Publications by authors named "Ilaria Iacobucci"

145 Publications

Phase Separation mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

Cancer Discov 2021 Dec 13. Epub 2021 Dec 13.

Structural Biology, St. Jude Children's Research Hospital

NUP98 fusion oncoproteins (FOs) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form, and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0674DOI Listing
December 2021

Biologic and Therapeutic Implications of Genomic Alterations in Acute Lymphoblastic Leukemia.

J Clin Med 2021 Aug 25;10(17). Epub 2021 Aug 25.

Department of Pathology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.

Acute lymphoblastic leukemia (ALL) is the most successful paradigm of how risk-adapted therapy and detailed understanding of the genetic alterations driving leukemogenesis and therapeutic response may dramatically improve treatment outcomes, with cure rates now exceeding 90% in children. However, ALL still represents a leading cause of cancer-related death in the young, and the outcome for older adolescents and young adults with ALL remains poor. In the past decade, next generation sequencing has enabled critical advances in our understanding of leukemogenesis. These include the identification of risk-associated ALL subtypes (e.g., those with rearrangements of , , , and ; the PAX5 P80R and IKZF1 N159Y mutations; and genomic phenocopies such as Ph-like ALL) and the genomic basis of disease evolution. These advances have been complemented by the development of novel therapeutic approaches, including those that are of mutation-specific, such as tyrosine kinase inhibitors, and those that are mutation-agnostic, including antibody and cellular immunotherapies, and protein degradation strategies such as proteolysis-targeting chimeras. Herein, we review the genetic taxonomy of ALL with a focus on clinical implications and the implementation of genomic diagnostic approaches.
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http://dx.doi.org/10.3390/jcm10173792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432032PMC
August 2021

What Antarctic Plants Can Tell Us about Climate Changes: Temperature as a Driver for Metabolic Reprogramming.

Biomolecules 2021 07 23;11(8). Epub 2021 Jul 23.

Department of Ecological and Biological Sciences, University of Tuscia, 01100 Viterbo, Italy.

Global warming is strongly affecting the maritime Antarctica climate and the consequent melting of perennial snow and ice covers resulted in increased colonization by plants. is a vascular plant highly adapted to the harsh environmental conditions of Antarctic Peninsula and understanding how the plant is responding to global warming is a new challenging target for modern cell physiology. To this aim, we performed differential proteomic analysis on plants grown in natural conditions compared to plants grown for one year inside open top chambers (OTCs) which determine an increase of about 4 °C at midday, mimicking the effect of global warming. A thorough analysis of the up- and downregulated proteins highlighted an extensive metabolism reprogramming leading to enhanced photoprotection and oxidative stress control as well as reduced content of cell wall components. Overall, OTCs growth seems to be advantageous for plants which could benefit from a better CO diffusion into the mesophyll and a reduced ROS-mediated photodamage.
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http://dx.doi.org/10.3390/biom11081094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392395PMC
July 2021

Lysosome purinergic receptor P2X4 regulates neoangiogenesis induced by microvesicles from sarcoma patients.

Cell Death Dis 2021 08 17;12(9):797. Epub 2021 Aug 17.

Department of Precision Medicine, University of Campania "LuigiVanvitelli", Naples, Italy.

The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.
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http://dx.doi.org/10.1038/s41419-021-04069-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371002PMC
August 2021

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Sci Signal 2021 07 6;14(690). Epub 2021 Jul 6.

CEINGE Biotecnologie Avanzate, Naples 80145, Italy.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO ) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.
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http://dx.doi.org/10.1126/scisignal.abe5040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432949PMC
July 2021

Therapeutic potential of ruxolitinib and ponatinib in patients with -rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

Haematologica 2021 10 1;106(10):2763-2767. Epub 2021 Oct 1.

Division of Oncology and Center for Childhood Cancer Research, Children's Hospital of Philadelphia; Philadelphia, Pennsylvania USA; Department of Pediatrics, University of Pennsylvania Perelman School of Medicine; Philadelphia, Pennsylvania USA; Abramson Cancer Center, University of Pennsylvania Perelman School of Medicine; Philadelphia, Pennsylvania USA.

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http://dx.doi.org/10.3324/haematol.2021.278697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485673PMC
October 2021

Integrated genomic-metabolic classification of acute myeloid leukemia defines a subgroup with NPM1 and cohesin/DNA damage mutations.

Leukemia 2021 10 30;35(10):2813-2826. Epub 2021 Jun 30.

MLL Munich Leukemia Laboratory, Munich, Germany.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.
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http://dx.doi.org/10.1038/s41375-021-01318-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478658PMC
October 2021

Enhancer Hijacking Drives Oncogenic Expression in Lineage-Ambiguous Stem Cell Leukemia.

Cancer Discov 2021 Nov 8;11(11):2846-2867. Epub 2021 Jun 8.

Department of Data Science, Dana-Farber Cancer Institute, Boston, Massachusetts.

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of , a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to . Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed allele and association of with activated hematopoietic progenitor cell -regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. SIGNIFICANCE: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of driven by diverse structural alterations, including superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries..
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http://dx.doi.org/10.1158/2159-8290.CD-21-0145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8563395PMC
November 2021

Genetic Alterations and Therapeutic Targeting of Philadelphia-Like Acute Lymphoblastic Leukemia.

Genes (Basel) 2021 05 1;12(5). Epub 2021 May 1.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a subgroup of B-cell precursor ALL which by gene expression analysis clusters with Philadelphia-positive ALL although lacking the pathognomonic BCR-ABL1 oncoprotein. Its prevalence increases with age and similar to -positive ALL, Ph-like ALL is characterized by or other B-lymphoid transcription factor gene deletions and by poor outcome to conventional therapeutic approaches. Genetic alterations are highly heterogenous across patients and include gene fusions, sequence mutations, DNA copy number changes and cryptic rearrangements. These lesions drive constitutively active cytokine receptor and kinase signaling pathways which deregulate ABL1 or JAK signaling and more rarely other kinase-driven pathways. The presence of activated kinase alterations and cytokine receptors has led to the incorporation of targeted therapy to the chemotherapy backbone which has improved treatment outcome for this high-risk subtype. More recently, retrospective studies have shown the efficacy of immunotherapies including both antibody drug-conjugates and chimeric antigen receptor T cell therapy and as they are not dependent on a specific genetic alteration, it is likely their use will increase in prospective clinical trials. This review summarizes the genomic landscape, clinical features, diagnostic assays, and novel therapeutic approaches for patients with Ph-like ALL.
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http://dx.doi.org/10.3390/genes12050687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147256PMC
May 2021

Protein-DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era.

J Proteome Res 2021 06 7;20(6):3018-3030. Epub 2021 May 7.

Department of Chemical Sciences, University Federico II of Naples, Strada Comunale Cinthia, 26, 80126 Naples, Italy.

The fields of application of functional proteomics are not limited to the study of protein-protein interactions; they also extend to those involving protein complexes that bind DNA or RNA. These interactions affect fundamental processes such as replication, transcription, and repair in the case of DNA, as well as transport, translation, splicing, and silencing in the case of RNA. Analytical or preparative experimental approaches, both and , have been developed to isolate and identify DNA/RNA binding proteins by exploiting the advantage of the affinity shown by these proteins toward a specific oligonucleotide sequence. The present review proposes an overview of the approaches most commonly employed in proteomics applications for the identification of nucleic acid-binding proteins, such as affinity purification (AP) protocols, EMSA, chromatin purification methods, and CRISPR-based chromatin affinity purification, which are generally associated with mass spectrometry methodologies for the unbiased protein identification.
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http://dx.doi.org/10.1021/acs.jproteome.1c00074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8280749PMC
June 2021

Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a.

Biomedicines 2021 Apr 6;9(4). Epub 2021 Apr 6.

IRCCS Istituto Romagnolo per lo Studio dei Tumori "Dino Amadori"-IRST, 47014 Meldola (FC), Italy.

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from , is a negative regulator of p53. We evaluated expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in -mutated (mut, irrespective of -ITD status) or -mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of -wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of -mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in -wt AML cell line and primary cells, but not in -mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated -wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 -wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in -wt AML.
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http://dx.doi.org/10.3390/biomedicines9040388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067413PMC
April 2021

A Comparative Study of the Effects of Platinum (II) Complexes on β-Amyloid Aggregation: Potential Neurodrug Applications.

Int J Mol Sci 2021 Mar 16;22(6). Epub 2021 Mar 16.

Department of Pharmacy, University of Naples "Federico II", 80131 Naples, Italy.

Herein the effects of three platinum complexes, namely (-4-2)-(2,2'-bipyridine)dichloridoplatinum(II), Pt-bpy, (-4-2)-dichlorido(1,10-phenanthroline) platinum(II), Pt-phen, and (-4-2)-chlorido(2,2':6',2''-terpyridine)platinum(II) chloride, Pt-terpy, on the aggregation of an amyloid model system derived from the C-terminal domain of Aβ peptide (Aβ) were investigated. Thioflavin T (ThT) binding assays revealed the ability of Pt(II) compounds to repress amyloid aggregation in a dose-dependent way, whereas the ability of Aβ peptide to interfere with ligand field of metal complexes was analyzed through UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. Spectroscopic data provided micromolar EC values and allowed to assess that the observed inhibition of amyloid aggregation is due to the formation of adducts between Aβ peptide and complexes upon the release of labile ligands as chloride and that they can explore different modes of coordination toward Aβ with respect to the entire Aβ polypeptide. In addition, conformational studies through circular dichroism (CD) spectroscopy suggested that Pt-terpy induces soluble β-structures of monomeric Aβ, thus limiting self-recognition. Noticeably, Pt-terpy demonstrated the ability to reduce the cytotoxicity of amyloid peptide in human SH-SY5Y neuroblastoma cells. Presented data corroborate the hypothesis to enlarge the application field of already known metal-based agents to neurodegenerative diseases, as potential neurodrugs.
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http://dx.doi.org/10.3390/ijms22063015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998721PMC
March 2021

Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A-Treated Stem Cells.

Int J Mol Sci 2021 Feb 19;22(4). Epub 2021 Feb 19.

Department of Chemical Sciences, University Federico II of Naples, Strada Comunale Cinthia, 26, 80126 Naples, Italy.

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.
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http://dx.doi.org/10.3390/ijms22042063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921975PMC
February 2021

ADAM10 hyperactivation acts on piccolo to deplete synaptic vesicle stores in Huntington's disease.

Hum Mol Genet 2021 06;30(13):1175-1187

Department of Biosciences, University of Milan, Milan 20133, Italy.

Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.
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http://dx.doi.org/10.1093/hmg/ddab047DOI Listing
June 2021

Modeling and targeting of erythroleukemia by hematopoietic genome editing.

Blood 2021 03;137(12):1628-1640

Department of Pathology, St Jude Children's Research Hospital, Memphis, TN.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.
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http://dx.doi.org/10.1182/blood.2020009103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995291PMC
March 2021

Prognostic mutation constellations in acute myeloid leukaemia and myelodysplastic syndrome.

Curr Opin Hematol 2021 03;28(2):101-109

Department of Pathology.

Purpose Of Review: In the past decade, numerous studies analysing the genome and transcriptome of large cohorts of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) patients have substantially improved our knowledge of the genetic landscape of these diseases with the identification of heterogeneous constellations of germline and somatic mutations with prognostic and therapeutic relevance. However, inclusion of integrated genetic data into classification schema is still far from a reality. The purpose of this review is to summarize recent insights into the prevalence, pathogenic role, clonal architecture, prognostic impact and therapeutic management of genetic alterations across the spectrum of myeloid malignancies.

Recent Findings: Recent multiomic-studies, including analysis of genetic alterations at the single-cell resolution, have revealed a high heterogeneity of lesions in over 200 recurrently mutated genes affecting disease initiation, clonal evolution and clinical outcome. Artificial intelligence and specifically machine learning approaches have been applied to large cohorts of AML and MDS patients to define in an unbiased manner clinically meaningful disease patterns including, disease classification, prognostication and therapeutic vulnerability, paving the way for future use in clinical practice.

Summary: Integration of genomic, transcriptomic, epigenomic and clinical data coupled to conventional and machine learning approaches will allow refined leukaemia classification and risk prognostication and will identify novel therapeutic targets for these still high-risk leukaemia subtypes.
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http://dx.doi.org/10.1097/MOH.0000000000000629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8174569PMC
March 2021

Rearrangements of ATP5L-KMT2A in acute lymphoblastic leukaemia.

Br J Haematol 2021 03 13;192(6):e139-e144. Epub 2020 Dec 13.

Scientific Directorate, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC), Italy.

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http://dx.doi.org/10.1111/bjh.17265DOI Listing
March 2021

Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.

Vet Pathol 2021 01 19;58(1):181-204. Epub 2020 Nov 19.

5417St. Jude Children's Research Hospital, Memphis, TN, USA.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.
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http://dx.doi.org/10.1177/0300985820970144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8414369PMC
January 2021

Osteopontin binds ICOSL promoting tumor metastasis.

Commun Biol 2020 10 26;3(1):615. Epub 2020 Oct 26.

Dipartimento di Scienze della Salute, Interdisciplinary Research Center of Autoimmune Diseases-IRCAD, Università del Piemonte Orientale, 28100, Novara, Italy.

ICOSL/ICOS are costimulatory molecules pertaining to immune checkpoints; their binding transduces signals having anti-tumor activity. Osteopontin (OPN) is here identified as a ligand for ICOSL. OPN binds a different domain from that used by ICOS, and the binding induces a conformational change in OPN, exposing domains that are relevant for its functions. Here we show that in vitro, ICOSL triggering by OPN induces cell migration, while inhibiting anchorage-independent cell growth. The mouse 4T1 breast cancer model confirms these data. In vivo, OPN-triggering of ICOSL increases angiogenesis and tumor metastatization. The findings shed new light on ICOSL function and indicate that another partner beside ICOS may be involved; they also provide a rationale for developing alternative therapeutic approaches targeting this molecular trio.
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http://dx.doi.org/10.1038/s42003-020-01333-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588454PMC
October 2020

From classical to new generation approaches: An excursus of -omics methods for investigation of protein-protein interaction networks.

J Proteomics 2021 01 20;230:103990. Epub 2020 Sep 20.

Department of Chemical Sciences, University Federico II of Naples, Strada Comunale Cinthia, 26, 80126 Naples, Italy; CEINGE Advanced Biotechnologies, Via G. Salvatore 486, 80145 Naples, Italy. Electronic address:

Functional Proteomics aims to the identification of in vivo protein-protein interaction (PPI) in order to piece together protein complexes, and therefore, cell pathways involved in biological processes of interest. Over the years, proteomic approaches used for protein-protein interaction investigation have relied on classical biochemical protocols adapted to a global overview of protein-protein interactions, within so-called "interactomics" investigation. In particular, their coupling with advanced mass spectrometry instruments and innovative analytical methods led to make great strides in the PPIs investigation in proteomics. In this review, an overview of protein complexes purification strategies, from affinity purification approaches, including proximity-dependent labeling techniques and cross-linking strategy for the identification of transient interactions, to Blue Native Gel Electrophoresis (BN-PAGE) and Size Exclusion Chromatography (SEC) employed in the "complexome profiling", has been reported, giving a look to their developments, strengths and weakness and providing to readers several recent applications of each strategy. Moreover, a section dedicated to bioinformatic databases and platforms employed for protein networks analyses was also included.
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http://dx.doi.org/10.1016/j.jprot.2020.103990DOI Listing
January 2021

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease.

PLoS One 2020 4;15(9):e0238037. Epub 2020 Sep 4.

Department of Chemical Sciences, University of Naples "Federico II", Naples, Italy.

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473538PMC
October 2020

Cisplatin binding to β-lactoglobulin: a structural study.

Dalton Trans 2020 Sep;49(35):12450-12457

Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone 16, 80134 Napoli, Italy.

β-Lactoglobulin is a major globular milk whey carrier with potential applications as an oral drug delivery system. Herein, the interactions between β-lactoglobulin and cisplatin are investigated by UV-Vis absorption spectroscopy, circular dichroism, X-ray crystallography and electrospray ionization mass spectrometry. Structural data indicate that the protein retains its conformation upon cisplatin binding. Pt-containing fragments bind the side chains of Met7, His146 and Lys8, with the number of binding sites increasing over time. Mass spectrometry data indicate that [Pt(NH3)2Cl+], [Pt(NH3)2OH22+] and [Pt(NH3)22+] fragments interact with β-lactoglobulin; up to 3 cisplatin fragments can bind the protein and the number of cisplatin binding sites increases over time. This work opens a new pathway in pharmaceutical studies based on a rational design of metal-based drug/β-lactoglobulin adducts as delivering vehicles of metallodrugs.
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http://dx.doi.org/10.1039/d0dt02582hDOI Listing
September 2020

Mutational landscape and patterns of clonal evolution in relapsed pediatric acute lymphoblastic leukemia.

Blood Cancer Discov 2020 Jul;1(1):96-111

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL, incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking and xenografting to formally define clonal structure, we identify 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. , and Ras signaling mutations rose from diagnosis subclones, whereas variants in and were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%) or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using deep digital PCR at levels comparable to orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones.
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http://dx.doi.org/10.1158/0008-5472.BCD-19-0041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418874PMC
July 2020

Modulation of Amyloidogenic Peptide Aggregation by Photoactivatable CO-Releasing Ruthenium(II) Complexes.

Pharmaceuticals (Basel) 2020 Jul 29;13(8). Epub 2020 Jul 29.

Department of Pharmacy, University of Naples Federico II, 80134 Napoli, Italy.

Three Ru(II)-based CO-releasing molecules featuring bidentate benzimidazole and terpyridine derivatives as ligands were investigated for their ability to modulate the aggregation process of the second helix of the C-terminal domain of nucleophosmin 1, namely nucleophosmin 1 (NPM1), a model amyloidogenic system, before and after irradiation at 365 nm. Thioflavin T (ThT) binding assays and UV/Vis absorption spectra indicate that binding of the compounds to the peptide inhibits its aggregation and that the inhibitory effect increases upon irradiation (half maximal effective concentration (EC) values in the high micromolar range). Electrospray ionization mass spectrometry data of the peptide in the presence of one of these compounds confirm that the modulation of amyloid aggregation relies on the formation of adducts obtained when the Ru compounds react with the peptide upon releasing of labile ligands, like chloride and carbon monoxide. This mechanism of action explains the subtle different behavior of the three compounds observed in ThT experiments. Overall, data support the hypothesis that metal-based CO releasing molecules can be used to develop metal-based drugs with potential application as anti-amyloidogenic agents.
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http://dx.doi.org/10.3390/ph13080171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464691PMC
July 2020

Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.

Blood 2020 08;136(6):698-714

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
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http://dx.doi.org/10.1182/blood.2019003062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215330PMC
August 2020

Pt(II) versus Pt(IV) in Carbene Glycoconjugate Antitumor Agents: Minimal Structural Variations and Great Performance Changes.

Inorg Chem 2020 Mar 4;59(6):4002-4014. Epub 2020 Mar 4.

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario di Monte S. Angelo, via Cintia 21, 80126 Napoli, Italy.

Octahedral Pt(IV) complexes () containing a glycoconjugate carbene ligand were prepared and fully characterized. These complexes are structural analogues to the trigonal bipyramidal Pt(II) species () recently described. Thus, an unprecedented direct comparison between the biological properties of Pt compounds with different oxidation states and almost indistinguishable structural features was performed. The stability profile of the novel Pt(IV) compounds in reference solvents was determined and compared to that of the analogous Pt(II) complexes. The uptake and antiproliferative activities of and were evaluated on the same panel of cell lines. DNA and protein binding properties were assessed using human serum albumin, the model protein hen egg white lysozyme, and double stranded DNA model systems by a variety of experimental techniques, including UV-vis absorption spectroscopy, fluorescence, circular dichroism, and electrospray ionization mass spectrometry. Although the compounds present similar structures, their in-solution stability, cellular uptake, and DNA binding properties are diverse. These differences may represent the basis of their different cytotoxicity and biological activity.
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http://dx.doi.org/10.1021/acs.inorgchem.9b03683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997382PMC
March 2020

The interaction between the F55 virus-encoded transcription regulator and the RadA host recombinase reveals a common strategy in Archaea and Bacteria to sense the UV-induced damage to the host DNA.

Biochim Biophys Acta Gene Regul Mech 2020 05 31;1863(5):194493. Epub 2020 Jan 31.

Department of Biology, University of Naples Federico II, 80126 Naples, Italy. Electronic address:

Sulfolobus spindle-shaped virus 1 is the only UV-inducible member of the virus family Fuselloviridae. Originally isolated from Saccharolobus shibatae B12, it can also infect Saccharolobus solfataricus. Like the CI repressor of the bacteriophage λ, the SSV1-encoded F55 transcription repressor acts as a key regulator for the maintenance of the SSV1 carrier state. In particular, F55 binds to tandem repeat sequences located within the promoters of the early and UV-inducible transcripts. Upon exposure to UV light, a temporally coordinated pattern of gene expression is triggered. In the case of the better characterized bacteriophage λ, the switch from lysogenic to lytic development is regulated by a crosstalk between the virus encoded CI repressor and the host RecA, which regulates also the SOS response. For SSV1, instead, the regulatory mechanisms governing the switch from the carrier to the induced state have not been completely unravelled. In this study we have applied an integrated biochemical approach based on a variant of the EMSA assay coupled to mass spectrometry analyses to identify the proteins associated with F55 when bound to its specific DNA promoter sequences. Among the putative F55 interactors, we identified RadA and showed that the archaeal molecular components F55 and RadA are functional homologs of bacteriophage λ (factor CI) and Escherichia coli (RecA) system.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194493DOI Listing
May 2020

Successes and Challenges for Diagnosis and Therapy of Acute Leukemia.

J Oncol 2019 22;2019:3408318. Epub 2019 Nov 22.

Pediatric Hematology-Oncology Unit, Department of Medical and Surgical Sciences DIMEC, University of Bologna, Bologna, Italy.

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http://dx.doi.org/10.1155/2019/3408318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893268PMC
November 2019

Novel and Rare Fusion Transcripts Involving Transcription Factors and Tumor Suppressor Genes in Acute Myeloid Leukemia.

Cancers (Basel) 2019 Dec 5;11(12). Epub 2019 Dec 5.

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 47014 Meldola (FC), Italy.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the chimera. We detected 7 different fusions with partner genes involving transcription factors (, ), tumor suppressors (, , ) and rearrangements associated with the loss of (, ). Notably, rearrangements co-occurred with mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.
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http://dx.doi.org/10.3390/cancers11121951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966504PMC
December 2019
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