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Despite high expression of PD-L1, around half of advanced non-small cell lung cancer (NSCLC) will not experience tumor response with pembrolizumab. There is an need for a better understanding of the resistance mechanisms in this setting.This bi-centric retrospective study included all consecutive patients with PDL1 ≥ 50% advanced NSCLC treated with pembrolizumab in first-line treatment between 2016 and 2020. We compared the clinical characteristics of patients with early progression (refractory) vs others. We performed a comprehensive gene expression profile screening by RNAseq capture on tumor samples.We included 46 patients. Twenty-two patients were refractory to pembrolizumab, mainly women, with poor performance status and lower albumin concentration. RNAseq analysis was performed on 19 samples. Hierarchical clustering allowed the identification of 3 clusters with various proportion of refractory tumors: intermediate (C1: 57%), high (C2: 71%) and low proportion (C3: 40%). Comparative analysis between C2 and C3 allowed the identification of overexpressed (n = 137) and underexpressed (n = 40) genes. Among the genes of interest, C2 exhibits higher activation of pathways associated with stemness phenotype (Hedgehog, Notch and Hippo pathways) and pathways associated with loss of PTEN and JAK2. In C2, genes associated with PD-1, toll-like receptor-9 (TLR-9), major histocompatibility complex (MHC) and interferon-γ pathways were underexpressed.This study gives an overview of activated and downregulated pathways in high PD-L1 NSCLC refractory to pembrolizumab. These tumors showed activation of pathways associated with cancer stem cells, loss of PTEN and JAK2, and inhibition of both priming and effector phases of the immune response.

Notch3 and Notch4 support survival of primary B-cell acute lymphoblastic leukemia (B-ALL) cells, suggesting a role for Notch signaling in drug response. Here we used in vitro, in silico, and in vivo mouse xenograft model-based approaches to define the role of the Notch pathway in B-ALL chemosensitivity. We observed significant Notch receptor and ligand expression in B-ALL primary cells and cell lines. Primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents alone or in combination with Notch signaling inhibitors. Gamma-secretase inhibitors (GSI) and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells by upregulating intracellular levels of reactive oxygen species, which in turn modulated mTOR, NF-κB, and ERK expression. In NOG-mouse-based xenograft models of B-ALL, co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden compared with DMSO or Ara-C alone, thus prolonging mouse survival. Overall, our results support the potential effectiveness of Notch inhibitors in patients with B-ALL.Significance: Inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, suggesting Notch inhibition as a potential therapeutic strategy to improve the outcome of patients with B-ALL.

Supplemental Methods describes methods that were not thoroughly described in the main manuscript as well as methods used to produce supplemental data. Table S1 describes characteristics of patients involved in the study. Table S2 describes genes potentially damages in 5 B-ALL patients following DNA sequencing. Table S3 describes, epigenetics patterns of ALL patients as obtained following the processing of methylation data publicly available under accession number GSE49031. Table S4 describes Chi Square analysis of the association between Notch expression level and response to therapy. Table S5 describes B-ALL cell survival upon treatment with Notch ligands. Figure S1 describes expression of Notch target genes and Notch active forms in B-ALL samples. This figure also displays the validation of L5C5 antibody in HEK 293T cells. Figures S2 describes the sensitivity (Annexin V) of B-ALL samples to Ara-C, Doxorubicin and Dexamethasone, each used alone or in combination with anti-Notch3 or anti-Notch4. Figure S3 describes the sensitivity (Annexin V) of B-ALL samples to Ara-C, Doxorubicin and Dexamethasone, each used alone or in combination with different Notch inhibitors. The aim is to screen the most active (in term of cell death) association in vitro, that will then be used in vivo. Figures S4 presents how anti-oxidants NAC and BME protect B-ALL cells from cell death induced by drug treatments.

Graft-vs-host-disease (GvHD) is currently the main complication of allogeneic hematopoietic stem cell transplantation. Mortality and morbidity rates are particularly high, especially in steroid-refractory acute GvHD (aGvHD). Immune regulatory human bone marrow mesenchymal stromal cells (hMB-MSCs) represent a therapeutic approach to address this issue. Unfortunately, their effect is hardly predictable in vivo due to several variables, that is, MSC tissue origin, concentration, dose number, administration route and timing, and inflammatory status of the recipient. Interestingly, human bone marrow MSC-derived extracellular vesicles (hBM-MSC-EVs) display many of the hBM-MSC immunoregulatory properties due to their content in paracrine factors that greatly varies according to the collection method. In this study, we focused on the immunological characterization of hBM-MSC-EVs on their capability of inducing regulatory T-cells (T-regs) both in vitro and in a xenograft mouse model of aGvHD. We correlated these data with the aGvHD incidence and degree following hBM-MSC-EV intravenous administration. Thus, we first quantified the EV immunomodulation in vitro in terms of EV immunomodulatory functional unit (EV-IFU), that is, the lowest concentration of EVs leading in vitro to at least threefold increase of the T-regs compared with controls. Second, we established the EV therapeutic dose in vivo (EV-TD) corresponding to 10-fold the in vitro EV-IFU. According to this approach, we observed a significant improvement of both mouse survival and control of aGvHD onset and progression. This study confirms that EVs may represent an alternative to whole MSCs for aGvHD prevention, once the effective dose is reproducibly identified according to EV-IFU and EV-TD definition.

Extracellular vesicles (EVs) are membrane-coated particles secreted by virtually all cell types in response to different stimuli, both in physiological and pathological conditions. Their content generally reflects their biological functions and includes a variety of molecules, such as nucleic acids, proteins and cellular components. The role of EVs as signaling vehicles has been widely demonstrated. In particular, they are actively involved in the pathogenesis of several hematological malignancies (HM), mainly interacting with a number of target cells and inducing functional and epigenetic changes. In this regard, by releasing their cargo, EVs play a pivotal role in the bilateral cross-talk between tumor microenvironment and cancer cells, thus facilitating mechanisms of immune escape and supporting tumor growth and progression. Recent advances in high-throughput technologies have allowed the deep characterization and functional interpretation of EV content. In this review, the current knowledge on the high-throughput technology-based characterization of EV cargo in HM is summarized.

Simple Summary Wnt and Hedgehog (Hh) pathways are associated with stemness profile and with resistance to anti-tumor therapies. Pre-clinical data have suggested a role of these pathways in resistance to immune checkpoint inhibitors (ICIs). Here we evaluated the expression and levels of Shh and Wnt molecules both in plasma and tumor samples in a prospective cohort of 63 consecutive NSCLC patients treated with ICIs. We found that baseline Hh activation as assessed by nuclear staining of Gli1 was associated with poor outcome and high primary resistance rate. Increase of plasma Wnt1 during treatment was associated with resistance to ICIs. This is the first study to our knowledge to find an association between the Hh and Wnt pathways and resistance to ICIs in advanced NSCLC patients. We believe that these results are important to understand the molecular mechanisms associated with resistance to ICIs, and ultimately to improve future treatment strategies. Abstract Hedgehog (Hh) and Wingless-type (Wnt) pathways are associated with resistance to immune checkpoint inhibitors (ICIs) in preclinical studies. This study aimed to assess the association between expression and activation levels of Wnt and Sonic Hedgehog (Shh) pathways and resistance to ICIs in advanced NSCLC patients treated with ICI. Hh and Wnt pathways activation was assessed by immunohistochemistry (Gli1 and beta-catenin) on corresponding tumor tissues, and by plasma concentrations of Shh and Wnt (Wnt1, Wnt2 and Wnt3) at ICI introduction and at the first clinical evaluation. Sixty-three patients were included, with 36 patients (57.1%) with available tissue. Response rate was lower in Gli1+ NSCLC (20.0%) compared to Gli1 negative (Gli-) NSCLC (55.6%) (p = 0.015). Rate of primary resistance was 69.8%, vs. 31.2%, respectively (p = 0.04), and median progression-free survival (PFS) was 1.9 months (interquartile range (IQR) 1.2–5.7) vs. 6.1 months (1.6–26.0), respectively (p = 0.08). Median PFS and overall survival were shorter in case of increase of Wnt1 concentration during ICI treatment compared to other patients: 3.9 months vs. 11.2 months (p = 0.008), and 15.3 months vs. not reached (p = 0.003). In conclusion, baseline activation of Hh pathway and increase of Wnt1 concentrations during ICI treatment were associated with poor outcome in NSCLC patients treated with ICIs.

Immune checkpoint inhibitors (ICIs) are commonly used in patients with advanced non-small cell lung cancer (NSCLC). An unmet need remains for new biomarkers associated with ICIs. In this study, consecutive patients with advanced NSCLC treated with nivolumab or pembrolizumab were included. Plasma at ICIs initiation was prospectively collected and a multiplex ELISA assay testing 48 cytokines and growth factors was performed. Exploratory endpoints were the association between plasma biomarkers with outcome and grade III&ndash, IV immune related adverse events (irAEs). Thirty-five patients were included. Patients without clinical benefit (n = 22) had higher pre-ICI soluble Hepatocyte Growth Factor (sHGF) (210.9 vs. 155.8 pg/mL, p = 0.010), lower pre-ICI soluble Fibroblast Growth Factor (sFGF) (4.0 vs. 4.8 pg/mL, p = 0.043) and lower pre-ICI interleukine-12 (IL-12) (1.3 vs. 2.2 pg/mL, p = 0.043) concentrations. Patients with early progression (n = 23) had higher pre-ICIs sHGF (206.2 vs. 155.8 pg/mL, p = 0.025) concentrations. Patients with low sHGF levels at ICIs initiation had longer progression-free survival and overall survival than those with high sHGF levels: respectively 2.5 vs. 8.0 months (p = 0.002), and 5.5 vs. 35.0 months (p = 0.001). TNF-&alpha, IL-16, IL-12p40 and MCP3 were associated with high grade irAEs. This study shows the potential association between several plasma biomarkers with outcome and grade 3&ndash, 4 IrAEs in advanced NSCLC treated with ICIs.

Wnt/&beta, catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied&mdash, in vitro and in vivo&mdash, the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of &beta, catenin, Ser675-phospho-&beta, catenin and GSK-3&alpha, (total and Ser 9) were found in AML cells from intermediate or poor risk patients, nevertheless, patients presenting high activity of Wnt/&beta, catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 (LiCl, AR-A014418), and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/&beta, catenin inhibition that may represent a potential new therapeutics strategy in AML.

BackgroundDespite prolonged tumor response to immune checkpoint inhibitors (ICIs) for a subset of patients with advanced non-small cell lung cancer (NSCLC), a secondary resistance will occur for a majority of these patients. The understanding of late progression mechanisms with ICIs is important to improve future treatment strategies.MethodsWe performed whole-exome sequencing (WES) on circulating tumor DNA and compared molecular profiles between the beginning of ICI treatment and tumor progression in patients with advanced NSCLC treated with ICIs and who had initial and prolonged tumor response with secondary progression, after at least 6 months of treatment.ResultsWe identified eight patients who experienced initial and durable tumor response, and secondary tumor progression after 6 months of treatment, with available paired blood samples (diagnosis and progression). All had lung adenocarcinoma, three had programmed-death ligand-1 expression ≥50% in immunohistochemistry and all presented low blood tumor mutational burden (bTMB). Seven patients received nivolumab in second-line or more, and one received pembrolizumab as first-line treatment. WES at progression showed clonal selection with molecular alterations of Wnt pathway-related genes, increase of copy number aberrations in cancer-related genes and loss of tumor-suppressor genes (such as PTEN) or of genes associated with immune response (such as B2M). No difference in term of bTMB was observed at progression.ConclusionsThis is the first study describing putative molecular mechanisms associated with late progression under ICI in lung cancer. Studies on treatment strategies adapted to these mechanisms are needed.

Protein tyrosine phosphatase receptor type &gamma, (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. &beta, catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between &beta, catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene &beta, Catenin. PTPRG was found to be capable of dephosphorylating &beta, catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of &beta, catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and &beta, catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and &beta, catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.

// Giorgio Malpeli 1, 2 , Stefano Barbi 2 , Gabriele Tosadori 3 , Corinna Greco 4 , Simonetta Zupo 5 , Serena Pedron 2 , Matteo Brunelli 2 , Anna Bertolaso 2 , Maria Teresa Scupoli 6 , Mauro Krampera 4 , Paul Takam Kamga 3 , Carlo Maria Croce 7 , George Adrian Calin 8 , Aldo Scarpa 2, 9 and Alberto Zamo 10 1 Department of Surgical Sciences, Dentistry, Gynecology and Pediatrics, Section of Surgery, University of Verona, Verona, Italy 2 Department of Diagnostics and Public Health, University of Verona, Verona, Italy 3 Center for BioMedical Computing, University of Verona, Verona, Italy 4 Department of Medicine, Section of Hematology, Stem Cell Research Laboratory, University of Verona, Verona, Italy 5 Laboratory of Molecular Diagnostics, IRCCS-AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 6 Department of Medicine, Section of Hematology, University of Verona, Verona, Italy 7 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 8 Department of Experimental Therapeutics and The Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 Applied Research on Cancer-Network (ARC-NET), University of Verona, Verona, Italy 10 Department of Oncology, University of Turin, Torino, Italy Correspondence to: Giorgio Malpeli, email: giorgio.malpeli@univr.it Keywords: microRNAs; MYC; non-Hodgkin B-cell lymphoma; cell lines; network analysis Received: October 26, 2017 Accepted: April 28, 2018 Published: July 03, 2018 ABSTRACT In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt’s lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC -translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.

// Paul Takam Kamga 1 , Giada Dal Collo 1 , Giulio Bassi 1 , Martina Midolo 1 , Massimo Delledonne 2, 3 , Marco Chilosi 4 , Massimiliano Bonifacio 1 and Mauro Krampera 1 1 Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona, Italy 2 Department of Biotechnology, University of Verona, Verona, Italy 3 Personal Genomics S.R.L., Verona, Italy 4 Section of Pathology, Department of Diagnostics and Public Health, University of Verona, Verona, Italy Correspondence to: Mauro Krampera, email: mauro.krampera@univr.it Keywords: Notch signaling; B-acute lymphoblastic leukemia; B-ALL; Alagille syndrome; ALGS Received: January 02, 2018 Accepted: March 11, 2018 Published: April 06, 2018 ABSTRACT Notch signaling contribution to B-cell acute lymphoblastic leukemia (B-ALL) development is still under investigation. The serendipitous onset of B-ALL in a patient affected by the germinal Notch mutation-dependent Alagille syndrome allowed us to establish a B-ALL cell line (VR-ALL) bearing a genetic loss of function in components of Notch signaling. VR-ALL is a common-type B-ALL cell line, grows in conventional culture medium supplemented with 10% serum, and gives rise, once injected into immunodeficient NOG mice, to a mouse xenograft model of B-ALL. Exome sequencing revealed deleterious mutations in some components of Notch signaling, including Jagged1, Notch1, and Notch2. In addition, VR-ALL is sensitive both in vitro and in vivo to γ-secretase inhibitors (GSIs) as well as conventional anti-leukemic drugs. For all these reasons, VR-ALL may help to gain more insights into the role of Notch signaling in B-ALL.

International audience; Mesenchymal stem/stromal cells (MSCs) are multipotent cells residing in the stromal tissues of the body and capable of promoting tissue repair and attenuating inflammatory processes through their immunomodulatory properties. Preclinical and clinical observations revealed that not only direct intercellular communication mediates MSC properties; in fact, a pivotal role is also played by the release of soluble and bioactive factors, such as cytokines, growth factor and extracellular vesicles (EVs). EVs are membrane-coated vesicles containing a large variety of bioactive molecules, including lipids, proteins, and nucleic acids, such as RNA. EVs release their contents into target cells, thus influencing cell fate through the control of intracellular processes. In addition, MSC-derived EVs can mediate modulatory effects toward different effector cells belonging to both innate and adaptive immunity. In this review, we will discuss the literature data concerning MSC-derived EVs, including the current standardized methods for their isolation and characterization, the mechanisms supporting their immunoregulatory properties, and their potential clinical application as alternative to MSC-based therapy for inflammatory reactions, such as graft-versus-host disease (GvHD).

The role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1&ndash, 4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0&ndash, M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan&ndash, Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML.

// Paul Takam Kamga 1 , Giulio Bassi 1 , Adriana Cassaro 1 , Martina Midolo 1 , Mariano Di Trapani 1 , Alessandro Gatti 1 , Roberta Carusone 1 , Federica Resci 1 , Omar Perbellini 1 , Michele Gottardi 2 , Massimiliano Bonifacio 1 , Armel Herve Nwabo Kamdje 3 , Achille Ambrosetti 1 and Mauro Krampera 1 1 Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona, Italy 2 Division of Hematology, Ca’ Foncello Hospital, Treviso, Italy 3 Department of Biomedical Sciences, University of Ngaoundere-Cameroon, Cameroon Correspondence to: Mauro Krampera, email: // Keywords : AML, MSC, Notch, chemoresistance Received : February 10, 2016 Accepted : February 25, 2016 Published : March 07, 2016 Abstract Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB. These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

Notch3 and Notch4 support survival of primary B-cell acute lymphoblastic leukemia (B-ALL) cells, suggesting a role for Notch signaling in drug response. Here we used in vitro, in silico, and in vivo mouse xenograft model-based approaches to define the role of the Notch pathway in B-ALL chemosensitivity. We observed significant Notch receptor and ligand expression in B-ALL primary cells and cell lines. Primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents alone or in combination with Notch signaling inhibitors. Gamma-secretase inhibitors (GSI) and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells by upregulating intracellular levels of reactive oxygen species, which in turn modulated mTOR, NF-κB, and ERK expression. In NOG-mouse-based xenograft models of B-ALL, co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden compared with DMSO or Ara-C alone, thus prolonging mouse survival. Overall, our results support the potential effectiveness of Notch inhibitors in patients with B-ALL. Significance: Inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, suggesting Notch inhibition as a potential therapeutic strategy to improve the outcome of patients with B-ALL.

// Giorgio Malpeli 1, 2 , Stefano Barbi 2 , Corinna Greco 3 , Simonetta Zupo 4 , Anna Bertolaso 2 , Maria Teresa Scupoli 5 , Mauro Krampera 3 , Paul Takam Kamga 3 , Carlo Maria Croce 6 , Aldo Scarpa 2, 7 and Alberto Zamo 2, 8 1 Department of Surgical Sciences, Dentistry, Gynecology and Pediatrics, Section of Surgery, University of Verona, Verona, Italy 2 Department of Diagnostics and Public Health, University of Verona, Verona, Italy 3 Department of Medicine, Section of Hematology, Stem Cell Research Laboratory, University of Verona, Italy 4 Laboratory of Molecular Diagnostics, IRCCS-AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 5 Department of Medicine, Section of Hematology, University of Verona, Verona, Italy 6 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 7 Applied Research on Cancer-Network, ARC-NET, University of Verona, Verona, Italy 8 Department of Oncology, University of Torino, Torino, Italy Correspondence to: Giorgio Malpeli, email: giorgio.malpeli@univr.it Keywords: follicular lymphoma; relapse; microRNAs; Foxp3; heterogeneity Received: August 23, 2017 Accepted: January 30, 2018 Published: April 13, 2018 ABSTRACT First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3 + cells number. Notably, Foxp3 + cells were significantly less in relapsed patients and lower Foxp3 + cell number associated with shorter time-to-relapse. Foxp3 + cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3 + cells might be predictive of disease relapse.

Background: Solid epidemiological evidences connect obesity with incidence, stage and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. Methods: We studied the role of factors secreted by two adipogenic model systems from primary human bone marrow stromal cells (hBMSCs) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS), Results: We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

Background : Cervical cancer is a major cause of women death worldwide. The reduction of the mortality and morbidity of this pathology depends on the early detection based on powerful suitable screening methods, that will lead to optimal treatment strategies. However in some rural region of developing countries, it is very difficult to get access to standard screening methods, alternative screening methods, cheaper and easy to handle are then useful. Objective : The aim of this work was to test the sensitivity and specificity of VIA (Visual inspection with acetic acid) and VILI (Visual inspection with lugol iodine) as a diagnostic test of cervical cancer compared to the Pap Smear, evaluating the feasibility in health formation in the North Cameroon region, of implementing epidemiological surveillance of cervical cancer based on early diagnosis using the VIA-VILI association Method : 309 women age 20 to 62 years were recruited in this study, 307 were included in the statistical analyzes. Each woman was screened for cervical cancer by a conventional Smear and visual inspection with acetic acid 5% and the lugol solution. Results : We found in our study a prevalence of precancerous lesions of cervix at 12.70%. The risks factors of cervical cancer identified are age, matrimonial status, age of first sexual intercourse and parity. The association of VIA and VILI showed a sensitivity, specificity, positive and negative predictive value respectively about 93.58%; 97.01%; 82.01%, 99.04%. Conclusion : Compared to PAP Smear, VIA or VILI could be used as an alternative screening methods for cervical cancer in developing countries, where it is difficult to access to more accurate test such as colposcopy and biopsy.

Immune checkpoint inhibitors (ICIs) have transformed the treatment landscape for patients with non-small cell lung cancer (NSCLC). Although some patients can experience important response rates and improved survival, many others do not benefit from ICIs developing hyper-progressive disease or immune-related adverse events. This underlines the need to select biomarkers for ICIs use in order to better select patients. There is currently no universally validated robust biomarker for daily use of ICIs. Programmed death-ligand 1 (PD-L1) or tumor mutational burden (TMB) are sometimes used but still have several limitations. Plasma biomarkers are a promising approach in ICI treatment. This review will describe the development of novel plasma biomarkers such as soluble proteins, circulating tumor DNA (ctDNA), blood TMB, and blood microbiome in NSCLC patients treated with ICIs and their potential use in predicting response and toxicity.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells which may reside as pericytes in various tissues and organs, where they may differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites, but also in tumors, which are known to behave like “wounds that do not heal.” Herein, we discuss the mechanisms used by MSCs to migrate, home, and repair injured tissues, and we review a number of reports showing that the tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and the therapeutic potential of MSCs are also discussed.

Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/β-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/β-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/β-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment. Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/β-catenin (PKF118-310, PNU-74654). Results: In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total β-catenin only in about 2/3 of primary samples analyzed, while . β-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of β-catenin, including (Ser675)β-catenin and non-phospho-(Ser33/37/Thr41) β-catenin. Notably, we found that active forms of β-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of β-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/β-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects. Disclosures Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

Leukemia is a cancer of the early blood-forming cells. Most often, leukemia is a cancer of the white blood cells, but some leukemias start in other blood cell types. Scientists in the U.S. believe they have identified a new pathway in the progression of chronic myelogenous leukemia (CML). They have also discovered that an extract from the root of a common ornamental plant can suppress the process. Drug discovery from natural sources involve a multifaceted approach combining botanical, phytochemical, biological, and molecular techniques. Accordingly, medicinal-plant-based drug discovery still remains an important area, hitherto unexplored, where a systematic search may definitely provide important leads against various pharmacological targets. Ironically, the potential benefits of plant-based medicines have led to unscientific exploitation of the natural resources, a phenomenon that is being observed globally. This decline in biodiversity is largely the result of the rise in the global population, rapid and sometimes unplanned industrialization, indiscriminate deforestation, and overexploitation of natural resources, pollution, and finally global climate change. Therefore, it is of utmost importance that plant biodiversity be preserved, to provide future structural diversity and lead compounds for the sustainable development of human civilization at large. This becomes even more important for developing nations, where well-planned bioprospecting coupled with nondestructive commercialization could help in the conservation of biodiversity, ultimately benefiting mankind in the long run. Based on these findings, the present review is an attempt to update our knowledge about the role of signaling pathways and medicinal plants in Leukemia therapy.

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAGGED-1 (ALGS type 1), and more rarely NOTCH-2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism. A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL). In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH-2 (E38K) and JAGGED-1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH-1 (K1821N), which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development that binds NOTCH-2 and likely altering its functionality. On the other hand, NOTCH-1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Introduction: Our group has recently shown that bone marrow-mesenchymal stromal cell (BM-MSCs)-mediated Notch signaling may control survival and chemoresistance of B-acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) cells. Conversely, the role of Notch signaling in acute myeloid leukemia (AML) remains controversial, as its contribution to the crosstalk between BM-MSCs and AML cells is still unknown. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML primary blast cells in co-culture with BM-MSCs. Methods: AML blast cells were obtained after informed consent from bone marrow samples (30) and peripheral blood (20) of AML patients, according to the Institutional guidelines. BM-MSCs were expanded from bone marrow of 12 healthy donors (BM-MSCs) and of 12 AML-patients (BM-MSCs*). PCR, FACS analysis and western immunoblotting were used to study the expression of Notch receptors and ligands, as well as Notch activation status, in AML cells and BM-MSCs. AML cells were co-cultured with BM-MSCs or BM-MSCs* at 10:1 (AML:BM-MSCs) ratio for 2 to 3 days in presence of Cytarabine, Etoposide, Idarubicin, as well as in presence or absence of anti-Notch-1, -2, -3, -4, anti-Jagged1, -2 and anti-DLL3 blocking antibodies or gamma secretase inhibitor-XII (GSI-XII). Cell viability was evaluated by Annexin-V/Propidium Iodide (PI) and MTT; proliferation and cell cycle were assessed through CFSE dilution and PI methods, respectively. Results: AML cells expressed Notch receptors and ligands, showing Notch-1, -2, Jagged-1, -2 and DLL-3 signature, while BM-MSCs/ BM-MSCs* showed expression of Notch-1, -2, -3, -4 and Jagged-1, -2 and DLL-1. We then analyzed Notch activation in different cell types by evaluating the expression of NICD-1, -2, -3 as well as Hes-1. We found that at least 50% of AML samples showed basal Notch activation while BM-MSCs/ BM-MSCs* showed slight Notch activation. The expression and activation pattern were modulated after 3 days of co-culture with either BM-MSCs or BM-MSCs*. The pan blockade of Notch signaling by GSI-XII were capable to inhibit AML cell proliferation as well as induce AML cell apoptosis in culture or in co-culture with BM-MSCs/ BM-MSCs*. The addition of chemotherapeutic agents decreased AML cell viability in culture, while a significant rescue from apoptosis was observed when cocultured with BM-MSCs or BM-MSCs*. Pan Notch signaling blockade by either GSI-XII or combination of Notch receptor-blocking antibodies in presence of chemotherapeutic agents significantly lowered the supportive role of BM-MSCs towards AML cell lines. The specific blockade of Notch-1, -2, -3 or Jagged-1, -2 rescued partially the chemosensibility, while blockade of Notch-4 or DLL-3 rescued totally the chemosensitivity of primary AML cells in co-culture with BM-MSCs. Conclusions: These results suggest that Notch signaling may represent a potential therapeutic strategy to overcome bone marrow stromal-mediated survival and chemoresistance of AML. Moreover Notch blocking antibodies were able to impair the survival benefit imparted by bone marrow stromal cells. Therefore blocking Notch antibodies could be a useful strategy to improve the efficiency of AML chemotherapy. Disclosures No relevant conflicts of interest to declare.

Stromal cells are essential components of the bone marrow (BM) microenvironment regulating and supporting the survival of different tumors, including B-cell acute and chronic lymphocytic leukemia (B-ALL and CLL), and acute myeloid leukemia (AML). In this study, we investigated the role of Notch signalling in human BM-mesenchymal stromal cell (hBM-MSC)-promoted ALL, CLL and AML survival and chemoresistance. The block of Notch signalling through γ-secretase inhibitor (GSI) XII reverted the protective effect mediated by co-culture with BM-MSC. The treatment with combinations of anti-Notch neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of BM-MSC from normal donors and B-ALL patients. The inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. The same Notch receptors are involved in CLL survival except for Notch-1 that, in CLL, mediates a synergistic effect with other Notch receptors in inducing the anti-apoptotic phenotype. Some preliminary data showed that Notch system is involved in survival and chemoresistance of acute myeloid leukemia blasts. Overall, our findings show that stromal cell-mediated Notch signaling has a role in promoting ALL, CLL and AML survival and resistance to chemotherapy. Therefore, the target of Notch pathway activation may represent a useful strategy to overcome drug resistance and improve the efficacy of conventional treatments. Disclosures: No relevant conflicts of interest to declare.

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes. Disclosures Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.