Expert Opinion on Investigational Drugs, Sep 10, 2015
In cancer patients, anemia is frequently observed, particularly as a consequence to chemotherapy ... more In cancer patients, anemia is frequently observed, particularly as a consequence to chemotherapy (chemotherapy-induced anemia, CIA). CIA is treated with Red Blood Cell transfusions and erythropoiesis-stimulating agents (ESAs). However, the use of ESAs in anemic cancer patients is associated with reduced survival time and time to progression. Consequently, new therapeutic options are needed. Areas covered: In this article, the authors discuss new erythroid-enhancing agents (EEAs) that act differently to erythropoietin. Specifically, the article summarizes the early clinical development of activin antagonists (Sotatercep [ACE-011] and ACE-536) and hepcidin antagonists [NOX-H94]). Expert opinion: Both Activin RIIA trap agents and hepcidin inhibitors are promising new EEAs, but their safety profile, and their impact on treating CIA, needs to be carefully assessed in controlled clinical trials over longer periods of time. It is also important to carefully evaluate CIA patients to properly assess the physiopathological mechanisms responsible for the development of their anemic condition and provide patients with the most appropriate treatment plan.
Abstract We report the serum levels of soluble interleukin2 receptor (siL2R), β-microglobulin (β-... more Abstract We report the serum levels of soluble interleukin2 receptor (siL2R), β-microglobulin (β-M) and interferon-γ (IFN-γ) in patients undergoing adoptive immunotherapy with rIL2 and lymphocyte-activated killer (LAK) cells. Our results indicate that rIL 2 induced a marked increase of the serum concentration of these markers, although this increase varied considerably for different individuals. Parallel studies with the same patients also showed a marked rise in the number of IL 2R + lymphocytes: the IL2Rs expressed on these cells were mainly of the «low affinity» type. We suggest that evaluation of these markers may allow the monitoring of immune system activation induced by rIL 2 in patients undergoing adoptive rIL 2 and LAK cell immunotherapy.
Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in norm... more Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies (“bursts”) generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose- related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.
Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent... more Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with selfrenewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,70O/cell), a 210-fold lower number of IL-6Rs (1 45/cell) and granulocyte-macrophage colonystimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/ cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and thekitligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at + 4°C. No crossreaction was observed between IL-6R. IL-3R. EpR, and the heterologous GFs, whereas the GM-CSFR showed crossreactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37%, followed by ligand-binding assay at 20°C. IL-6, IL-3. GM-CSF, and Ep induce a marked down-EMATOPOIESIS is a multistep cell proliferation/dif-H ferentiation process that is sustained by a pool of stem cells. The stem elements can self-renew and differentiate into progenitors.'-4 These progenitors are committed to specific lineages and are functionally defined as colony-or burstforming units (CFUs or BFUs), ie, progenitors ofthe erythroid series (BFU-E or CFU-E), the granulocyte-monocytic lineage (CFU-GM), and multipotent CFU for the GM, erythroid, and megakaryocytic series (CFU-GEMM).I4 The progenitors in turn differentiate into morphologically recognizable precursors that mature to terminal elements circulating in peripheral blood. A group of glycoprotein hematopoietic growth factors (HGFs), termed colony stimulating factors (CSFs) or interleukins (ILs), control the survival, proliferation, and differentiation of stem and/or progenitor cells. In addition, they affect a variety of functional activities of mature/terminal cells.'-4
We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitr... more We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of H 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV ؉ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, Ϸ 5 ؋ 10 3 tissue culture infectious dose 50 /mL/10 6 cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that Ϸ 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.
Prostate cancer is the most frequent nonskin cancer and second most common cause of cancer-relate... more Prostate cancer is the most frequent nonskin cancer and second most common cause of cancer-related deaths in man. Prostate cancer is a clinically heterogeneous disease with many patients exhibiting an aggressive disease with progression, metastasis, and other patients showing an indolent disease with low tendency to progression. Three stages of development of human prostate tumors have been identified: intraepithelial neoplasia, adenocarcinoma androgen-dependent, and adenocarcinoma androgen-independent or castration-resistant. Advances in molecular technologies have provided a very rapid progress in our understanding of the genomic events responsible for the initial development and progression of prostate cancer. These studies have shown that prostate cancer genome displays a relatively low mutation rate compared with other cancers and few chromosomal loss or gains. The ensemble of these molecular studies has led to suggest the existence of two main molecular groups of prostate cancers: one characterized by the presence of ERG rearrangements (~50% of prostate cancers harbor recurrent gene fusions involving ETS transcription factors, fusing the 5 untranslated region of the androgen-regulated gene TMPRSS2 to nearly the coding sequence of the ETS family transcription factor ERG) and features of chemoplexy (complex gene rearrangements developing from a coordinated and simultaneous molecular event), and a second one characterized by the absence of ERG rearrangements and by the frequent mutations in the E3 ubiquitin ligase adapter SPOP and/or deletion of CDH1, a chromatin remodeling factor, and interchromosomal rearrangements and SPOP mutations are early events during prostate cancer development. During disease progression, genomic and epigenomic abnormalities accrued and converged on prostate cancer pathways, leading to a highly heterogeneous transcriptomic landscape, characterized by a hyperactive androgen receptor signaling axis.
Mediterranean Journal of Hematology and Infectious Diseases, Oct 29, 2022
Despite recent progress, acute myeloid leukemia (AML) remains a disease associated with poor prog... more Despite recent progress, acute myeloid leukemia (AML) remains a disease associated with poor prognosis, particularly in older AML patients unfit to tolerate intensive chemotherapy treatment. The development and introduction in the therapy of Venetoclax (VEN), a potent BH3 mimetic targeting the antiapoptotic protein BCL-2, inducing apoptosis of leukemic cells, has shown to be a promising treatment for newly diagnosed, relapsed, and refractory AML patients ineligible for induction chemotherapy. Combination treatments using Ventoclax and a hypomethylating agent (azacitidine or decitabine) or low-intensity chemotherapy have shown in newly diagnosed patients variable response rates, with highly responsive patients with NPM1, IDH1-IDH2, TET2, and RUNX1 mutations and with scarcely responsive patients with FLT3, TP53 and ASXL1 mutations, complex karyotypes, and secondary AMLs. Patients with refractory/relapsing disease are less responsive to Venetoclax-based regimens. However, in the majority of patients, the responses have only a limited duration, and the development of resistance is frequently observed. Therefore, understanding the resistance mechanisms is crucial for developing new strategies and identifying rational drug combination regimens. In this context, two strategies seem to be promising: (i) triplet therapies based on the combined administration of Venetoclax, a hypomethylating agent (or low-dose chemotherapy), and an agent targeting a specific genetic alteration of leukemic cells (i.e., FLT3 inhibitors in FLT3mutated AMLs) or an altered signaling pathway; (ii) combination therapies based on the administration of two BH3 mimetics (i.e., BCL-2 +MCL-1 mimetics) and a hypomethylating agent.
Methodology has been developed that enables virtually complete purification and recovery of early... more Methodology has been developed that enables virtually complete purification and recovery of early hematopoietic progenitors from human adult blood, a minority of which is multipotent and endowed with self-renewal capacities, i.e., exhibits stem cell properties. This report briefly reviews: (i) the key steps involved in the progenitor purification and assay procedure; (ii) the characterization of "pure" progenitors at the level of membrane antigen pattern and response to HGFs; (iii) the development of a liquid suspension culture for the pure progenitors, which allows synchronized and selective erythroid or GM differentiation, and hence may be utilized for the analysis of molecular mechanisms underlying early and late stages of hematopoiesis; (iv) the study of the expression and modulation of HGFRs expressed on progenitors.
The burden of cancer is increasing worldwide, with a continuous rise of the annual total cases. A... more The burden of cancer is increasing worldwide, with a continuous rise of the annual total cases. Although mortality rates due to cancer are declining in developed countries, the total number of cancer deaths continues to rise due to the increase in the number of aged people. Three main causes of cancer have been described, represented by environmental factors, hereditary factors and random factors related to defects originated during cell replication. The frequency of cancers is very different for the various tissues and there is great debate on the extent of the specific contribution of environmental factors and random factors (due to "bad luck") to cancer development. However, there is consensus that about 50% of all cases of cancer are related to environment and are preventable. Although a part of cancers is related to intrinsic mechanisms non preventable of genetic instability, it is evident that implementation of primary and secondary prevention measures is the only affordable strategy to meet from a medical and economic point of view the tremendous pressure created on healthcare structures by the increased cancer burden. It is time to bypass the paradox of disease prevention: celebrated in principle, resisted in practice.
Minimal or measurable residual disease (MRD) is a term that refers to the submicroscopic tumor di... more Minimal or measurable residual disease (MRD) is a term that refers to the submicroscopic tumor disease persisting after therapy. Sensitive immunophenotypic and molecular techniques are used to detect the small amount of residual tumor cells, conferring a detection capacity clearly more sensitive of common cytomorphologic techniques. MRD evaluation now represents an important tool in the study of solid tumors and of hematological malignancies. Concerning hematological malignancies, MRD evaluation was particularly developed in the study of multiple myeloma and acute myeloid leukemia, representing in these diseases a precious biomarker to quantify response to treatment, to evaluate the chemosensitivity/chemoresistance of the disease and to have a prognostic prediction on disease outcome. The finding that MRD evaluation may have a prognostic value, predicting the risk of relapse, stimulated interest in the introduction of MRD in clinical trials, either as a clinical endpoint or as a tool to guide treatment decisions. However, the clinical use of MRD requires a standardization of the techniques used for its detection, the use of multiple techniques and the development of a consistent accuracy and reproducibility. Finally, prospective clinical trials are required to assess the real clinical benefit potentially deriving from the introduction of MRD evaluation into clinical studies.
Transferrin receptor (TfR) is a membrane receptor involved in the control of iron supply to the c... more Transferrin receptor (TfR) is a membrane receptor involved in the control of iron supply to the cell through the binding of transferrin, the major iron-carrier protein. This receptor plays a key role in the control of cell proliferation because iron is essential for sustaining ribonucleotide reductase activity, and is the only enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Furthermore, TfR is overexpressed in several tumors. The synthesis of the receptor is controlled through an iron-dependent negative feedback in cells where the receptor is involved in the control of cell proliferation, but through a positive feedback in cell types involved in iron storage (monocytes-macrophages). Recent studies have shed light on the molecular basis by which iron regulates TfR expression. Thus, the 3' untranslated region of TfR mRNA contains a cis-acting RNA element, termed the iron-regulatory element (IRE), that interacts with an IRE-binding protein (IRE-BP). The high-affinity interaction between IRE-BP and IRE in the TfR mRNA leads to repression of mRNA degradation and thus to higher synthesis of TfR. Hence, the study of IRE-BP activity will elucidate the mechanisms that modulate TfR expression in normal and malignant cells.
Cholangiocarcinomas (CCAs) are a group of heterogeneous epithelial malignancies that can originat... more Cholangiocarcinomas (CCAs) are a group of heterogeneous epithelial malignancies that can originate at the level of any location of the biliary tree. These tumors are relatively rare but associated with a high rate of mortality. CCAs are morphologically and molecularly heterogeneous and for their location can be distinguished as intracellular and extracellular, subdivided into perihilar and distal. Recent epidemiological, molecular, and cellular studies have supported that the consistent heterogeneity observed for CCAs may result from the convergence of various key elements mainly represented by risk factors, heterogeneity of the associated molecular abnormalities at genetic and epigenetic levels and by different potential cells of origin. These studies have consistently contributed to better defining the pathogenesis of CCAs and to identify in some instances new therapeutic targets. Although the therapeutic progress were still limited, these observations suggest that a better understanding of the molecular mechanisms underlying CCA in the future will help to develop more efficacious treatment strategies.
or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(... more or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU-E. as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS1 methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 1O cells/dish) purified embryonic BFU-E generated
Mediterranean Journal of Hematology and Infectious Diseases, Jun 29, 2023
TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) form a distinct and ... more TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) form a distinct and heterogeneous group of myeloid malignancies associated with poor outcomes. Studies carried out in the last years have in part elucidated the complex role played by TP53 mutations in the pathogenesis of these myeloid disorders and in the mechanisms of drug resistance. A consistent number of studies has shown that some molecular parameters, such as the presence of a single or multiple TP53 mutations, the presence of concomitant TP53 deletions, the association with co-occurring mutations, the clonal size of TP53 mutations, the involvement of a single (monoallelic) or of both TP53 alleles (biallelic) and the cytogenetic architecture of concomitant chromosome abnormalities are major determinants of outcomes of patients. The limited response of these patients to standard treatments, including induction chemotherapy, hypomethylating agents and venetoclax-based therapies and the discovery of an immune dysregulation have induced a shift to new emerging therapies, some of which being associated with promising efficacy. The main aim of these novel immune and nonimmune strategies consists in improving survival and in increasing the number of TP53-mutated MDS/AML patients in remission amenable to allogeneic stem cell transplantation.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
MicroRNAs (miRs) are a class of a small (~ 22nt) RNAs, which play an important role in the negati... more MicroRNAs (miRs) are a class of a small (~ 22nt) RNAs, which play an important role in the negative regulation of gene expression by base-pairing to complementary sites on the target mRNAs. While it is established that miRs are involved in a variety of basic processes, e.g., cell proliferation and apoptosis, neural development, fat metabolism and stress response, little is known on their expression and function in hematopoiesis. In order to investigate miR expression in erythropoietic (E), megakaryocytic (Mk), granulopoietic (G) and monocytopoietic (Mo) lineages, we have assayed their level at discrete sequential stages of the E, Mk, G or Mo series in unilineage differentiation/maturation cultures of cord blood (CB) CD34+ cells. The analysis was performed using a microarray chip containing as probes gene-specific 40mer oligonucleotides, generated from 161 human and 84 mouse precursors miRs (Liu GC et al., PNAS, 2004). Northern blot analysis confirmed the microarray data. The results indicate that the majority of the analyzed miRs is not expressed in CB hematopoietic cells. However, 49 miRs are expressed at significant levels in CD34+ cells: in most cases the expression level declines during hematopoietic differentiation according to diverse patterns, i.e., the decline may be more or less pronounced, more or less rapid and differ in the diverse hematopoietic lineages. As expression pattern examples, we observed that: (a) miR 223 is strongly downmodulated in the E lineage, whereas its level is not affected or increased in the other series; (b) miR 221 and 222 level sharply declines in the E lineage, while the drop is less pronounced in the Mk, G and Mo series; conversely, (c) miR 17, 20, 106 are downmodulated prevalentely in the G/Mo series, as compared to the E/Mk lineages. Interestingly, cluster analysis indicates that miR expression in hematopoietic cells is sharply different from that observed in CB T lymphocytes. The lineage- and stage-specific pattern of miR expression is of functional relevance. As an example, transfection of miR 222 oligonucleotide into CD34+ cells grown in multilineage clonogenic culture causes a pronounced shift from E to GM colony formation, indicating modulation of the lineage commitment of hematopoietic progenitors. The target genes of miRs expressed in hematopoietic cells are often of pivotal functional significance, e.g., miR 222 targets the kit receptor (N. Felli et al., this Meeting). A single miR may target diverse mRNAs, e.g., miR 222 targets kit, Ets1 and Fli1. Conversely, a single mRNA may be targeted by different miRs, e.g.,, kit is targeted by miR 146, 221 and 222. Noterworthily, the miR expression pattern in primitive hematopoietic cells and their progeny is fully distinct from that observed in primitive mesenchymal and neural cells (i.e., “neurospheres”) and their progeny: this suggests that miR downmodulation during differentiation of primitive cells contributes to tissue-specific gene expression by unblocking translational repression of the target mRNAs.
Expert Opinion on Investigational Drugs, Sep 10, 2015
In cancer patients, anemia is frequently observed, particularly as a consequence to chemotherapy ... more In cancer patients, anemia is frequently observed, particularly as a consequence to chemotherapy (chemotherapy-induced anemia, CIA). CIA is treated with Red Blood Cell transfusions and erythropoiesis-stimulating agents (ESAs). However, the use of ESAs in anemic cancer patients is associated with reduced survival time and time to progression. Consequently, new therapeutic options are needed. Areas covered: In this article, the authors discuss new erythroid-enhancing agents (EEAs) that act differently to erythropoietin. Specifically, the article summarizes the early clinical development of activin antagonists (Sotatercep [ACE-011] and ACE-536) and hepcidin antagonists [NOX-H94]). Expert opinion: Both Activin RIIA trap agents and hepcidin inhibitors are promising new EEAs, but their safety profile, and their impact on treating CIA, needs to be carefully assessed in controlled clinical trials over longer periods of time. It is also important to carefully evaluate CIA patients to properly assess the physiopathological mechanisms responsible for the development of their anemic condition and provide patients with the most appropriate treatment plan.
Abstract We report the serum levels of soluble interleukin2 receptor (siL2R), β-microglobulin (β-... more Abstract We report the serum levels of soluble interleukin2 receptor (siL2R), β-microglobulin (β-M) and interferon-γ (IFN-γ) in patients undergoing adoptive immunotherapy with rIL2 and lymphocyte-activated killer (LAK) cells. Our results indicate that rIL 2 induced a marked increase of the serum concentration of these markers, although this increase varied considerably for different individuals. Parallel studies with the same patients also showed a marked rise in the number of IL 2R + lymphocytes: the IL2Rs expressed on these cells were mainly of the «low affinity» type. We suggest that evaluation of these markers may allow the monitoring of immune system activation induced by rIL 2 in patients undergoing adoptive rIL 2 and LAK cell immunotherapy.
Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in norm... more Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies (“bursts”) generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose- related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.
Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent... more Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with selfrenewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,70O/cell), a 210-fold lower number of IL-6Rs (1 45/cell) and granulocyte-macrophage colonystimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/ cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and thekitligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at + 4°C. No crossreaction was observed between IL-6R. IL-3R. EpR, and the heterologous GFs, whereas the GM-CSFR showed crossreactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37%, followed by ligand-binding assay at 20°C. IL-6, IL-3. GM-CSF, and Ep induce a marked down-EMATOPOIESIS is a multistep cell proliferation/dif-H ferentiation process that is sustained by a pool of stem cells. The stem elements can self-renew and differentiate into progenitors.'-4 These progenitors are committed to specific lineages and are functionally defined as colony-or burstforming units (CFUs or BFUs), ie, progenitors ofthe erythroid series (BFU-E or CFU-E), the granulocyte-monocytic lineage (CFU-GM), and multipotent CFU for the GM, erythroid, and megakaryocytic series (CFU-GEMM).I4 The progenitors in turn differentiate into morphologically recognizable precursors that mature to terminal elements circulating in peripheral blood. A group of glycoprotein hematopoietic growth factors (HGFs), termed colony stimulating factors (CSFs) or interleukins (ILs), control the survival, proliferation, and differentiation of stem and/or progenitor cells. In addition, they affect a variety of functional activities of mature/terminal cells.'-4
We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitr... more We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of H 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV ؉ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, Ϸ 5 ؋ 10 3 tissue culture infectious dose 50 /mL/10 6 cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that Ϸ 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.
Prostate cancer is the most frequent nonskin cancer and second most common cause of cancer-relate... more Prostate cancer is the most frequent nonskin cancer and second most common cause of cancer-related deaths in man. Prostate cancer is a clinically heterogeneous disease with many patients exhibiting an aggressive disease with progression, metastasis, and other patients showing an indolent disease with low tendency to progression. Three stages of development of human prostate tumors have been identified: intraepithelial neoplasia, adenocarcinoma androgen-dependent, and adenocarcinoma androgen-independent or castration-resistant. Advances in molecular technologies have provided a very rapid progress in our understanding of the genomic events responsible for the initial development and progression of prostate cancer. These studies have shown that prostate cancer genome displays a relatively low mutation rate compared with other cancers and few chromosomal loss or gains. The ensemble of these molecular studies has led to suggest the existence of two main molecular groups of prostate cancers: one characterized by the presence of ERG rearrangements (~50% of prostate cancers harbor recurrent gene fusions involving ETS transcription factors, fusing the 5 untranslated region of the androgen-regulated gene TMPRSS2 to nearly the coding sequence of the ETS family transcription factor ERG) and features of chemoplexy (complex gene rearrangements developing from a coordinated and simultaneous molecular event), and a second one characterized by the absence of ERG rearrangements and by the frequent mutations in the E3 ubiquitin ligase adapter SPOP and/or deletion of CDH1, a chromatin remodeling factor, and interchromosomal rearrangements and SPOP mutations are early events during prostate cancer development. During disease progression, genomic and epigenomic abnormalities accrued and converged on prostate cancer pathways, leading to a highly heterogeneous transcriptomic landscape, characterized by a hyperactive androgen receptor signaling axis.
Mediterranean Journal of Hematology and Infectious Diseases, Oct 29, 2022
Despite recent progress, acute myeloid leukemia (AML) remains a disease associated with poor prog... more Despite recent progress, acute myeloid leukemia (AML) remains a disease associated with poor prognosis, particularly in older AML patients unfit to tolerate intensive chemotherapy treatment. The development and introduction in the therapy of Venetoclax (VEN), a potent BH3 mimetic targeting the antiapoptotic protein BCL-2, inducing apoptosis of leukemic cells, has shown to be a promising treatment for newly diagnosed, relapsed, and refractory AML patients ineligible for induction chemotherapy. Combination treatments using Ventoclax and a hypomethylating agent (azacitidine or decitabine) or low-intensity chemotherapy have shown in newly diagnosed patients variable response rates, with highly responsive patients with NPM1, IDH1-IDH2, TET2, and RUNX1 mutations and with scarcely responsive patients with FLT3, TP53 and ASXL1 mutations, complex karyotypes, and secondary AMLs. Patients with refractory/relapsing disease are less responsive to Venetoclax-based regimens. However, in the majority of patients, the responses have only a limited duration, and the development of resistance is frequently observed. Therefore, understanding the resistance mechanisms is crucial for developing new strategies and identifying rational drug combination regimens. In this context, two strategies seem to be promising: (i) triplet therapies based on the combined administration of Venetoclax, a hypomethylating agent (or low-dose chemotherapy), and an agent targeting a specific genetic alteration of leukemic cells (i.e., FLT3 inhibitors in FLT3mutated AMLs) or an altered signaling pathway; (ii) combination therapies based on the administration of two BH3 mimetics (i.e., BCL-2 +MCL-1 mimetics) and a hypomethylating agent.
Methodology has been developed that enables virtually complete purification and recovery of early... more Methodology has been developed that enables virtually complete purification and recovery of early hematopoietic progenitors from human adult blood, a minority of which is multipotent and endowed with self-renewal capacities, i.e., exhibits stem cell properties. This report briefly reviews: (i) the key steps involved in the progenitor purification and assay procedure; (ii) the characterization of "pure" progenitors at the level of membrane antigen pattern and response to HGFs; (iii) the development of a liquid suspension culture for the pure progenitors, which allows synchronized and selective erythroid or GM differentiation, and hence may be utilized for the analysis of molecular mechanisms underlying early and late stages of hematopoiesis; (iv) the study of the expression and modulation of HGFRs expressed on progenitors.
The burden of cancer is increasing worldwide, with a continuous rise of the annual total cases. A... more The burden of cancer is increasing worldwide, with a continuous rise of the annual total cases. Although mortality rates due to cancer are declining in developed countries, the total number of cancer deaths continues to rise due to the increase in the number of aged people. Three main causes of cancer have been described, represented by environmental factors, hereditary factors and random factors related to defects originated during cell replication. The frequency of cancers is very different for the various tissues and there is great debate on the extent of the specific contribution of environmental factors and random factors (due to "bad luck") to cancer development. However, there is consensus that about 50% of all cases of cancer are related to environment and are preventable. Although a part of cancers is related to intrinsic mechanisms non preventable of genetic instability, it is evident that implementation of primary and secondary prevention measures is the only affordable strategy to meet from a medical and economic point of view the tremendous pressure created on healthcare structures by the increased cancer burden. It is time to bypass the paradox of disease prevention: celebrated in principle, resisted in practice.
Minimal or measurable residual disease (MRD) is a term that refers to the submicroscopic tumor di... more Minimal or measurable residual disease (MRD) is a term that refers to the submicroscopic tumor disease persisting after therapy. Sensitive immunophenotypic and molecular techniques are used to detect the small amount of residual tumor cells, conferring a detection capacity clearly more sensitive of common cytomorphologic techniques. MRD evaluation now represents an important tool in the study of solid tumors and of hematological malignancies. Concerning hematological malignancies, MRD evaluation was particularly developed in the study of multiple myeloma and acute myeloid leukemia, representing in these diseases a precious biomarker to quantify response to treatment, to evaluate the chemosensitivity/chemoresistance of the disease and to have a prognostic prediction on disease outcome. The finding that MRD evaluation may have a prognostic value, predicting the risk of relapse, stimulated interest in the introduction of MRD in clinical trials, either as a clinical endpoint or as a tool to guide treatment decisions. However, the clinical use of MRD requires a standardization of the techniques used for its detection, the use of multiple techniques and the development of a consistent accuracy and reproducibility. Finally, prospective clinical trials are required to assess the real clinical benefit potentially deriving from the introduction of MRD evaluation into clinical studies.
Transferrin receptor (TfR) is a membrane receptor involved in the control of iron supply to the c... more Transferrin receptor (TfR) is a membrane receptor involved in the control of iron supply to the cell through the binding of transferrin, the major iron-carrier protein. This receptor plays a key role in the control of cell proliferation because iron is essential for sustaining ribonucleotide reductase activity, and is the only enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Furthermore, TfR is overexpressed in several tumors. The synthesis of the receptor is controlled through an iron-dependent negative feedback in cells where the receptor is involved in the control of cell proliferation, but through a positive feedback in cell types involved in iron storage (monocytes-macrophages). Recent studies have shed light on the molecular basis by which iron regulates TfR expression. Thus, the 3' untranslated region of TfR mRNA contains a cis-acting RNA element, termed the iron-regulatory element (IRE), that interacts with an IRE-binding protein (IRE-BP). The high-affinity interaction between IRE-BP and IRE in the TfR mRNA leads to repression of mRNA degradation and thus to higher synthesis of TfR. Hence, the study of IRE-BP activity will elucidate the mechanisms that modulate TfR expression in normal and malignant cells.
Cholangiocarcinomas (CCAs) are a group of heterogeneous epithelial malignancies that can originat... more Cholangiocarcinomas (CCAs) are a group of heterogeneous epithelial malignancies that can originate at the level of any location of the biliary tree. These tumors are relatively rare but associated with a high rate of mortality. CCAs are morphologically and molecularly heterogeneous and for their location can be distinguished as intracellular and extracellular, subdivided into perihilar and distal. Recent epidemiological, molecular, and cellular studies have supported that the consistent heterogeneity observed for CCAs may result from the convergence of various key elements mainly represented by risk factors, heterogeneity of the associated molecular abnormalities at genetic and epigenetic levels and by different potential cells of origin. These studies have consistently contributed to better defining the pathogenesis of CCAs and to identify in some instances new therapeutic targets. Although the therapeutic progress were still limited, these observations suggest that a better understanding of the molecular mechanisms underlying CCA in the future will help to develop more efficacious treatment strategies.
or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(... more or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU-E. as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS1 methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 1O cells/dish) purified embryonic BFU-E generated
Mediterranean Journal of Hematology and Infectious Diseases, Jun 29, 2023
TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) form a distinct and ... more TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) form a distinct and heterogeneous group of myeloid malignancies associated with poor outcomes. Studies carried out in the last years have in part elucidated the complex role played by TP53 mutations in the pathogenesis of these myeloid disorders and in the mechanisms of drug resistance. A consistent number of studies has shown that some molecular parameters, such as the presence of a single or multiple TP53 mutations, the presence of concomitant TP53 deletions, the association with co-occurring mutations, the clonal size of TP53 mutations, the involvement of a single (monoallelic) or of both TP53 alleles (biallelic) and the cytogenetic architecture of concomitant chromosome abnormalities are major determinants of outcomes of patients. The limited response of these patients to standard treatments, including induction chemotherapy, hypomethylating agents and venetoclax-based therapies and the discovery of an immune dysregulation have induced a shift to new emerging therapies, some of which being associated with promising efficacy. The main aim of these novel immune and nonimmune strategies consists in improving survival and in increasing the number of TP53-mutated MDS/AML patients in remission amenable to allogeneic stem cell transplantation.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
MicroRNAs (miRs) are a class of a small (~ 22nt) RNAs, which play an important role in the negati... more MicroRNAs (miRs) are a class of a small (~ 22nt) RNAs, which play an important role in the negative regulation of gene expression by base-pairing to complementary sites on the target mRNAs. While it is established that miRs are involved in a variety of basic processes, e.g., cell proliferation and apoptosis, neural development, fat metabolism and stress response, little is known on their expression and function in hematopoiesis. In order to investigate miR expression in erythropoietic (E), megakaryocytic (Mk), granulopoietic (G) and monocytopoietic (Mo) lineages, we have assayed their level at discrete sequential stages of the E, Mk, G or Mo series in unilineage differentiation/maturation cultures of cord blood (CB) CD34+ cells. The analysis was performed using a microarray chip containing as probes gene-specific 40mer oligonucleotides, generated from 161 human and 84 mouse precursors miRs (Liu GC et al., PNAS, 2004). Northern blot analysis confirmed the microarray data. The results indicate that the majority of the analyzed miRs is not expressed in CB hematopoietic cells. However, 49 miRs are expressed at significant levels in CD34+ cells: in most cases the expression level declines during hematopoietic differentiation according to diverse patterns, i.e., the decline may be more or less pronounced, more or less rapid and differ in the diverse hematopoietic lineages. As expression pattern examples, we observed that: (a) miR 223 is strongly downmodulated in the E lineage, whereas its level is not affected or increased in the other series; (b) miR 221 and 222 level sharply declines in the E lineage, while the drop is less pronounced in the Mk, G and Mo series; conversely, (c) miR 17, 20, 106 are downmodulated prevalentely in the G/Mo series, as compared to the E/Mk lineages. Interestingly, cluster analysis indicates that miR expression in hematopoietic cells is sharply different from that observed in CB T lymphocytes. The lineage- and stage-specific pattern of miR expression is of functional relevance. As an example, transfection of miR 222 oligonucleotide into CD34+ cells grown in multilineage clonogenic culture causes a pronounced shift from E to GM colony formation, indicating modulation of the lineage commitment of hematopoietic progenitors. The target genes of miRs expressed in hematopoietic cells are often of pivotal functional significance, e.g., miR 222 targets the kit receptor (N. Felli et al., this Meeting). A single miR may target diverse mRNAs, e.g., miR 222 targets kit, Ets1 and Fli1. Conversely, a single mRNA may be targeted by different miRs, e.g.,, kit is targeted by miR 146, 221 and 222. Noterworthily, the miR expression pattern in primitive hematopoietic cells and their progeny is fully distinct from that observed in primitive mesenchymal and neural cells (i.e., “neurospheres”) and their progeny: this suggests that miR downmodulation during differentiation of primitive cells contributes to tissue-specific gene expression by unblocking translational repression of the target mRNAs.
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