves were compared with the log-rank test. <i>P</i> values are indicated in the graphs.</p