THE PIVOTAL ROLE OF NOTCH SIGNALING IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL) CHEMOSENSITIVITY

Growing evidence suggests that Notch signaling pathway can modulate drug response in hematological malignancies including T-ALL, B-CLL and AML. In B-ALL we have previously demonstrated that Notch3 and Notch4 support survival of primary B-ALL cells, suggesting a role for Notch signaling in drug response. Here, we used in vitro, in silico, and in vivo approaches to comprehensively the role of Notch pathway in B-ALL pathogenesis in terms of prognosis, proliferation, survival and drug response. B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were isolated from bone marrow or peripheral blood of 51 B-ALL patients. Flow cytometry and western immunoblotting analyses showed that primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy, suggesting that Notch signaling may be critical to drug response in B-ALL. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents (Cytarabine, Ara-C; Dexamethasone, Dexa; Doxorubicin, Doxo) alone or in combination with Notch signaling modulators, including anti-Notch blocking antibodies, gamma secretase inhibitors (GSIs), and Notch transcription factor inhibitor (SAHM1). GSIs and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells, up-regulating intracellular levels of reactive oxygen species (ROS) that were then capable to modulate pro-survival protein levels such as mTor, Akt, NF\u3ba-B and Erk. In vitro observations were successfully translated in mouse-based xenograft models of B-ALL, obtained by injecting the B-ALL line RS4;11 in NOG mice. The in vivo co-administration of Notch inhibitor GSI-XII or anti-Notch4 with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden, thus prolonging mouse survival, compared with DMSO or Ara-C alone. Overall, our results highlighted the prognostic value of Notch expression in B-ALL as well as its critical role in B-ALL cell survival and response to chemotherapy in vitro and in vivo. We demonstrated that inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, improving Ara-C-mediated reduction of blast cells in bone marrow, suggesting that Notch signaling is a possible therapeutic strategy to eradicate the minimal residual disease in B-ALL

Functional dosing of mesenchymal stromal cell-derived extracellular vesicles for the prevention of acute graft-versus-host-disease

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

Targeting the Endothelin-1 Receptors Curtails Tumor Growth and Angiogenesis in Multiple Myeloma

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1 alpha pathways, respectively. HIF-1 alpha silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1 alpha and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches

Unlocking the Potential of Biomarkers for Immune Checkpoint Inhibitors in Cancer Therapy

Immune checkpoint inhibitors (ICIs) are pharmaceutical agents capable of disrupting immune checkpoint signaling, leading to T-cell activation and a robust anti-tumor response [...

Novel Anti-CD117 Antibodies for Rapid and Efficient Hematopoietic Stem Cell Depletion and Safe Bone Marrow Conditioning

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) has proven to be effective for the treatment of hematopoietic disorders, including immune deficiencies, bone marrow failure syndromes (BMFS) and leukemia. However, current preparation for HSCT carries significant toxicity due to radiation and/or chemotherapy that put the patients at high risk of life-threatening infections and toxicity. Severe infections frequently present in the immediate post-HSCT period, while complications caused by chemotoxicity and irradiation damage affect multiple organs at the long term. The high mortality and morbidity rates, although acceptable for malignant diseases, make the risk-benefit ratio too high to apply HSCT for young patients with for instance BMFS and immune deficiencies. The low-risk solution that we propose in this report is the engineering and pre-clinically testing of novel antibodies that rapidly and transiently deplete hematopoietic stem cells (HSCs) to create the desired bone marrow (BM) space that allows for sufficient HSC engraftment. Highly effective HSC depleting antibodies that have a controllable activity in the body would be ideal for rapid and efficient treatment of patients. Such antibodies would facilitate BM conditioning without the risk of infections and toxicity. In this project, we developed and tested novel antibodies that target the c-KIT/CD117 receptor, which is a characteristic molecule on HSCs. METHODS: We developed novel anti-CD117 reagents including anti-CD117 IgG Fc-engineered (anti-CD117) antibody and bispecific T-cell engager (BiTE, CD117xCD3). For our experiments we used an anti-CD117 reference antibody, which is currently tested in clinical trials. We first determined the binding capacity and affinity of these novel reagents to human CD117 expressing TF-1 cell line. The in vitro depletion capacity of our anti-CD117 antibody was tested with an antibody-dependent cytotoxicity reporter Bioassay and NK-cell-mediated cell depletion assays using TF-1 cells and human primary BM-CD34+ cells as targets. The cytotoxicity of the BiTE was shown in vitro in a T-cell-mediated cell depletion assay with the same type of target cells. In addition, we determined T-cell activation, T-cell proliferation and inflammatory cytokine levels. Next, we tested our novel reagents in a CD34+ transplanted humanized mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) for their ability to deplete human CD117+ HSCs. Mice were treated with our BiTE reagents for three days by multiple injections. The percentage of depleted human HSCs in the BM was determined at various time points by flow cytometry. Finally, the levels of the inflammatory cytokines were tested in the serum with ELLA Simple Plex assays. RESULTS: We demonstrated that our novel anti-CD117 reagents bind specifically to CD117 expressed on TF-1 and human CD34+ cells. Our novel anti-CD117 antibody showed an enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), with a more than 2.5 improved killing of TF-1 cells compared to the reference antibody in vitro. Furthermore, we observed a greater than 4-fold improved killing of human CD34+ cells. We demonstrated that our CD117xCD3 BiTE also binds specifically to human T cells leading to a strong T-cell activation, T-cell proliferation, and upregulation of cytokines, in the presence of human CD117+ target cells. In in vitro cytotoxicity assays using our CD117xCD3 BiTE, we observed an 80% killing of TF-1 within 6 hours and 60% depletion of human CD34+ cells at 24 hours, whereas control BiTE did not induce cell death. We observed up to 80% depletion of human CD117+ HSCs in the BM of humanized mice already at 1 day post-treatment, whereas control-treated mice did not show depletion of CD117+ cells. This depletion was even more pronounced and on average 95% at 5 days post-treatment. Notably, no inflammatory cytokines in the serum or obvious toxicities were found in the treated mice. CONCLUSION: We showed that our novel anti-CD117 antibody and CD117xCD3 BiTE exhibit improved cell depletion in vitro. Furthermore, we showed that the CD117xCD3 BiTE rapidly depletes human HSCs in the BM of humanized mice. Our novel anti-CD117 reagents are promising for further development of safe BM conditioning protocols prior to HSCT

The Role of Notch and Wnt Signaling in MSC Communication in Normal and Leukemic Bone Marrow Niche

International audienceNotch and Wnt signaling are highly conserved intercellular communication pathways involved in developmental processes, such as hematopoiesis. Even though data from literature support a role for these two pathways in both physiological hematopoiesis and leukemia, there are still many controversies concerning the nature of their contribution. Early studies, strengthened by findings from T-cell acute lymphoblastic leukemia (T-ALL), have focused their investigation on the mutations in genes encoding for components of the pathways, with limited results except for B-cell chronic lymphocytic leukemia (CLL); in because in other leukemia the two pathways could be hyper-expressed without genetic abnormalities. As normal and malignant hematopoiesis require close and complex interactions between hematopoietic cells and specialized bone marrow (BM) niche cells, recent studies have focused on the role of Notch and Wnt signaling in the context of normal crosstalk between hematopoietic/leukemia cells and stromal components. Amongst the latter, mesenchymal stromal/stem cells (MSCs) play a pivotal role as multipotent non-hematopoietic cells capable of giving rise to most of the BM niche stromal cells, including fibroblasts, adipocytes, and osteocytes. Indeed, MSCs express and secrete a broad pattern of bioactive molecules, including Notch and Wnt molecules, that support all the phases of the hematopoiesis, including self-renewal, proliferation and differentiation. Herein, we provide an overview on recent advances on the contribution of MSC-derived Notch and Wnt signaling to hematopoiesis and leukemia development

Characterization of a new B-ALL cell line with constitutional defect of the Notch signaling pathway

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 \u3b3-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

Small molecule inhibitors of microenvironmental wnt/β-catenin signaling enhance the chemosensitivity of acute myeloid leukemia

International audienceWnt/β-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—in vitro and in vivo—the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of β-catenin, Ser675-phospho-β-catenin and GSK-3α (total and Ser 9) were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/β-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/β-catenin inhibition that may represent a potential new therapeutics strategy in AML

Role of Wnt/\u3b2-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy

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/\u3b2-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/\u3b2-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/\u3b2-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/\u3b2-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 \u3b2-catenin only in about 2/3 of primary samples analyzed, while . \u3b2-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of \u3b2-catenin, including (Ser675)\u3b2-catenin and non-phospho-(Ser33/37/Thr41) \u3b2-catenin. Notably, we found that active forms of \u3b2-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 \u3b2-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/\u3b2-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects