PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells.

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity.

Advances in Azorella glabra Wedd. Extract Research: In Vitro Antioxidant Activity, Antiproliferative Effects on Acute Myeloid Leukemia Cells and Bioactive Compound Characterization.

Azorella glabra Wedd. (AG) is traditionally used to treat gonorrhea or kidney's problems. The antioxidant, antidiabetic, anticholinesterase and in vitro antitumor activities of AG extracts were recently reported. The aim of this work was to investigate anti-leukemic properties of AG chloroform fraction (AG CHCl3) and of its ten sub-fractions (I-X) and to identify their possible bioactive compounds. We determined their in vitro antioxidant activity using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), nitric oxide (NO) and superoxide anion (SO) assays, and their phytochemical profile by spectrophotometric and LC-MS/MS techniques. I-X action on two acute myeloid leukemia (AML) cell lines viability, apoptosis and cell cycle were evaluated by MTS, western blotting and cytofluorimetric assays. Different polyphenol, flavonoid and terpenoid amount, and antioxidant activity were found among all samples. Most of I-X induced a dose/time dependent reduction of cell viability higher than parent extract. IV and VI sub-fractions showed highest cytotoxic activity and, of note, a negligible reduction of healthy cell viability. They activated intrinsic apoptotic pathway, induced a G0/G1 block in leukemic cells and, interestingly, led to apoptosis in patient AML cells. These activities could be due to mulinic acid or azorellane terpenoids and their derivatives, tentatively identified in both IV and VI. In conclusion, our data suggest AG plant as a source of potential anti-AML agents.

Smenamide A Analogues. Synthesis and Biological Activity on Multiple Myeloma Cells.

Smenamides are an intriguing class of peptide/polyketide molecules of marine origin showing antiproliferative activity against lung cancer Calu-1 cells at nanomolar concentrations through a clear pro-apoptotic mechanism. To probe the role of the activity-determining structural features, the 16-epi-analogue of smenamide A and eight simplified analogues in the 16-epi series were prepared using a flexible synthetic route. The synthetic analogues were tested on multiple myeloma (MM) cell lines showing that the configuration at C-16 slightly affects the activity, since the 16-epi-derivative is still active at nanomolar concentrations. Interestingly, it was found that the truncated compound 8, mainly composed of the pyrrolinone terminus, was not active, while compound 13, essentially lacking the pyrrolinone moiety, was 1000-fold less active than the intact substance and was the most active among all the synthesized compounds.

MicroRNAs as New Biomarkers for Diagnosis and Prognosis, and as Potential Therapeutic Targets in Acute Myeloid Leukemia.

Acute myeloid leukemias (AML) are clonal disorders of hematopoietic progenitor cells which are characterized by relevant heterogeneity in terms of phenotypic, genotypic, and clinical features. Among the genetic aberrations that control disease development there are microRNAs (miRNAs). miRNAs are small non-coding RNAs that regulate, at post-transcriptional level, translation and stability of mRNAs. It is now established that deregulated miRNA expression is a prominent feature in AML. Functional studies have shown that miRNAs play an important role in AML pathogenesis and miRNA expression signatures are associated with chemotherapy response and clinical outcome. In this review we summarized miRNA signature in AML with different cytogenetic, molecular and clinical characteristics. Moreover, we reviewed the miRNA regulatory network in AML pathogenesis and we discussed the potential use of cellular and circulating miRNAs as biomarkers for diagnosis and prognosis and as therapeutic targets.

Future in the Past: Azorella glabra Wedd. as a Source of New Natural Compounds with Antiproliferative and Cytotoxic Activity on Multiple Myeloma Cells.

Multiple myeloma (MM) is the second most common hematologic malignancy and, although the development of novel agents has improved survival of patients, to date, it remains incurable. Thus, newer and more effective therapeutic strategies against this malignancy are necessary. Plant extracts play an important role in anti-tumor drug discovery. For this reason, in the investigation of novel natural anti-MM agents, we evaluated the phytochemical profiles, in vitro antioxidant activity, and effects on MM cells of Azorella glabra (AG) Wedd. Total polyphenols (TPC), flavonoids (TFC), and terpenoids (TTeC) contents were different among samples and the richest fractions in polyphenols demonstrated a higher antioxidant activity in in vitro assays. Some fractions showed a dose and time dependent anti-proliferative activity on MM cells. The chloroform fraction (CHCl3) showed major effects in terms of reduction of cell viability, induction of apoptosis, and cell cycle arrest on MM cells. The apoptosis induction was also confirmed by the activation of caspase-3. Importantly, the CHCl3 fraction exhibited a negligible effect on the viability of healthy cells. These results encourage further investigations on AG extracts to identify specific bioactive compounds and to define their potential applications in MM.

Mesenchymal Stem Cell Derived Extracellular Vesicles: A Role in Hematopoietic Transplantation?

Mesenchymal stem cells (MSCs) are a heterogeneous cellular population containing different progenitors able to repair tissues, support hematopoiesis, and modulate immune and inflammatory responses. Several clinical trials have used MSCs in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent hematopoietic stem cell (HSC) engraftment failure, reduce aplasia post chemotherapy, and to control graft versus host disease (GvHD). The efficacy of MSCs is linked to their immune suppressive and anti-inflammatory properties primarily due to the release of soluble factors. Recent studies indicate that most of these effects are mediated by extracellular vesicles (EVs). MSC-EVs have therefore therapeutic effects in regenerative medicine, tumor inhibition, and immune-regulation. MSC-EVs may offer specific advantages for patient safety, such as lower propensity to trigger innate and adaptive immune responses. It has been also shown that MSC-EVs can prevent or treat acute-GvHD by modulating the immune-response and, combined with HSCs, may contribute to the hematopoietic microenvironment reconstitution. Finally, MSC-EVs may provide a new potential therapeutic option (e.g., transplantation, gene therapy) for different diseases, particularly hematological malignancies. In this review, we will describe MSC and MSC-EVs role in improving allo-HSCT procedures and in treating GvHD.

Extracellular Vesicles in Hematological Malignancies: From Biology to Therapy.

Extracellular vesicles (EVs) are a heterogeneous group of particles, between 15 nanometers and 10 microns in diameter, released by almost all cell types in physiological and pathological conditions, including tumors. EVs have recently emerged as particularly interesting informative vehicles, so that they could be considered a true "cell biopsy". Indeed, EV cargo, including proteins, lipids, and nucleic acids, generally reflects the nature and status of the origin cells. In some cases, EVs are enriched of peculiar molecular cargo, thus suggesting at least a degree of specific cellular packaging. EVs are identified as important and critical players in intercellular communications in short and long distance interplays. Here, we examine the physiological role of EVs and their activity in cross-talk between bone marrow microenvironment and neoplastic cells in hematological malignancies (HMs). In these diseases, HM EVs can modify tumor and bone marrow microenvironment, making the latter "stronger" in supporting malignancy, inducing drug resistance, and suppressing the immune system. Moreover, EVs are abundant in biologic fluids and protect their molecular cargo against degradation. For these and other "natural" characteristics, EVs could be potential biomarkers in a context of HM liquid biopsy and therapeutic tools. These aspects will be also analyzed in this review.

What Does Atypical Chronic Lymphocytic Leukemia Really Mean? A Retrospective Morphological and Immunophenotypic Study.

Atypical chronic lymphocytic leukemia (CLL) is still defined according to morphological criteria. However, deviance from the typical surface immunological profile suggests an atypical immunological-based CLL. A large cohort of patients with CLL was retrospectively evaluated aiming at assessing morphological (FAB criteria), immunophenotypical (two or more discordances from the typical profile), and clinical-biological features of atypical CLL. Compared to typical cases, morphologically atypical CLL showed a greater percentage of unmutated IgVH and CD38 positivity, and a higher expression of CD20. Immunophenotypically atypical CLL was characterized by more advanced clinical stages, higher expression of CD20, higher rate of FMC7, CD79b and CD49d positivity, and by an intermediate-high expression of membrane surface immunoglobulin, compared to typical cases. When patients were categorized based on immunophenotypic and morphologic concordance or discordance, no difference emerged. Finally, morphological features better discriminated patients' prognosis in terms of time-to-first treatment, while concordant atypical cases showed overall a worse prognosis. Discordant cases by immunophenotype and/or morphology did not identify specific prognostic groups. Whether-in the era of molecular markers used as prognostic indicators-it does make sense to focus on morphology and immunophenotype features in CLL is still matter of debate needing further research.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. METHODS: A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. RESULTS: Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. CONCLUSION: These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Acute Myeloid Leukemia Cells Functionally Compromise Hematopoietic Stem/Progenitor Cells Inhibiting Normal Hematopoiesis Through the Release of Extracellular Vesicles.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). It is not well known how leukemia cells alter hematopoiesis promoting tumor growth and leukemic niche formation. In this study, we investigated how AML deregulates the hematopoietic process of HSPCs through the release of extracellular vesicles (EVs). First, we found that AML cells released a heterogeneous population of EVs containing microRNAs involved in AML pathogenesis. Notably, AML-EVs were able to influence the fate of HSPCs modifying their transcriptome. In fact, gene expression profile of AML-EV-treated HSPCs identified 923 down- and 630 up-regulated genes involved in hematopoiesis/differentiation, inflammatory cytokine production and cell movement. Indeed, most of the down-regulated genes are targeted by AML-EV-derived miRNAs. Furthermore, we demonstrated that AML-EVs were able to affect HSPC phenotype, modifying several biological functions, such as inhibiting cell differentiation and clonogenicity, activating inflammatory cytokine production and compromising cell movement. Indeed, a redistribution of HSPC populations was observed in AML-EV treated cells with a significant increase in the frequency of common myeloid progenitors and a reduction in granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors. This effect was accompanied by a reduction in HSPC colony formation. AML-EV treatment of HSPCs increased the levels of CCL3, IL-1B and CSF2 cytokines, involved in the inflammatory process and in cell movement, and decreased CXCR4 expression associated with a reduction of SDF-1 mediated-migration. In conclusion, this study demonstrates the existence of a powerful communication between AML cells and HSPCs, mediated by EVs, which suppresses normal hematopoiesis and potentially contributes to create a leukemic niche favorable to neoplastic development.

Case report: Hematologic malignancies concomitant diagnosis of hairy cell leukemia and chronic lymphocytic leukemia: A rare association.

A case of concomitant hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) in a 50- year-old man was reported. Flow cytometry and droplet digital PCR (ddPCR) were used to detect the B-Raf proto-oncogene (BRAF) V600E mutation. The HCL population was the predominant component. The patient was first treated with cladribine and then with rituximab and achieved HCL partial remission. Importantly, the high sensitivity of our flow cytometric approach allowed the detection of a small population "P3," in addition to the typical HCL and CLL clones. The P3 clone changed over time, from an HCL-like to a CLL-like immunophenotype. This case is added to the few other cases of synchronous HCL and CLL already reported in the literature and underlines the importance of analyzing chronic lymphoproliferative disorders by highly sensitive diagnostic techniques, like the multicolor flow cytometry and ddPCR, to evaluate the possible association between HCL and CLL at diagnosis.

Clinical relevance of extracellular vesicles in hematological neoplasms: from liquid biopsy to cell biopsy.

In the era of precision medicine, liquid biopsy is becoming increasingly important in oncology. It consists in the isolation and analysis of tumor-derived biomarkers, including extracellular vesicles (EVs), in body fluids. EVs are lipid bilayer-enclosed particles, heterogeneous in size and molecular composition, released from both normal and neoplastic cells. In tumor context, EVs are valuable carriers of cancer information; in fact, their amount, phenotype and molecular cargo, including proteins, lipids, metabolites and nucleic acids, mirror nature and origin of parental cells rendering EVs appealing candidates as novel biomarkers. Translation of these new potential diagnostic tools into clinical practice could deeply revolutionize the cancer field mainly for solid tumors but for hematological neoplasms, too.

Multiple Myeloma-Derived Extracellular Vesicles Impair Normal Hematopoiesis by Acting on Hematopoietic Stem and Progenitor Cells.

Multiple myeloma (MM) is characterized by the abnormal proliferation of clonal plasma cells (PCs) in bone marrow (BM). MM-PCs progressively occupy and likely alter BM niches where reside hematopoietic stem and progenitor cells (HSPCs) whose viability, self-renewal, proliferation, commitment, and differentiation are essential for normal hematopoiesis. Extracellular vesicles (EVs) are particles released by normal and neoplastic cells, such as MM cells. They are important cell-to-cell communicators able to modify the phenotype, genotype, and the fate of the recipient cells. Investigation of mechanisms and mediators underlying HSPC-MM-PC crosstalk is warranted to better understand the MM hematopoietic impairment and for the identification of novel therapeutic strategies against this incurable malignancy. This study is aimed to evaluate whether EVs released by MM-PCs interact with HSPCs, what effects they exert, and the underlying mechanisms involved. Therefore, we investigated the viability, cell cycle, phenotype, clonogenicity, and microRNA profile of HSPCs exposed to MM cell line-released EVs (MM-EVs). Our data showed that: (i) MM cells released a heterogeneous population of EVs; (ii) MM-EVs caused a dose-dependent reduction of HSPCs viability; (iii) MM-EVs caused a redistribution of the HSPC pool characterized by a significant increase in the frequency of stem and early precursors accompanied by a reduction of late precursor cells, such as common myeloid progenitors (CMPs), megakaryocyte erythroid progenitors (MEPs), B and NK progenitors, and a slight increase of granulocyte macrophage progenitors (GMPs); (iv) MM-EVs caused an increase of stem and early precursors in S phase with a decreased number of cells in G0/G1 phase in a dose-dependent manner; (v) MM-EVs reduced the HSPC colony formation; and (vi) MM-EVs caused an increased expression level of C-X-C motif chemokine receptor type 4 (CXCR4) and activation of miRNAs. In conclusion, MM cells through the release of EVs, by acting directly on normal HSPCs, negatively dysregulate normal hematopoiesis, and this could have important therapeutic implications.

Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma.

INTRODUCTION: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established. METHODS: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR. RESULTS: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters. CONCLUSION: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.

CD200 Baseline Serum Levels Predict Prognosis of Chronic Lymphocytic Leukemia.

Membrane-bound CD200 is overexpressed in chronic lymphocytic leukemia (CLL), and there is some evidence that its soluble ectodomain (sCD200) could also be involved in the pathophysiology and the disease. However, very little is known about sCD200's prognostic significance. sCD200 was tested at diagnosis in 272 patients with CLL and in 78 age- and sex-matched healthy subjects using a specific human CD200 (OX-2 membrane glycoprotein) ELISA kit. A significantly higher concentration of sCD200 was found in CLL patients compared to controls. In our cohort, sCD200 was significantly higher in patients who were older than 66 years, with Binet stage C, unmutated IgVH and unfavorable (del11q or del17p) FISH. Time-to-first treatment and overall survival were significantly shorter in patients with higher sCD200 concentration, using as a cut-off 1281 pg/mL, the median value for sCD200 concentration in the whole CLL cohort. However, the prognostic impact of sCD200 was not confirmed in multivariate analysis. Baseline sCD200 values appeared to have an impact on the response to chemotherapy or chemo-immunotherapy, but not to targeted agents. Collectively, our data show that sCD200 serum levels correlate with more aggressive clinical and biological features and are able to predict a worse prognosis. This work supports the relevant role of CD200 not only as a diagnostic tool but also as a prognostic indicator and a potential therapeutic target in CLL.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

An update on extracellular vesicles in multiple myeloma: a focus on their role in cell-to-cell cross-talk and as potential liquid biopsy biomarkers.

INTRODUCTION: Multiple myeloma (MM) is characterized by a clonal proliferation of neoplastic plasma cells (PCs) in bone marrow (BM) and the interplay between MM PCs and the BM microenvironment, which plays a relevant role in its pathogenesis. In this important cross-talk, extracellular vesicles (EVs) are active. EVs, including small and medium/large EVs, are lipid bi-layer particles released in circulation by normal and neoplastic cells. A selected cargo of lipids, proteins, and nucleic acids is loaded into EVs, and delivered locally and to distant sites, thus influencing the physiology of recipient cells. In the 'liquid biopsy' context, EVs can be isolated from human biofluids proving to be powerful markers in cancer. Areas covered: Here, we summarize the recent advances on EVs in MM field. Expert commentary: EVs from MM PCs: i) enhance malignant cell proliferation and aggressiveness through an autocrine loop; ii) are able to transfer drug resistance in sensitive-drug cells; iii) stimulate angiogenesis; iv) increase the activity of osteoclasts; v) have immunosuppressive effects. In addition, EVs from MM stromal cells also promote MM cell proliferation and drug resistance. Finally, we underline the importance of EVs as MM potential biomarkers in 'cancer liquid biopsy' and as a potential new therapeutic target.

Extracellular Vesicles: A New Prospective in Crosstalk between Microenvironment and Stem Cells in Hematological Malignancies.

The bone marrow (BM) microenvironment in hematological malignancies (HMs) comprises heterogeneous populations of neoplastic and nonneoplastic cells. Cancer stem cells (CSCs), neoplastic cells, hematopoietic stem cells (HSCs), and mesenchymal stromal/stem cells (MSCs) are all components of this microenvironment. CSCs are the HM initiators and are associated with neoplastic growth and drug resistance, while HSCs are able to reconstitute the entire hematopoietic system; finally, MSCs actively support hematopoiesis. In some HMs, CSCs and neoplastic cells compromise the normal development of HSCs and perturb BM-MSCs. In response, "reprogrammed" MSCs generate a favorable environment to support neoplastic cells. Extracellular vesicles (EVs) are an important cell-to-cell communication type in physiological and pathological conditions. In particular, in HMs, EV secretion participates to unidirectional and bidirectional interactions between neoplastic cells and BM cells. The transfer of EV molecular cargo triggers different responses in target cells; in particular, malignant EVs modify the BM environment in favor of neoplastic cells at the expense of normal HSCs, by interfering with antineoplastic immunity and participating in resistance to treatment. Here, we review the role of EVs in BM cell communication in physiological conditions and in HMs, focusing on the effects of BM niche EVs on HSCs and MSCs.