Papers by alessandro morotti
A very sensitive and rapid method for BCR-ABL T315Imutation detection by peptide nucleic acid directed PCR clamping
Haematologica, 2009
Interleukin-2 Receptor Alpha Chain, Also Called CD25, Is a Potential Target in Acute Lymphoblastic Leukemia
Background: Acute lymphoblastic leukemia (ALL) is a molecularly heterogeneous disease originating... more Background: Acute lymphoblastic leukemia (ALL) is a molecularly heterogeneous disease originating from clonal proliferation of precursor B-lineage cells. In adults, ALL diagnosis is still associated with a dismal prognosis due to the lack of specific targeted therapies. This study was designed to investigate the expression of interleukin-2 receptor alpha chain CD25 in B-ALL and its biological significance, especially following the availability of specific CD25 targeting compounds. Methods:The expression of IL2RA (CD25 gene) was detected by flow cytometry (FC), immunohistochemistry and Western blot analysis, in 25 newly diagnosed ALL patients, both Philadelphia positive (12 patients) and Philadelphia negative (13 patients). Similarly, CD25 expression was assessed in four B-ALL commercially available cell lines. Infection with shRNA specifically directed against CD25 was used to evaluate apoptosis induction and cell cycle arrest in primary B-ALL cells established from two patients. Re...
Journal of Experimental & Clinical Cancer Research
Background Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen speci... more Background Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy. Methods We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions. Results Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung ...
International Journal of Molecular Sciences
It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet... more It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of athero...
Targeting the Extracellular HSP90 Co-Chaperone Morgana Inhibits Cancer Cell Migration and Promotes Anticancer Immunity
Cancer Research
This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaper... more This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaperone Morgana to inhibit metastasis formation and enhance the CD8+ T-cell–mediated antitumor immune response. HSP90 is secreted by cancer cells into the extracellular milieu, where it exerts protumoral activities by activating extracellular substrate proteins and triggering autocrine signals through cancer cell surface receptors. Emerging evidence indicates that HSP90 co-chaperones are also secreted and may direct HSP90 extracellular activities. In this study, we found that the HSP90 co-chaperone Morgana is released by cancer cells and, in association with HSP90, induces cancer cell migration through TLR2, TLR4, and LRP1. In syngeneic cancer mouse models, a mAb targeting Morgana extracellular activity reduced primary tumor growth via macrophage-dependent recruitment of CD8+ T lymphocytes, blocked cancer cell migration, and inhibited metastatic spreading. Overall, these data define Morgana as a new player in the HSP90 extracellular interactome and suggest that Morgana may regulate HSP90 activity to promote cancer cell migration and suppress antitumor immunity. Significance: This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaperone Morgana to inhibit metastasis formation and enhance the CD8+ T-cell–mediated antitumor immune response. Graphical Abstract
Oncology Letters
Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) is driven by the p190 ... more Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) is driven by the p190 breakpoint cluster region (BCR)-ABL isoform. Although effectively targeted by BCR-ABL tyrosine kinase inhibitors (TKIs), ALL is associated with a less effective response to TKIs compared with chronic myeloid leukemia. Therefore, the identification of additional genes required for ALL maintenance may provide possible therapeutic targets to aid the eradication of this cancer. The present study demonstrated that p190 BCR-ABL is able to interact with the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which in turn affects p53 protein stability. Notably, the inhibition of HAUSP with small molecule inhibitors promoted the upregulation of p53 protein levels. These results suggest that HAUSP inhibitors may harbor clinically relevant implications in the treatment of Ph + ALL.
Pharmaceuticals
For many years in the field of onco-hematology much attention has been given to mutations in prot... more For many years in the field of onco-hematology much attention has been given to mutations in protein-coding genes or to genetic alterations, including large chromosomal losses or rearrangements. Despite this, biological and clinical needs in this sector remain unmet. Therefore, it is not surprising that recent studies have shifted from coded to non-coded matter. The discovery of non-coding RNAs (ncRNAs) has influenced several aspects related to the treatment of cancer. In particular, in chronic lymphocytic leukemia (CLL) the knowledge of ncRNAs and their contextualization have led to the identification of new biomarkers used to follow the course of the disease, to the anticipation of mechanisms that support resistance and relapse, and to the selection of novel targeted treatment regimens. In this review, we will summarize the main ncRNAs discovered in CLL and the molecular mechanisms by which they are affected and how they influence the development and the progression of the disease.
International Journal of Molecular Sciences
Platelet hyperactivation is involved in the established prothrombotic condition of metabolic dise... more Platelet hyperactivation is involved in the established prothrombotic condition of metabolic diseases such as Type 2 Diabetes Mellitus (T2DM) and familial hypercholesterolemia (HC), justifying the therapy with aspirin, a suppressor of thromboxane synthesis through the irreversible inhibition of cyclooxygenase-1 (COX-1), to prevent cardiovascular diseases. However, some patients on aspirin show a higher than expected platelet reactivity due, at least in part, to a pro-oxidant milieu. The aim of this study was to investigate platelet reactivity in T2DM (n = 103) or HC (n = 61) patients (aspirin, 100 mg/day) and its correlation with biomarkers of redox function including the superoxide anion scavenger superoxide dismutase (SOD) and the in vivo marker of oxidative stress urinary 8-iso-prostaglandin F2α. As results, in T2DM and HC subjects the prevalence of high on-aspirin platelet reactivity was comparable when both non-COX-1-dependent and COX-1-dependent assays were performed, and plat...
The Synergism Between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia
BackgroundDihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynth... more BackgroundDihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors were previously investigated in solid tumors, where they showed promising antiproliferative activity, both in vitro and in vivo. However, their effectiveness was not confirmed in clinical trials, probably due to the pyrimidine salvage pathway that cancer cells could exploit to survive. In this study we investigated the pro-apoptotic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we challenged our model mimicking in vivo conditions, and looked for synergic combination to boost apoptosis.MethodsWe evaluated the apoptotic rate of multiple AML cell lines and AML primary cells treated with MEDS433 or other DHODH inhibitors, alone and in combination with classical antileukemic drugs or with dipyridamo...
Clinical characteristics and risk factors associated with COVID-19 severity in patients with haematological malignancies in Italy: a retrospective, multicentre, cohort study
The Lancet Haematology
The Non Receptor Transmembrane Tyrosine Kinase STYK1 Represents a Novel Lymphocyte Marker, Whose Expression Is Deregulated in Leukemia, and a Potential Molecular Druggable Target
Blood
Introduction Tyrosine kinases (TKs) play different roles either in physiological or in pathologic... more Introduction Tyrosine kinases (TKs) play different roles either in physiological or in pathological cellular processes ranging from the regulation of intracellular signaling pathways leading for example to proliferation, differentiation and/or DNA syntheisis. The whole human genome encodes for approximately 95 TKs classified in two major families: transmembrane and non-transmembrane TKs. Transmembrane TKs are further splitted in two groups: Receptor Tyrosine Kinases (RTKs) and Non-Receptor Transmembrane Tyrosine Kinases. Despite in the last decade the role of RTKs has been deeply investigated very little is known about the second class of TKs, Non-Receptor Transmembrane Tyrosine Kinases. Recently, we began to investigate the roles played by these peculiar molecules in hematological tissues and preliminary data are herein presented and discussed. Material & Methods and Results STYK1 is a Non-Receptor Transmembrane TK characterized by a very short extracellular domain, of approximatel...
Single Base Pair Mutation Detection by PNA Direct PCR Clamping: Application to Bcr-Abl T315I Mutation
Blood
Background: Mutations occurring within the kinase domain of the tyrosine kinase c-Abl, and its on... more Background: Mutations occurring within the kinase domain of the tyrosine kinase c-Abl, and its oncogenic counterpart Bcr-Abl, strongly affects the outcome of clinical treatment with tyrosine kinase inhibitors (TKIs). Although a second and third generation of TKIs has been developed and these molecules are currently available some mutations (i.e. T315I) confer insensitivity to the drugs. Therefore, a method allowing quick detection of these mutations still represents a challenge for most of clinicians because it would allow them to rapidly switch to alternative therapies instead of TKIs. Preliminary data of a novel method allowing easy and rapid detection of the mutation affecting the codon 315 (T315I), which is nowadays the most severe and resistant mutation to TKIs, is presented. Rationale & Methods: The innovative strategy we propose is based on a variant of a single tube PNA-PCR clamping. Peptide Nucleic Acid (PNA) are synthetic molecules sharing physical-chemical features with D...
The IKK Inhibitor PS1145 Allows to Overcome Imatinib Resistance
Blood
Since a number of CML patients are resistant to Imatinib, additional molecular defects should be ... more Since a number of CML patients are resistant to Imatinib, additional molecular defects should be identified and targeted to improve the therapeutic strategies. A constitutive NF-kB activity has been demonstrated in several hematologic malignancies, therefore NF-kB blocking approaches have been introduced as antineoplastic strategies. The phosphorilation of IKB by IKK leads to IKB degradation by the proteasome, so freeing NF-kB to enter the nucleus and activate transcription. IKK may therefore represent an attractive target for molecular therapies. The aim of the study was to evaluate the effects of the IKK inhibitor PS1145 (Millenium) in CML cell lines and patients sensitive and resistant to Imatinib. K562 and KCl cells both sensitive (s) and resistant (r) to Imatinib and the BM cells collected from 13 CML patients were incubated with PS1145 10 μM, with Imatinib 1 μM and with the combination of the two drugs for 24 and 48 hrs. 11 out of 13 patients were in chronic phase, 1 in accele...
Over-Expression of the P65 Subunit of NF-kB and Increased DNA Binding Activity of NF-kB Is a Common Event Both in Philadelphia Positive and Negative Chronic Myeloproliferative Disorders
Blood
The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased pr... more The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased proliferation of cells from one or more myeloid lineages. The most common CMPD is Chronic Myeloid Leukemia which is characterized by the Philadelphia t(9;22) chromosomal translocation. The pathogenesis of Philadelphia negative CMPD is poorly understood, although the activation of tyrosine kinases appears to be an essential feature. For example, a constitutively activated PDGF receptor tyrosine kinase (FIP1L1-PDGFRA) is involved in some cases of the hypereosinophilic syndrome (HES), which is a form of CMPD characterized by increased proliferation of eosinophils. Different reports have demonstrated that the transcription factor NF-kB is essential for Bcr-Abl mediated transformation. NF-kB is a transcription factor which is composed of two subunits (generally p65 and p50). NF-kB dimers are retained into the cytoplasm by the inhibitory protein IkB. Different stimuli trigger the Serine phosphor...
Absence of Spred1, a Negative Regulator of Ras/MAPK Pathway, as a Mechanism Responsible for the Constitutive Activation of RTK Mediated Signalling in Acute Leukemias
Blood
Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They ... more Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They are implicated in negative feedback interactions that regulate intracellular pathways. The repressive function of Spred proteins targets several TK receptors so resulting in a variety of biological effects. Spred proteins, after growth factor stimulation, translocate to the plasma membrane, become tyrosine phosphorilated and interact with components of the Ras/MAPK and Ras/Raf/Erk pathways. Abnormal activation of Ras/MAPK pathway has been demonstrated in many haematological disorders including AML. In order to assess whether the activation of this pathway, leading to the disruption of many biological process, could be ascribed not only to the constitutive activation of TKs but also to defective signalling inhibition, we studied the function of Spred1 in AML. Using a Real Time PCR we studied the expression level of Spred1 in 80 AML samples (15 PB and 65 BM), 26 normal controls (16 PB and ...
Overexpression of the p65 Subunit of NF-kB and IkB Mediated Nuclear Sequestration of p53 as Common Events in Philadelphia Positive and Negative Chronic Myeloid Leukemia
Blood
Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder caused by the Phila... more Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia translocation. Rare patients with a clinical presentation of CML are negative for the Ph chromosome. The pathogenesis of Ph negative CML is still unknown. Different reports have demonstrated that the transcription factor NF-kappaB is essential for Bcr-Abl mediated transformation. NF-kappaB is composed of two subunits (mainly p65 and p50) which are retained into the cytoplasm by the inhibitory protein IkappaB-alpha. Different stimuli trigger IkappaB degradation and nuclear translocation of NF-kappaB, where it mediates the transcription of different genes involved in cellular proliferation, transformation and resistance to apoptosis. Aim of the work. We have evaluated the role of NF-kappaB in primary chronic myeloid leukemia samples both positive and negative for the Philadelphia chromosome. Methods. Bone marrow samples of 10 chronic myeloproliferative disorders (6 Ph positive and ...
NPM1 Mutations Are Responsible for Better Response to Induction Therapy in AML Patients through the Inactivation of NF-kB
Blood
Mutations in NPM1 exon 12 and the resulting shift of NPM1 into the cytoplasm are the most specifi... more Mutations in NPM1 exon 12 and the resulting shift of NPM1 into the cytoplasm are the most specific and frequent events in acute myeloid leukaemia patients with normal karyotype. Cytoplasmatic NPM is associated with responsiveness to induction chemotherapy, although its role in predicting outcome after remission remains to be defined. The aim of the study was to identify the molecular mechanisms responsible for chemosensitivity in leukemic cells carrying the mutation of NPM1. NF-kB is a transcription factor involved in many intracellular pathways including apoptosis. NF-kB is a heterodimer of p50 and p65 subunits sequestered in the cytoplasm in its inactive form through interaction with inhibitory IKB proteins and activated in the nucleous after degradation of IKB. The activation of NF-kB is responsible for chemoresistance to different drugs including anthracyclines. Based on this assumption, we analyzed the possible cytoplasmatic interaction between the mutated form of NPM1 (NPM+) a...
Journal of Blood Medicine
The reversal of low-molecular-weight heparin (LMWH) and the management of bleeding patients on LM... more The reversal of low-molecular-weight heparin (LMWH) and the management of bleeding patients on LMWH remain highly challenging. Even if LMWH is very extensively administered in the prophylaxis and treatment of venous thrombosis, specific antidotes are lacking, and reversal strategies have very weak grade of evidences on clinical effectiveness. We here describe a reversal strategy with protamine and FVIIa in a patient presenting with hemorrhagic shock and cardiocirculatory arrest.
Cancers
Tumor suppressors play an important role in cancer pathogenesis and in the modulation of resistan... more Tumor suppressors play an important role in cancer pathogenesis and in the modulation of resistance to treatments. Loss of function of the proteins encoded by tumor suppressors, through genomic inactivation of the gene, disable all the controls that balance growth, survival, and apoptosis, promoting cancer transformation. Parallel to genetic impairments, tumor suppressor products may also be functionally inactivated in the absence of mutations/deletions upon post-transcriptional and post-translational modifications. Because restoring tumor suppressor functions remains the most effective and selective approach to induce apoptosis in cancer, the dissection of mechanisms of tumor suppressor inactivation is advisable in order to further augment targeted strategies. This review will summarize the role of tumor suppressors in chronic lymphocytic leukemia and attempt to describe how tumor suppressors can represent new hopes in our arsenal against chronic lymphocytic leukemia (CLL).
Journal of Cellular and Molecular Medicine
The newest drugs to target chronic lymphocytic leukaemia (CLL) include the inhibitors of the intr... more The newest drugs to target chronic lymphocytic leukaemia (CLL) include the inhibitors of the intracellular B-cell receptor signalling (BCR inhibitor) 1 and the BCL2 inhibitor venetoclax. 2,3 B-cell receptor signalling inhibitors incorporate the direct BTK inhibitor ibrutinib and the inhibitor of PI3K-delta, 4 a BTK downstream effector, idelalisib. Both ibrutinib and idelalisib have entered the clinical field with impressive results in chemotherapy-refractory CLL patients. However, both drugs are less effective with p53 mutated/deleted CLL cells. This type of CLL remains highly challenging form which should better benefit from the treatment with the BCL2 inhibitor, venetoclax, which acts as a pro-apoptotic trigger. 5 With such a remarkable option of drugs and the possibility to target p53 mutated/deleted clones, CLL should be considered as an easily treatable cancer and the intent to eradicate the disease no longer a fleeting mirage. Unfortunately, cases of resistance to each of these novel drugs have already been reported and mechanisms of resistance deeply investigated. 2,6-8 Interestingly, however, no recurrent abnormalities or mutations have been associated with a specific pattern of resistance, posing some concerns on how resistant patients can be further treated.
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Papers by alessandro morotti