DNA methylation of hematopoietic stem/progenitor cells from donor peripheral blood to patient bone marrow: implications for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) is a life-saving treatment for selected hematological malignancies. So far, it remains unclear whether transplanted hematopoietic stem/progenitor cells (HSPCs) undergo epigenetic changes upon engraftment in recipient bone marrow (BM) after AHSCT and whether these changes might be useful in the transplant diagnostics. The purpose of this study was to characterize the whole genome methylation profile of HSPCs following AHSCT. Moreover, the relationship between the observed methylation signature and patient outcome was analyzed. Mobilized peripheral blood (mPB)-HSPCs from seven donors and BM-HSPCs longitudinally collected from transplanted patients with hematological malignancies up to one year from AHSCT (a total of twenty-eight samples) were analyzed using DNA methylation based-arrays. The obtained data showed that DNA methylation of mPB-HSPCs differs between young and adult donors and changes following HSPC engraftment in the BM of recipient patients. Looking at methylation in promoter regions, at 30 days post-AHSCT, BM-HSPCs showed a higher number of differentially methylated genes (DMGs) compared to those of mPB-HSPCs, with a prevalent hyper-methylation. These changes were maintained during all the analyzed time points, and methylation became like the donors after one year from transplant. Functional analysis of these DMGs showed an enrichment in cell adhesion, differentiation and cytokine (interleukin-2, -5 and -7) production and signaling pathways. Of note, DNA methylation analysis allowed to identify a potential "cancer/graft methylation signature" of transplant failure. It was evident in the latest available post-transplant BM-HSPC sample (at 160 days) and surprisingly already in early phase (at 30 days) in patients whose transplant was doomed to fail. Overall, the analysis of HSPC methylation profile could offer useful prognostic information to potentially assess engraftment success and predict graft failure in AHSCT.

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.

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.

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.

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.

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.

A case of acute promyelocytic leukemia variant with derivative chromosome 3 der(3)t(3;8) associated with 8q partial gain.

BACKGROUND: Acute promyelocytic leukemia (APL) is characterized by fusion of PML/RARα genes as a result of t(15;17)(q24;q21). APL is now one of the curable hematological malignancies thanks to molecularly targeted therapies based on all-trans retinoic acid (ATRA) and arsenic trioxide (ATX). Extramedullary (EM) relapse is a rare event in APL, ear involvement being even more infrequent, with only six cases so far described. About 30-35% of patients with newly diagnosed APL have additional cytogenetics abnormalities, whose prognostic significance is still controversial. The most common additional aberration is trisomy 8 or partial gain 8q. CASE PRESENTATION: We describe here a novel unbalanced translocation der(3)t(3;8)(q29;q23.3-q24.3) associated with 8q partial gain in a 41 year-old man affected by APL in molecular remission after first line treatment, who had a responsive EM relapse in the auditory canal. CONCLUSIONS: EM relapse is a rare event in APL and ear involvement is even more infrequent. To our knowledge, this is the first reported case of APL with a new der(3)t(3;8)(q29;q23.3-q24.3) and 8q partial gain associated with t(15;17)(q24;q21). Despite the recurrence of the disease at EM level, the clinical outcome of this patients was favorable.

DNA methylation dynamic of bone marrow hematopoietic stem cells after allogeneic transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative therapeutic approach for different hematological malignancies (HMs), and epigenetic modifications, including DNA methylation, play a role in the reconstitution of the hematopoietic system after AHSCT. This study aimed to explore global DNA methylation dynamic of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) from donors and their respective recipients affected by acute myeloid leukemia (AML), acute lymphoid leukemia (ALL) and Hodgkin lymphoma (HL) during the first year after transplant. METHODS: We measured DNA methylation profile by Illumina HumanMethylationEPIC in BM HSPC of 10 donors (t0) and their matched recipients at different time points after AHSCT, at day + 30 (t1), + 60 (t2), + 120 (t3), + 180 (t4), and + 365 (t5). Differential methylation analysis was performed by using R software and CRAN/Bioconductor packages. Gene set enrichment analysis was carried out on promoter area of significantly differentially methylated genes by clusterProfiler package and the mSigDB genes sets. RESULTS: Results show significant differences in the global methylation profile between HL and acute leukemias, and between patients with mixed and complete chimerism, with a strong methylation change, with prevailing hyper-methylation, occurring 30 days after AHSCT. Functional analysis of promoter methylation changes identified genes involved in hematopoietic cell activation, differentiation, shaping, and movement. This could be a consequence of donor cell "adaptation" in recipient BM niche. Interestingly, this epigenetic remodeling was reversible, since methylation returns similar to that of donor HSPCs after 1 year. Only for a pool of genes, mainly involved in dynamic shaping and trafficking, the DNA methylation changes acquired after 30 days were maintained for up to 1 year post-transplant. Finally, preliminary data suggest that the methylation profile could be used as predictor of relapse in ALL. CONCLUSIONS: Overall, these data provide insights into the DNA methylation changes of HSPCs after transplantation and a new framework to investigate epigenetics of AHSCT and its outcomes.

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.

An exploratory study by DMET array identifies a germline signature associated with imatinib response in gastrointestinal stromal tumor.

Imatinib represents the standard therapy for gastrointestinal stromal tumor (GIST) patients with metastatic/unresectable disease. Despite  the excellent results achieved with its introduction, the majority of patients quite invariably experience disease progression. The aim of this study was to understand the contribution of germline DNA polymorphisms in discriminating between imatinib clinical response [evaluated as progression free survival (PFS)] and toxicity. In particular, a discovery cohort (34 GIST with a KIT exon 11 primary mutation, and no toxicity) was analyzed through DMET array that interrogates 1936 variants in 231 genes of the ADME process. We further confirmed the genotype of selected variants in an extended cohort of 49 patients (the original cohort and 15 new cases, all with exon 11 primary mutation), identifying 6 SNPs- ABCB4 rs1202283, ABCC2 rs2273697, ABCG1 rs1541290, CYP11B1 rs7003319, CYP7B1 rs6987861, and NQO1 rs10517-significantly associated with response to imatinib. Three SNPs, ABCB4 rs1202283, ABCC2 rs2273697, and NQO1 rs10517, which had a significant association after adjusted multivariate analysis, were included in a genetic prediction model. We confirmed that these SNPs could stratify the cohort of 49 patients according to the risk of developing progression under imatinib treatment. In conclusion, we identified a genetic signature of response to imatinib therapy in GIST patients able to stratify patients at low and high risk to progress, according to their genotype.

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.

Global methylation patterns in primary plasma cell leukemia.

Primary plasma cell leukemia (pPCL) is a rare and very aggressive variant of multiple myeloma (MM). Specific clinical, biological and molecular patterns distinguish pPCL from MM. Here, we performed a genome-wide methylation analysis by high-density array in 14 newly diagnosed pPCL patients along with 60 MMs, and 5 patients affected by monoclonal gammopathy of uncertain significance (MGUS). Our analysis revealed a global hypomethylation profile associated with pPCL. Additionally, differential methylation patterns were found related to distinct chromosomal aberrations and DIS3 mutations, affecting genes with roles in bone metabolism, cell migration, transcription regulation or DNA damage response. When compared with MM patients, pPCL showed a distinct methylation profile mostly characterized by hypomethylated probes specific for genes involved in several processes like cell adhesion and migration. Furthermore, decreasing methylation levels were evidenced for genes significantly modulated in the progressive phases of plasma cell dyscrasias, from MGUS to MM and pPCL. Overall, our data provide new insights into the molecular characterization of pPCL, thus being potentially useful in the prognostic stratification or identification of novel molecular targets.

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.

IL6/STAT3 axis mediates resistance to BRAF inhibitors in thyroid carcinoma cells.

Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 26-53% of human thyroid malignancies and, differently from melanomas, are poorly sensitive to BRAF inhibitors (BRAFi), and develop acquired resistance through activation of alternative signaling pathways. A whole-genome gene expression analysis of TC BRAF V600E cells exposed to PLX4032 identified JAK/STAT among the most significantly modulated signaling pathways. Interestingly, both transient exposure and chronic adaptation to PLX4032 resulted in upregulation of IL6/STAT3 axis and this impaired the cytostatic activity of PLX4032. Mechanistically, exposure to PLX4032 enhanced IL6 secretion and this, in turn, was responsible for STAT3 upregulation, activation of ERK signaling and poor sensitivity to BRAF inhibition. Consistently, the dual blockade of STAT3 (by siRNA or pharmacological inhibition) or IL6 signaling (by the humanized anti-human IL6 receptor antibody, tocilizumab) and BRAF (by PLX4032) improved the inhibition of cell cycle progression compared to PLX4032 single agent. These data support the role of IL6/STAT3 signaling pathway in modulating TC cell response to PLX4032 and candidate IL6 targeting as a strategy to improve the activity of PLX4032 in BRAF V600E TC cells.

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.

EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells.

PURPOSE: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. METHODS: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. RESULTS: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. CONCLUSIONS: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

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.

Knockdown of miR-128a induces Lin28a expression and reverts myeloid differentiation blockage in acute myeloid leukemia.

Lin28A is a highly conserved RNA-binding protein that concurs to control the balance between stemness and differentiation in several tissue lineages. Here, we report the role of miR-128a/Lin28A axis in blocking cell differentiation in acute myeloid leukemia (AML), a genetically heterogeneous disease characterized by abnormally controlled proliferation of myeloid progenitor cells accompanied by partial or total inability to undergo terminal differentiation. First, we found Lin28A underexpressed in blast cells from AML patients and AML cell lines as compared with CD34+ normal precursors. In vitro transfection of Lin28A in NPM1-mutated OCI-AML3 cell line significantly triggered cell-cycle arrest and myeloid differentiation, with increased expression of macrophage associate genes (EGR2, ZFP36 and ANXA1). Furthermore, miR-128a, a negative regulator of Lin28A, was found overexpressed in AML cells compared with normal precursors, especially in acute promyelocytic leukemia (APL) and in 'AML with maturation' (according to 2016 WHO classification of myeloid neoplasms and acute leukemia). Its forced overexpression by lentiviral infection in OCI-AML3 downregulated Lin28A with ensuing repression of macrophage-oriented differentiation. Finally, knockdown of miR-128a in OCI-AML3 and in APL/AML leukemic cells (by transfection and lentiviral infection, respectively) induced myeloid cell differentiation and increased expression of Lin28A, EGR2, ZFP36 and ANXA1, reverting myeloid differentiation blockage. In conclusion, our findings revealed a new mechanism for AML differentiation blockage, suggesting new strategies for AML therapy based upon miR-128a inhibition.

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.