508 results on '"Pg, Pelicci"'
Search Results
2. Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia
- Author
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Ff, Ferrara, Fazi F, Bianchini A, Padula F, Gelmetti V, Minucci S, Mancini M, Pg, Pelicci, Lo Coco F, and Clara NERVI
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Transcriptional Activation ,Oncogene Proteins, Fusion ,Gene Expression Regulation, Leukemic ,Acetylation ,Antineoplastic Agents ,Cell Differentiation ,Tretinoin ,Hydroxamic Acids ,Histone Deacetylases ,Leukemia, Myelomonocytic, Acute ,Histone Deacetylase Inhibitors ,Histones ,Leukemia, Myeloid, Acute ,RUNX1 Translocation Partner 1 Protein ,Core Binding Factor Alpha 2 Subunit ,Tumor Cells, Cultured ,Humans ,Enzyme Inhibitors ,Signal Transduction ,Transcription Factors - Abstract
Histone deacetylase (HDAC)-dependent transcriptional repression of the retinoic acid (RA)-signaling pathway underlies the differentiation block of acute promyelocytic leukemia. RA treatment relieves transcriptional repression and triggers differentiation of acute promyelocytic leukemia blasts, leading to disease remission. We report that transcriptional repression of RA signaling is a common mechanism in acute myeloid leukemias (AMLs). HDAC inhibitors restored RA-dependent transcriptional activation and triggered terminal differentiation of primary blasts from 23 AML patients. Accordingly, we show that AML1/ETO, the commonest AML-associated fusion protein, is an HDAC-dependent repressor of RA signaling. These findings relate alteration of the RA pathway to myeloid leukemogenesis and underscore the potential of transcriptional/differentiation therapy in AML.
- Published
- 2001
3. Selective expression and constitutive phosphorylation of SHC proteins [corrected] in the CD34+ fraction of chronic myelogenous leukemias
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Bonati A, Carmelo Carlo-Stella, Lunghi P, Albertini R, Pinelli S, Migliaccio E, Sammarelli G, Savoldo B, Tabilio A, Pp, Dall Aglio, and Pg, Pelicci
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src Homology Domains ,Adaptor Proteins, Vesicular Transport ,Src Homology 2 Domain-Containing, Transforming Protein 1 ,Shc Signaling Adaptor Proteins ,Bone Marrow ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Granulocyte Colony-Stimulating Factor ,Humans ,Proteins ,Antigens, CD34 ,Phosphorylation ,Adaptor Proteins, Signal Transducing - Abstract
The BCR/ABL fusion protein is a constitutively active tyrosine kinase that is responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinically, CML is characterized by a chronic phase (CP) that eventually terminates into a blast crisis (BC). BC transformation is associated with accumulation of CD34+ blasts. We investigated the expression and phosphorylation of Src-homology-2 and collagen-homology domains (SHC) [corrected] proteins in subpopulations of CML primary cells. Shc polypeptides are tyrosine kinase substrates that are constitutively tyrosine-phosphorylated in continuous cell lines of CML origin. High levels of Shc expression were found in the CD34+ cells from CML-BC, CML-CP and normal bone marrow. In contrast, CD34- fractions from CML-CP and normal bone marrow expressed low levels of p46Shc. Shc proteins were constitutively phosphorylated in the CD34+ fractions from CML cells (both CP and BC), but not in normal CD34+ cells. These data bear implications for the role of Shc in normal hemopoiesis and CML leukemogenesis: (a) dramatic changes of Shc expression during terminal differentiation of hemopoietic cells adds a further level of regulation to the signal transduction function of Shc; and (b) constitutive Shc tyrosine-phosphorylation in the rare CD34+ cells of CML-CP might contribute to the selection of this subpopulation during the blast crisis transformation of CMLs.
- Published
- 2000
4. Constitutive degradation of PML/RARalpha through the proteasome pathway mediates retinoic acid resistance
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Fanelli M, Minucci S, Gelmetti V, Clara NERVI, Gambacorti-Passerini C, and Pg, Pelicci
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Cysteine Endopeptidases ,Proteasome Endopeptidase Complex ,Leukemia, Promyelocytic, Acute ,Oncogene Proteins, Fusion ,Drug Resistance, Neoplasm ,Multienzyme Complexes ,Humans ,Antineoplastic Agents ,Tretinoin ,Neoplasm Proteins - Abstract
PML/RARalpha is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RARalpha, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RARalpha protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RARalpha is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RARalpha protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RARalpha, but not RARalpha, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RARalpha protein may lead to RA resistance and that PML/RARalpha expression is crucial to convey RA sensitivity to APL cells.
- Published
- 1999
5. Detection of normal and chimeric nucleophosmin in human cells
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Jl, Cordell, Ka, Pulford, Bigerna B, Roncador G, Alison Banham, Colombo E, Pg, Pelicci, Dy, Mason, and Falini B
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Cell Nucleus ,Cytoplasm ,Recombinant Fusion Proteins ,Blotting, Western ,Antibodies, Monoclonal ,Nuclear Proteins ,Immunohistochemistry ,Peptide Fragments ,Translocation, Genetic ,Neoplasm Proteins ,Mice ,Chromosomes, Human, Pair 2 ,Animals ,Chromosomes, Human, Pair 5 ,Humans ,Lymphoma, Large B-Cell, Diffuse ,Nucleophosmin - Abstract
In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RARalpha and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.
- Published
- 1999
6. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein
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Clara NERVI, Ff, Ferrara, Fanelli M, Rippo MR, Tomassini B, Pf, Ferrucci, Ruthardt M, Gelmetti V, Gambacorti-Passerini C, Diverio D, Grignani F, Pg, Pelicci, and Testi R
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Cell Nucleus ,Proteasome Endopeptidase Complex ,Oncogene Proteins, Fusion ,Caspase 3 ,Gene Expression Regulation, Leukemic ,Recombinant Fusion Proteins ,Tumor Suppressor Proteins ,Nuclear Proteins ,Antineoplastic Agents ,Cell Differentiation ,Tretinoin ,Cysteine Proteinase Inhibitors ,Promyelocytic Leukemia Protein ,Protein Structure, Secondary ,Neoplasm Proteins ,Enzyme Activation ,Cysteine Endopeptidases ,Leukemia, Promyelocytic, Acute ,Multienzyme Complexes ,Caspases ,Enzyme Induction ,Tumor Cells, Cultured ,Humans ,Transcription Factors - Abstract
All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.
- Published
- 1998
7. The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein
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Pelicci G, Giordano S, Zhen Z, Ae, Salcini, Lanfrancone L, Alberto BARDELLI, Panayotou G, Md, Waterfield, Ponzetto C, and Pg, Pelicci
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Hepatocyte Growth Factor ,Molecular Sequence Data ,Proteins ,Receptor Protein-Tyrosine Kinases ,Haplorhini ,Proto-Oncogene Proteins c-met ,Mice ,Cell Movement ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Cell Division ,Adaptor Proteins, Signal Transducing ,GRB2 Adaptor Protein - Abstract
The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates that phosphotyrosines Y1349VHV and Y1356VNV can work as docking sites for Shc. The Kd of this interaction, measured in real time using synthetic phosphopeptides and recombinant Shc on a BIAcore biosensor, is 150 nm for both sites. After stimulation of the HGF receptor, Shc is phosphorylated on Y317VNV, generating an high affinity binding site for Grb2 (Kd = 15 nM). This duplicates the high affinity binding site for Grb2 present on the HGF receptor (Y1356VNV). Thus HGF stimulation can trigger the Ras pathway by recruiting Grb2 both directly through the receptor, and indirectly, through Shc. Overexpression of wild-type Shc, but not of the Y317--F mutant, enhances cell migration and growth in response to HGF. These data show that Shc is a relevant substrate of the HGF receptor, and works as an 'amplifier' of the motogenic as well as of the mitogenic response.
- Published
- 1995
8. Effect of the acute promyelocytic leukemia PML/RAR alpha protein on differentiation and survival of myeloid precursors
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Fagioli M, Grignani F, Pier Francesco Ferrucci, Alcalay M, Mencarelli A, Nicoletti I, and Pg, Pelicci
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Cell Survival ,Receptors, Retinoic Acid ,Sulfates ,Tumor Suppressor Proteins ,Nuclear Proteins ,Cell Differentiation ,Tretinoin ,DNA ,Promyelocytic Leukemia Protein ,Hematopoietic Stem Cells ,Zinc Sulfate ,Neoplasm Proteins ,Leukemia, Promyelocytic, Acute ,Zinc Compounds ,Tumor Cells, Cultured ,Humans ,Cell Division ,Transcription Factors - Abstract
Acute promyelocytic leukaemia is characterized by an expansion of haematopoietic precursors arrested at the promyelocytic stage (1). The differentiation block can be reversed by retinoic acid, which induces blast differentiation both in vitro (2) and in vivo (3-4). Acute promyelocytic leukaemia is also characterized by a 15;17 chromosome translocation (5) with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and within the PML gene, that encodes a putative transcription factor of unknown function (6-7), on 15 (8-10). As a consequence of the translocation a PML/RAR alpha gene is formed. It is transcriptionally active and encodes a PML/RAR alpha fusion protein detectable in all APL cases (11-14). We expressed the PML/RAR alpha protein in U937 myeloid precursor cell line and show that they: 1) lose the capacity to differentiate under the action of different stimuli (vitamin D3, transforming growth factor beta 1); ii) acquire enhanced sensitivity to retinoic acid; iii) exhibit a higher growth rate that is due to a reduction in apoptotic cell death. These results provide the first evidence of biological activity of PML/RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.
- Published
- 1994
9. Results of T-depleted BMT in chronic myelogenous leukaemia after a conditioning regimen that included thiotepa
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Franco Aversa, Pg, Pelicci, Terenzi A, Carotti A, Felicini R, Mencarelli A, Donti E, Latini P, Aristei C, and Mf, Martelli
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Adult ,Male ,Adolescent ,T-Lymphocytes ,Fusion Proteins, bcr-abl ,Middle Aged ,Polymerase Chain Reaction ,Lymphocyte Depletion ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,result chronic ,Humans ,Female ,RNA, Messenger ,RNA, Neoplasm ,Thiotepa ,Bone Marrow Transplantation - Published
- 1991
10. Characterization of human and mouse c-fes cDNA clones and identification of the 5' end of the gene
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Myriam Alcalay, Antolini F, Wj, Ven, Lanfrancone L, Grignani F, and Pg, Pelicci
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Base Sequence ,Placenta ,Molecular Sequence Data ,Restriction Mapping ,DNA ,Exons ,Protein-Tyrosine Kinases ,Methylation ,Cell Line ,Mice ,Proto-Oncogene Proteins c-fes ,Pregnancy ,Proto-Oncogene Proteins ,Sequence Homology, Nucleic Acid ,Proto-Oncogenes ,Animals ,Humans ,Female ,Amino Acid Sequence ,RNA, Messenger ,Gene Library - Abstract
cDNA clones of the human c-fes mRNA were isolated. Nucleotide analysis showed that c-fes mRNA contains a 2514 nucleotide open reading frame, which could encode for a 93 kDa protein, and both 5' and 3' v-fes nonhomologous sequences. Primer extension experiments confirmed that the longest isolated cDNAs are about the same length as the entire human c-fes mRNA. Sequence comparison between human c-fes cDNA and the corresponding genomic regions identified a 5' viral-non homologous exon (exon 1) located 491 bp upstream from the first v-fes homologous exon. The genomic region surrounding c-fes exon 1 contains a CpG island and acts as a promoter in vitro. Analysis of the 5' end of mouse c-fes cDNA suggested that the 5' human and mouse gene structure are similar.
- Published
- 1990
11. Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties
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Pp, Pandolfi, Grignani F, Myriam Alcalay, Mencarelli A, Biondi A, LoCoco F, and Pg, Pelicci
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Chloramphenicol O-Acetyltransferase ,Transcriptional Activation ,Chromosomes, Human, Pair 15 ,Binding Sites ,Base Sequence ,Transcription, Genetic ,Receptors, Retinoic Acid ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Restriction Mapping ,Tretinoin ,DNA ,Transfection ,Translocation, Genetic ,Retinoids ,Gene Expression Regulation ,Leukemia, Promyelocytic, Acute ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Carrier Proteins ,Chromosomes, Human, Pair 17 ,Transcription Factors - Abstract
Acute promyelocytic leukemia (APL) is characterized by the 15;17 chromosomal translocation. Cloning experiments have established that the chromosome 17 breakpoint maps to the RAR alpha and the 15 to the myl locus. The resulting chimeric gene is transcribed as a myl/RAR alpha fusion mRNA. By isolating both normal myl and APL myl/RAR alpha cDNAs, we showed that the myl/RAR alpha mRNA encodes for a putative fusion protein with a molecular weight of about 103 kDa, which is made up of 530 amino acids derived from the myl N-terminus and 402 amino acids originating from the RAR alpha C-terminus. The protein includes the RAR alpha DNA and retinoid-binding regions but lacks the A portion of the N-terminal region (A/B region) which is thought to contain one of the RAR alpha transactivation domains. The myl/RAR alpha protein acted as a retinoid-inducible transcription factor with both ligand-independent repressor and ligand-dependent activator functions in transactivation experiments of a retinoic acid-responsive gene. Myl/RAR alpha exerted this dual function three times more effectively than RAR alpha and had about 10-fold greater affinity for RA than RAR alpha. Comparison of myl/RAR alpha genomic and cDNA sequences from the same case demonstrated that both chromosome 15 and 17 breakpoints occurred within introns and the myl and RAR alpha sequences are spliced in the same polyadenylated RNA.
12. Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms
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Fagioli M, Myriam Alcalay, Pp, Pandolfi, Venturini L, Mencarelli A, Simeone A, Acampora D, Grignani F, and Pg, Pelicci
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Chromosomes, Human, Pair 15 ,Binding Sites ,Base Sequence ,Transcription, Genetic ,RNA Splicing ,Tumor Suppressor Proteins ,Molecular Sequence Data ,Nuclear Proteins ,DNA, Neoplasm ,Exons ,Promyelocytic Leukemia Protein ,Translocation, Genetic ,Cell Line ,Neoplasm Proteins ,Leukemia, Promyelocytic, Acute ,Humans ,Amino Acid Sequence ,RNA, Neoplasm ,Cloning, Molecular ,Chromosomes, Human, Pair 17 ,Transcription Factors - Abstract
The acute promyelocytic leukaemia (APL)-specific chromosome 15;17 translocation leads to the fusion of a newly identified putative transcription factor, PML, and the retinoic acid receptor alpha. We have characterized the structure of the PML genomic locus and preliminarily characterized its expression pattern. The PML locus spans a minimum of 35 kb and is subdivided into nine exons. The putative PML DNA binding site is encoded by exons 2 and 3. We isolated a large number of alternatively spliced PML transcripts that encode numerous PML isoforms. Two groups of isoforms were identified that differed either in their C-terminal region or in the length of their central region, but retained the putative DNA-binding and dimerization domains. RNAase protection experiments revealed that the different PML isoforms are equally expressed in established cell lines of different histological origin.
13. PML/RAR alpha+ U937 mutant and NB4 cell lines: retinoic acid restores the monocytic differentiation response to vitamin D3
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Testa U, Grignani F, Barberi T, Fagioli M, Masciulli R, Pier Francesco Ferrucci, Seripa D, Camagna A, Alcalay M, and Pg, Pelicci
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Lipopolysaccharides ,Gene Expression Regulation, Leukemic ,Tumor Suppressor Proteins ,Lipopolysaccharide Receptors ,Antigens, Differentiation, Myelomonocytic ,Nuclear Proteins ,Cell Differentiation ,Tretinoin ,Promyelocytic Leukemia Protein ,Transfection ,Neoplasm Proteins ,Zinc ,Leukemia, Promyelocytic, Acute ,Antigens, CD ,Transforming Growth Factor beta ,Tumor Cells, Cultured ,Humans ,Cell Division ,Cholecalciferol ,Transcription Factors - Abstract
We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha protein as high as fresh acute promyelocytic leukemia blasts. In the absence of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydroxyvitamin D3 (D3) and particularly D3 plus transforming growth factor beta 1 (TGF-beta 1) induced terminal differentiation of MTPR9 cells (as observed in "wild-type" U937 cells), on the basis of morphology, membrane antigen pattern, and functional criteria. Conversely, in the presence of Zn2+, D3 and D3 plus TGF-beta 1 failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by high level PML-RAR alpha protein; indeed, Zn(2+)-treated MTPR9 cells incubated with RA plus D3 exhibited significant terminal monocytic maturation, comparable to that of cells treated with D3 alone or combined with RA in absence of Zn2+. Similar observations were made in NB4, a PML-RAR+ human acute leukemic line. As expected RA treatment of NB4 cells causes granulocytic differentiation. Interestingly, the cell line is only scarcely induced to mature monocytic cells by D3 or D3 plus TGF-beta 1 treatment, whereas it is effectively induced to monocytic maturation by combined treatment with D3 and RA. Accordingly, the rate of NB4 cell proliferation is only slightly affected by D3 or D3 plus TGF-beta 1 treatment, mildly inhibited by RA, and markedly decreased by D3 plus RA. These results indicate that in both U937 and NB4 cells high level PML/RAR alpha expression inhibits the monocytic terminal differentiation program triggered by D3 or D3 plus TGF-beta 1, whereas RA treatment effectively antagonizes this inhibitory PML-RAR alpha action and restores the D3 differentiative effect.
14. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP multicenter 'AIDA' trial. GIMEMA-AIEOP Multicenter 'AIDA' Trial
- Author
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Diverio D, Rossi V, Avvisati G, De Santis S, Alessandra PISTILLI, Pane F, Saglio G, Martinelli G, Mc, Petti, Santoro A, Pg, Pelicci, Mandelli F, Biondi A, Lo Coco F, Diverio, D, Rossi, V, Avvisati, G, De Santis, S, Pistilli, A, Pane, Fabrizio, Saglio, G, Martinelli, G, Petti, M. C, Santoro, A, Pelicci, P. G, Mandelli, F, Biondi, A, and Lo Coco, F.
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Adult ,Male ,Risk ,Neoplasm, Residual ,Adolescent ,Oncogene Proteins, Fusion ,Reproducibility of Result ,Sensitivity and Specificity ,Polymerase Chain Reaction ,Neoplasm Protein ,Leukemia, Promyelocytic, Acute ,Antineoplastic Combined Chemotherapy Protocols ,Biomarkers, Tumor ,Humans ,Life Tables ,Prospective Studies ,Child ,Proportional Hazards Models ,Salvage Therapy ,Antineoplastic Combined Chemotherapy Protocol ,Life Table ,Remission Induction ,Reproducibility of Results ,Middle Aged ,Survival Analysis ,Neoplasm Proteins ,Prospective Studie ,Treatment Outcome ,Italy ,Proportional Hazards Model ,Female ,Survival Analysi ,Neoplasm Recurrence, Local ,Human - Abstract
Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARalpha fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARalpha+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARalpha, as assessed by serial dilution experiments, was 10(-4). All patients were in hematologic remission and tested PCR- at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR- in >/=2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARalpha during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up.
15. Characterization of phorbol esters binding to K 562 cells
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Louache F, Jean-Luc Villeval, Pg, Pelicci, Rouis M, Titeux M, Henri A, Rochant H, Thomopoulos P, and Testa U
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Leukemia, Myeloid, Acute ,Receptors, Drug ,Phorbol Esters ,Humans ,Receptors, Cell Surface ,Leukemia, Erythroblastic, Acute ,Caenorhabditis elegans Proteins ,Carrier Proteins ,Tritium ,Binding, Competitive ,Phorbol 12,13-Dibutyrate ,Protein Kinase C ,Cell Line - Abstract
Binding of 20-[3H]-phorbol 2, 13-dibutyrate [( 3H] PDB) to intact human K 562 cells was characterized. Specific binding of [3H] PDB to K 562 cells at 20 degrees C or 37 degrees C reached a maximum within 15-20 min. Maximal specific [3H] PDB binding to K 562 cells was followed by a decline (down regulation) of radioacticity. This down regulation was temperature dependent; no loss of radioactivity occurred by 1 hour at 4 degrees C. When [3H] PDB binding was carried out a 4 degrees C, [3HDB bound to K 562 cells in a rapid, specific, and reversible manner. Phorbol esters which lack tumor-promoting activity, did not inhibit [3H] PDB binding. A Scatchard analysis was compatible with one class of binding sites, Kd = 50 nM and about 2 X 10(5) binding sites per cell. Human serum inhibited specific binding of [3H] PDB. The effect of several chemical compounds on [3H] PDB binding was also investigated. Most of the compounds tested such as butyrate, hemin, gamma-globulins, transferrin, insulin, EGF, and albumin failed to significantly affect the binding of [3H] PDB. In contrast, retinoic acid and quinacrine significantly affected the binding of [3H] PDB: retinoic acid induced a marked increase of [3H] PDB binding which was dose dependent; quinacrine induced a decrease of [3H] PDB binding, even at low concentration.
16. Characterization of the PML-RAR alpha chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation
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Clara NERVI, Ec, Poindexter, Grignani F, Pp, Pandolfi, Lo Coco F, Avvisati G, Pg, Pelicci, and Am, Jetten
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Receptors, Retinoic Acid ,Recombinant Fusion Proteins ,Immunoblotting ,Retinoic Acid ,Translocation ,Tretinoin ,Acute ,Translocation, Genetic ,Chromosomes ,Leukemia, Promyelocytic, Acute ,Genetic ,Receptors ,Humans ,Chromatography, High Pressure Liquid ,Promyelocytic ,Chromosomes, Human, Pair 15 ,Chromatography ,Leukemia ,Pair 17 ,Pair 15 ,Carrier Proteins ,Transcription Factors ,Chromosomes, Human, Pair 17 ,High Pressure Liquid ,Settore MED/15 - Malattie del Sangue ,Human - Abstract
The acute promyelocytic leukemia 15;17 chromosomal translocation fuses the PML gene to the RAR alpha locus. The resulting chimeric gene encodes for a putative PML-RAR alpha fusion protein. PML is a putative transcriptional factor and RAR alpha is one of the nuclear retinoic acid receptors through which retinoic acid regulates gene expression. In this study, we investigated the retinoid binding and biochemical properties of the PML-RAR alpha protein by size exclusion high-performance liquid chromatography and immunoblot analysis and compared them with those of normal RAR alpha. The introduction of the expression vector PSG5/PML-RAR alpha into COS-1 cells led to high levels of expression of the PML-RAR alpha fusion protein. This protein was primarily localized in the nucleus and bound retinoids with the same affinity and specificity as the wild type RAR alpha receptor. The PML-RAR alpha fusion protein, but not the RAR alpha, was found in high molecular weight complexes with either itself or other nuclear factors. In the acute promyelocytic leukemia-derived cell line NB4, which contains the t(15;17) chromosomal marker, the PML-RAR alpha product was also found as a high molecular complex. The interaction of the PML-RAR alpha with itself or with other nuclear proteins may be important in understanding the role of the PML-RAR alpha fusion protein in promyelocytic leukemogenesis.
17. Molecular genetics of the t(15;17) translocation in acute promyelocytic leukemia
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Biondi A, Rambaldi A, Pp, Pandolfi, Myriam Alcalay, Longo L, Rossi V, Giudici G, Lo Coco F, and Pg, Pelicci
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Gene Rearrangement ,Chromosomes, Human, Pair 15 ,Base Sequence ,Receptors, Retinoic Acid ,Tumor Suppressor Proteins ,Molecular Sequence Data ,Chromosome Mapping ,Nuclear Proteins ,Promyelocytic Leukemia Protein ,Translocation, Genetic ,Neoplasm Proteins ,Leukemia, Promyelocytic, Acute ,Humans ,Amino Acid Sequence ,Chromosomes, Human, Pair 17 ,Transcription Factors
18. PIK3CA mutation analysis in circulating tumor cells of patients with hormone receptor positive metastatic breast cancer.
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Marino E, Mauro C, Belloni E, Picozzi M, Favalli V, Cassatella MC, Zorzino L, Giacò L, Pelicci PG, Barberis M, Sandri MT, and Bernard L
- Abstract
In metastatic breast cancer (MBC), blood is a source of circulating tumor cells (CTCs). CTCs may serve as a ''real-time liquid biopsy" as they represent metastatic tumor genetics better than primary tumor. PIK3CA is one of the most important oncogenes in treatment-unresponsive breast cancers. The aim of this study was to detect PIK3CA mutations and hereditary cancer variants in CTCs from MBC patients. Forty-seven blood samples were obtained from 20 MBC patients from at least 1/3 consecutive time points. CTCs were quantified using the CellSearch system and isolated from 11/20 patients with ≥5/7.5 ml CTCs (14/47 blood samples) using the DEPArray system. DNA was extracted and amplified to perform Sanger sequencing on PIK3CA gene. Sequencing revealed a pathogenic PIK3CA mutation in 2/11 (18 %) cases. Subsequently, we evaluated a 26-target hereditary gene panel by Next Generation Sequencing and identified a concomitant pathogenic mutation in the TP53 gene in a patient with a PIK3CA mutation. No pathogenic germline variants were found. Our data support the conclusion that CTCs analysis may be used to identify mutations in patients to identify those more likely to metastasize., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)
- Published
- 2024
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19. Identification of Novel Potential Predisposing Variants in Familial Acute Myeloid Leukemia.
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Ronchini C, Gigli F, Fontanini M, Furgi R, Amato V, Giglio F, Gregato G, Bertolini F, Rondoni M, Lanza F, Billio A, Derenzini E, Tarella C, Pelicci PG, Alcalay M, and Todisco E
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- Humans, Female, Male, Middle Aged, Adult, DEAD-box RNA Helicases genetics, Aged, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute pathology, Leukemia, Myeloid, Acute diagnosis, Genetic Predisposition to Disease, Germ-Line Mutation, Pedigree
- Abstract
Background: Myeloid neoplasms, including acute myeloid leukemia, have been traditionally among the less investigated cancer types concerning germline predisposition. Indeed, myeloid neoplasms with germline predisposition are challenging to identify because often display similar clinical and morphological characteristics of sporadic cases and have similar age at diagnosis. However, a misidentifications of familiarity in myeloid neoplasms have a critical impact on clinical management both for the carriers and their relatives., Aims: We conducted a family segregation study, in order to identify novel cancer predisposing genes in myeloid neoplasms and classify novel identified variants., Methods and Results: We performed a thorough genomic analysis using a large custom gene panel (256 genes), the Myelo-Panel, targeted on cancer predisposing genes. In particular, we assessed both germline and somatic variants in four families, each with two siblings, who developed hematological neoplasms: seven acute myeloid leukemia and one Philadelphia-positive chronic myeloid leukemia. In each family, we identified at least one novel potentially predisposing variant, affecting also genes not included in the current European LeukemiaNet guidelines for AML management. Moreover, we suggest reclassification of two germline variants as pathogenic: likely pathogenic p.S21Tfs*139 in CEPBA and VUS p.K392Afs*66 in DDX41., Conclusion: We believe that predisposition to hematological neoplasms is still underestimated and particularly difficult to diagnosed. Considering that misidentification of familiarity in myeloid neoplasms have a critical impact on the clinical management both for the carriers and their relatives, our study highlights the importance of revision, in this clinical context, of clinical practices that should include thorough reconstruction of family history and in-depth genetic testing., (© 2024 The Author(s). Cancer Reports published by Wiley Periodicals LLC.)
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- 2024
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20. Recent Advances in Immune-Based Therapies for Acute Myeloid Leukemia.
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Restelli C, Ruella M, Paruzzo L, Tarella C, Pelicci PG, and Colombo E
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- Humans, Cancer Vaccines therapeutic use, Cancer Vaccines immunology, Tumor Microenvironment immunology, Tumor Microenvironment drug effects, Immune Checkpoint Inhibitors therapeutic use, Immune Checkpoint Inhibitors pharmacology, Leukemia, Myeloid, Acute immunology, Leukemia, Myeloid, Acute therapy, Immunotherapy methods, Immunotherapy trends
- Abstract
Despite advancements, acute myeloid leukemia (AML) remains unconquered by current therapies. Evidence of immune evasion during AML progression, such as HLA loss and T-cell exhaustion, suggests that antileukemic immune responses contribute to disease control and could be harnessed by immunotherapy. In this review, we discuss a spectrum of AML immunotherapy targets, encompassing cancer cell-intrinsic and surface antigens as well as targeting in the leukemic milieu, and how they can be tailored for personalized approaches. These targets are overviewed across major immunotherapy modalities applied to AML: immune checkpoint inhibitors, antibody-drug conjugates, therapeutic vaccines, bispecific/trispecific antibodies, and chimeric antigen receptor (CAR)-T and CAR-NK cells. Significance: Immune therapies in AML treatment show evolving promise. Ongoing research aims to customize approaches for varied patient profiles and clinical scenarios. This review covers immune surveillance mechanisms, therapy options like checkpoint inhibitors, antibodies, CAR-T/NK cells, and vaccines, as well as resistance mechanisms and microenvironment considerations., (©2024 American Association for Cancer Research.)
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- 2024
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21. From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.
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Pelicci S, Furia L, Pelicci PG, and Faretta M
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- Humans, Single Molecule Imaging methods, Microscopy, Fluorescence methods, Protein Binding, Cell Line, Tumor, Mitochondria metabolism, Tumor Suppressor Protein p53 metabolism, Tumor Suppressor p53-Binding Protein 1 metabolism
- Abstract
Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.
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- 2024
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22. EU-LIFE charter of independent life science research institutes.
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Superti-Furga G, Agostinho M, Bury J, Cook S, Durinx C, Ender A, van Luenen H, Lund AH, Medema RH, Miączyńska M, Nickel D, Pelicci PG, Puisieux A, Ripatti S, Sander M, Schubeler D, Serrano L, Sommer T, Sonne-Hansen K, Tomančák P, Vives J, Vontas J, and Bettencourt-Dias M
- Subjects
- Academies and Institutes, Biological Science Disciplines, Biomedical Research
- Abstract
The diverse range of organizations contributing to the global research ecosystem is believed to enhance the overall quality and resilience of its output. Mid-sized autonomous research institutes, distinct from universities, play a crucial role in this landscape. They often lead the way in new research fields and experimental methods, including those in social and organizational domains, which are vital for driving innovation. The EU-LIFE alliance was established with the goal of fostering excellence by developing and disseminating best practices among European biomedical research institutes. As directors of the 15 EU-LIFE institutes, we have spent a decade comparing and refining our processes. Now, we are eager to share the insights we've gained. To this end, we have crafted this Charter, outlining 10 principles we deem essential for research institutes to flourish and achieve ground-breaking discoveries. These principles, detailed in the Charter, encompass excellence, independence, training, internationality and inclusivity, mission focus, technological advancement, administrative innovation, cooperation, societal impact, and public engagement. Our aim is to inspire the establishment of new institutes that adhere to these principles and to raise awareness about their significance. We are convinced that they should be viewed a crucial component of any national and international innovation strategies., (© 2024 Federation of European Biochemical Societies.)
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- 2024
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23. High-Fat Diet Promotes Acute Promyelocytic Leukemia through PPARδ-Enhanced Self-renewal of Preleukemic Progenitors.
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Mazzarella L, Falvo P, Adinolfi M, Tini G, Gatti E, Piccioni R, Bonetti E, Gavilán E, Valli D, Gruszka A, Bodini M, Gallo B, Orecchioni S, de Michele G, Migliaccio E, Duso BA, Roerink S, Stratton M, Bertolini F, Alcalay M, Dellino GI, and Pelicci PG
- Subjects
- Animals, Mice, Cathepsin G, Diet, High-Fat adverse effects, Obesity complications, Oncogene Proteins, Fusion genetics, Leukemia, Promyelocytic, Acute drug therapy, Leukemia, Promyelocytic, Acute genetics, Leukemia, Promyelocytic, Acute pathology, PPAR delta therapeutic use
- Abstract
Risk and outcome of acute promyelocytic leukemia (APL) are particularly worsened in obese-overweight individuals, but the underlying molecular mechanism is unknown. In established mouse APL models (Ctsg-PML::RARA), we confirmed that obesity induced by high-fat diet (HFD) enhances leukemogenesis by increasing penetrance and shortening latency, providing an ideal model to investigate obesity-induced molecular events in the preleukemic phase. Surprisingly, despite increasing DNA damage in hematopoietic stem cells (HSC), HFD only minimally increased mutational load, with no relevant impact on known cancer-driving genes. HFD expanded and enhanced self-renewal of hematopoietic progenitor cells (HPC), with concomitant reduction in long-term HSCs. Importantly, linoleic acid, abundant in HFD, fully recapitulates the effect of HFD on the self-renewal of PML::RARA HPCs through activation of peroxisome proliferator-activated receptor delta, a central regulator of fatty acid metabolism. Our findings inform dietary/pharmacologic interventions to counteract obesity-associated cancers and suggest that nongenetic factors play a key role., Prevention Relevance: Our work informs interventions aimed at counteracting the cancer-promoting effect of obesity. On the basis of our study, individuals with a history of chronic obesity may still significantly reduce their risk by switching to a healthier lifestyle, a concept supported by evidence in solid tumors but not yet in hematologic malignancies. See related Spotlight, p. 47., (©2023 American Association for Cancer Research.)
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- 2024
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24. Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion.
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Pallavi R, Gatti E, Durfort T, Stendardo M, Ravasio R, Leonardi T, Falvo P, Duso BA, Punzi S, Xieraili A, Polazzi A, Verrelli D, Trastulli D, Ronzoni S, Frascolla S, Perticari G, Elgendy M, Varasi M, Colombo E, Giorgio M, Lanfrancone L, Minucci S, Mazzarella L, and Pelicci PG
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- Humans, Animals, Mice, Caloric Restriction, Histone Demethylases genetics, Neoplastic Stem Cells pathology, Cell Line, Tumor, Leukemia, Myeloid, Acute pathology, Insulins
- Abstract
Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in ~90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors., (© 2024. The Author(s).)
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- 2024
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25. UNCAN.eu: Toward a European Federated Cancer Research Data Hub.
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Boutros M, Baumann M, Bigas A, Chaabane L, Guérin J, Habermann JK, Jobard A, Pelicci PG, Stegle O, Tonon G, Valencia A, Winkler EC, Blanc P, De Maria R, Medema RH, Nagy P, Tabernero J, and Solary E
- Subjects
- Humans, Research, European Union, Neoplasms
- Abstract
To enable a collective effort that generates a new level of UNderstanding CANcer (UNCAN.eu) [Cancer Discov (2022) 12 (11): OF1], the European Union supports the creation of a sustainable platform that connects cancer research across Member States. A workshop hosted in Heidelberg gathered European cancer experts to identify ongoing initiatives that may contribute to building this platform and discuss the governance and long-term evolution of a European Federated Cancer Data Hub., (©2023 The Authors; Published by the American Association for Cancer Research.)
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- 2024
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26. High-sensitivity analysis of clonal hematopoiesis reveals increased clonal complexity of potential-driver mutations in severe COVID-19 patients.
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Ronchini C, Caprioli C, Tunzi G, D'Amico FF, Colombo E, Giani M, Foti G, Conconi D, Lavitrano M, Passerini R, Pase L, Capizzi S, Mastrilli F, Alcalay M, Orecchia R, Natoli G, and Pelicci PG
- Subjects
- Humans, RNA, Viral, SARS-CoV-2 genetics, Mutation, Clonal Hematopoiesis genetics, COVID-19 genetics
- Abstract
Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ronchini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2024
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27. Comprehensive genomic profiling on metastatic Melanoma: results from a network screening from 7 Italian Cancer Centres.
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Pallocca M, Molineris I, Berrino E, Marcozzi B, Betti M, Levati L, D'Atri S, Menin C, Madonna G, Ghiorzo P, Bulgarelli J, Ferraresi V, Venesio T, Rodolfo M, Rivoltini L, Lanfrancone L, Ascierto PA, Mazzarella L, Pelicci PG, De Maria R, Ciliberto G, Medico E, and Russo G
- Subjects
- Humans, Proto-Oncogene Proteins B-raf, Genomics, Italy, Early Detection of Cancer, Melanoma genetics
- Abstract
Background: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma., Methods: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting., Results: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints., Conclusions: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research., (© 2023. The Author(s).)
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- 2024
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28. Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.
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Mazzarella L, Santoro F, Ravasio R, Fumagalli V, Massa PE, Rodighiero S, Gavilán E, Romanenghi M, Duso BA, Bonetti E, Manganaro L, Pallavi R, Trastulli D, Pallavicini I, Gentile C, Monzani S, Leonardi T, Pasqualato S, Buttinelli G, Di Martino A, Fedele G, Schiavoni I, Stefanelli P, Meroni G, de Francesco R, Steinkuhler C, Fossati G, Iannacone M, Minucci S, and Pelicci PG
- Subjects
- Animals, Humans, Mice, Antiviral Agents pharmacology, COVID-19 Drug Treatment, Cytokines metabolism, SARS-CoV-2 metabolism, COVID-19, Lysine
- Abstract
Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.
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- 2023
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29. GASOLINE: detecting germline and somatic structural variants from long-reads data.
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Magi A, Mattei G, Mingrino A, Caprioli C, Ronchini C, Frigè G, Semeraro R, Baragli M, Bolognini D, Colombo E, Mazzarella L, and Pelicci PG
- Subjects
- Humans, Sequence Analysis, Genome, Germ Cells, High-Throughput Nucleotide Sequencing, Genome, Human, Sequence Analysis, DNA methods, Gasoline, Software
- Abstract
Long-read sequencing allows analyses of single nucleic-acid molecules and produces sequences in the order of tens to hundreds kilobases. Its application to whole-genome analyses allows identification of complex genomic structural-variants (SVs) with unprecedented resolution. SV identification, however, requires complex computational methods, based on either read-depth or intra- and inter-alignment signatures approaches, which are limited by size or type of SVs. Moreover, most currently available tools only detect germline variants, thus requiring separate computation of sample pairs for comparative analyses. To overcome these limits, we developed a novel tool (Germline And SOmatic structuraL varIants detectioN and gEnotyping; GASOLINE) that groups SV signatures using a sophisticated clustering procedure based on a modified reciprocal overlap criterion, and is designed to identify germline SVs, from single samples, and somatic SVs from paired test and control samples. GASOLINE is a collection of Perl, R and Fortran codes, it analyzes aligned data in BAM format and produces VCF files with statistically significant somatic SVs. Germline or somatic analysis of 30[Formula: see text] sequencing coverage experiments requires 4-5 h with 20 threads. GASOLINE outperformed currently available methods in the detection of both germline and somatic SVs in synthetic and real long-reads datasets. Notably, when applied on a pair of metastatic melanoma and matched-normal sample, GASOLINE identified five genuine somatic SVs that were missed using five different sequencing technologies and state-of-the art SV calling approaches. Thus, GASOLINE identifies germline and somatic SVs with unprecedented accuracy and resolution, outperforming currently available state-of-the-art WGS long-reads computational methods., (© 2023. The Author(s).)
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- 2023
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30. A Rare Subset of Primary Tumor Cells with Concomitant Hyperactivation of Extracellular Matrix Remodeling and dsRNA-IFN1 Signaling Metastasizes in Breast Cancer.
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Roda N, Cossa A, Hillje R, Tirelli A, Ruscitto F, Cheloni S, Priami C, Dalmasso A, Gambino V, Blandano G, Polazzi A, Falvo P, Gatti E, Mazzarella L, Luzi L, Migliaccio E, and Pelicci PG
- Subjects
- Humans, Cell Line, Tumor, Prognosis, Extracellular Matrix genetics, Neoplasm Metastasis, Gene Expression Regulation, Neoplastic, Signal Transduction genetics, Gene Expression Profiling
- Abstract
Metastatic breast cancer has a poor prognosis and is largely considered incurable. A better understanding of the molecular determinants of breast cancer metastasis could facilitate development of improved prevention and treatment strategies. We used lentiviral barcoding coupled to single-cell RNA sequencing to trace clonal and transcriptional evolution during breast cancer metastasis and showed that metastases derive from rare prometastatic clones that are underrepresented in primary tumors. Both low clonal fitness and high metastatic potential were independent of clonal origin. Differential expression and classification analyses revealed that the prometastatic phenotype was acquired by rare cells characterized by the concomitant hyperactivation of extracellular matrix remodeling and dsRNA-IFN signaling pathways. Notably, genetic silencing of key genes in these pathways (KCNQ1OT1 or IFI6, respectively) significantly impaired migration in vitro and metastasis in vivo, with marginal effects on cell proliferation and tumor growth. Gene expression signatures derived from the identified prometastatic genes predict metastatic progression in patients with breast cancer, independently of known prognostic factors. This study elucidates previously unknown mechanisms of breast cancer metastasis and provides prognostic predictors and therapeutic targets for metastasis prevention., Significance: Transcriptional lineage tracing coupled with single-cell transcriptomics defined the transcriptional programs underlying metastatic progression in breast cancer, identifying prognostic signatures and prevention strategies., (©2023 American Association for Cancer Research.)
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- 2023
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31. Barcode demultiplexing of nanopore sequencing raw signals by unsupervised machine learning.
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Papetti DM, Spolaor S, Nazari I, Tirelli A, Leonardi T, Caprioli C, Besozzi D, Vlachou T, Pelicci PG, Cazzaniga P, and Nobile MS
- Abstract
Introduction: Oxford Nanopore Technologies (ONT) is a third generation sequencing approach that allows the analysis of individual, full-length nucleic acids. ONT records the alterations of an ionic current flowing across a nano-scaled pore while a DNA or RNA strand is threading through the pore. Basecalling methods are then leveraged to translate the recorded signal back to the nucleic acid sequence. However, basecall generally introduces errors that hinder the process of barcode demultiplexing, a pivotal task in single-cell RNA sequencing that allows for separating the sequenced transcripts on the basis of their cell of origin. Methods: To solve this issue, we present a novel framework, called UNPLEX, designed to tackle the barcode demultiplexing problem by operating directly on the recorded signals. UNPLEX combines two unsupervised machine learning methods: autoencoders and self-organizing maps (SOM). The autoencoders extract compact, latent representations of the recorded signals that are then clustered by the SOM. Results and Discussion: Our results, obtained on two datasets composed of in silico generated ONT-like signals, show that UNPLEX represents a promising starting point for the development of effective tools to cluster the signals corresponding to the same cell., Competing Interests: TL received reimbursement of expenses from ONT to speak at an event. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Papetti, Spolaor, Nazari, Tirelli, Leonardi, Caprioli, Besozzi, Vlachou, Pelicci, Cazzaniga and Nobile.)
- Published
- 2023
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32. Author Correction: High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.
- Author
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Magi A, Mattei G, Mingrino A, Caprioli C, Ronchini C, Frigè G, Semeraro R, Bolognini D, Rambaldi A, Candoni A, Colombo E, Mazzarella L, and Pelicci PG
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- 2023
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33. High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.
- Author
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Magi A, Mattei G, Mingrino A, Caprioli C, Ronchini C, Frigè G, Semeraro R, Bolognini D, Rambaldi A, Candoni A, Colombo E, Mazzarella L, and Pelicci PG
- Subjects
- Humans, DNA genetics, DNA metabolism, Transcription Factors genetics, Transcription Factors metabolism, Gene Regulatory Networks genetics, Gene Regulatory Networks physiology, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, CpG Islands genetics, CpG Islands physiology, DNA Methylation genetics, DNA Methylation physiology, Drug Resistance, Neoplasm genetics, Drug Resistance, Neoplasm physiology, Epigenome genetics, Epigenome physiology, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute genetics, Nanopores
- Abstract
Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution., (© 2023. The Author(s).)
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- 2023
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34. Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications.
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Pelicci S, Furia L, Pelicci PG, and Faretta M
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- Macromolecular Substances, Microscopy, Fluorescence methods
- Abstract
The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.
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- 2023
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35. Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.
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Priami C, Montariello D, De Michele G, Ruscitto F, Polazzi A, Ronzoni S, Bertalot G, Binelli G, Gambino V, Luzi L, Mapelli M, Giorgio M, Migliaccio E, and Pelicci PG
- Subjects
- Animals, Mice, Cell Proliferation, Src Homology 2 Domain-Containing, Transforming Protein 1 genetics, Src Homology 2 Domain-Containing, Transforming Protein 1 metabolism, Stem Cells metabolism, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Mammary Glands, Animal cytology
- Abstract
Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc
-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cell polarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cell cycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)- Published
- 2022
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36. Hematological disorders after salvage PARPi treatment for ovarian cancer: Cytogenetic and molecular defects and clinical outcomes.
- Author
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Todisco E, Gigli F, Ronchini C, Amato V, Sammassimo S, Pastano R, Parma G, Lapresa MT, Bertolini F, Corsini C, Gregato G, Poletti C, Pelicci PG, Alcalay M, Colombo N, and Tarella C
- Subjects
- Carcinoma, Ovarian Epithelial drug therapy, Cytogenetic Analysis, Female, Humans, Poly(ADP-ribose) Polymerase Inhibitors therapeutic use, Poly(ADP-ribose) Polymerases therapeutic use, Salvage Therapy, Neoplasms, Second Primary drug therapy, Ovarian Neoplasms drug therapy, Ovarian Neoplasms genetics, Ovarian Neoplasms pathology
- Abstract
Inhibitors of poly(ADP-ribose) polymerase (PARPi) are increasingly employed as salvage therapy in epithelial ovarian cancer (EOC), but cytotoxic drug exposure along with PARP inhibition may favor development of hematological disorders. In our study, of 182 women with EOC treated with PARPi, 16 (8.7%) developed therapy-related myeloid neoplasms (t-MNs), with 12 cases of myelodysplasia and 4 of acute myeloid leukemia. All experienced persistent cytopenia after PARPi discontinuation. Seven patients had del(5q)/-5 and/or del(7q)/-7, nine had a complex karyotype and TP53 mutations, recently reported as risk factor for t-MNs in EOC post-PARPi, were found in 12 out of 13 tested patients. Four patients had a rapid and fatal outcome, one had stable disease, eleven underwent induction therapy, followed by allogeneic hematopoietic cell transplantation in seven. Three of these 11 patients experienced refractory disease, and 8 had complete remission. During a 6.8 months (range 2.3-49) median observation time, 3 out of 16 patients were alive, with one surviving patient free of both solid and hematological tumors. Ten patients died because of leukemia, two because of transplant-related events, one from heart failure. Five more patients experienced persistent cell blood count abnormalities following PARPi discontinuation, without reaching MDS diagnostic criteria. A customized Myelo-panel showed clonal hematopoiesis in all five patients. These findings confirm the actual risk of t-MNs in EOC patients after chemotherapy and prolonged PARPi therapy. The management of these patients is complex and outcomes are extremely poor. Careful diagnostic procedures are strongly recommended whenever unusual cytopenias develop in patients receiving PARPi therapy., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)
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- 2022
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37. Alterations induced by the PML-RARα oncogene revealed by image cross correlation spectroscopy.
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Cerutti E, D'Amico M, Cainero I, Pelicci PG, Faretta M, Dellino GI, Diaspro A, and Lanzanò L
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- Humans, Oncogenes, Spectrum Analysis, Oncogene Proteins, Fusion genetics, Oncogene Proteins, Fusion metabolism, Leukemia, Promyelocytic, Acute genetics, Leukemia, Promyelocytic, Acute metabolism
- Abstract
The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To understand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replication and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by lifetime tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon activation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARα. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARα microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARα could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
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- 2022
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38. Automated multimodal fluorescence microscopy for hyperplex spatial-proteomics: Coupling microfluidic-based immunofluorescence to high resolution, high sensitivity, three-dimensional analysis of histological slides.
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Furia L, Pelicci S, Perillo F, Bolognesi MM, Pelicci PG, Facciotti F, Cattoretti G, and Faretta M
- Abstract
In situ multiplexing analysis and in situ transcriptomics are now providing revolutionary tools to achieve the comprehension of the molecular basis of cancer and to progress towards personalized medicine to fight the disease. The complexity of these tasks requires a continuous interplay among different technologies during all the phases of the experimental procedures. New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity. We propose here a new experimental pipeline to obtain an optimized costs-to-benefits ratio thanks to the alternate employment of automated and manual procedures during all the phases of a multiplexing experiment from sample preparation to image collection and analysis. A comparison between ultra-fast and automated immunofluorescence staining and standard staining protocols has been carried out to compare the performances in terms of antigen saturation, background, signal-to-noise ratio and total duration. We then developed specific computational tools to collect data by automated analysis-driven fluorescence microscopy. Computer assisted selection of targeted areas with variable magnification and resolution allows employing confocal microscopy for a 3D high resolution analysis. Spatial resolution and sensitivity were thus maximized in a framework where the amount of stored data and the total requested time for the procedure were optimized and reduced with respect to a standard experimental approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Furia, Pelicci, Perillo, Bolognesi, Pelicci, Facciotti, Cattoretti and Faretta.)
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- 2022
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39. From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action.
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Furia L, Pelicci S, Scanarini M, Pelicci PG, and Faretta M
- Subjects
- DNA metabolism, DNA Damage, DNA Repair, Image Cytometry, Tumor Suppressor p53-Binding Protein 1 metabolism, DNA Breaks, Double-Stranded, Tumor Suppressor Protein p53 metabolism
- Abstract
53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53-53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.
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- 2022
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40. Deconstructing PML-induced premature senescence.
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Bischof O, Kirsh O, Pearson M, Itahana K, Pelicci PG, and Dejean A
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- 2022
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41. Corrigendum: Single-cell technologies to decipher the immune microenvironment in myeloid neoplasms: Perspectives and opportunities.
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Caprioli C, Nazari I, Milovanovic S, and Pelicci PG
- Abstract
[This corrects the article DOI: 10.3389/fonc.2021.796477.]., (Copyright © 2022 Caprioli, Nazari, Milovanovic and Pelicci.)
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- 2022
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42. Beyond Genetics: Metastasis as an Adaptive Response in Breast Cancer.
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Ruscitto F, Roda N, Priami C, Migliaccio E, and Pelicci PG
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- Female, Humans, Neoplasm Metastasis, Breast Neoplasms metabolism
- Abstract
Metastatic disease represents the primary cause of breast cancer (BC) mortality, yet it is still one of the most enigmatic processes in the biology of this tumor. Metastatic progression includes distinct phases: invasion, intravasation, hematogenous dissemination, extravasation and seeding at distant sites, micro-metastasis formation and metastatic outgrowth. Whole-genome sequencing analyses of primary BC and metastases revealed that BC metastatization is a non-genetically selected trait, rather the result of transcriptional and metabolic adaptation to the unfavorable microenvironmental conditions which cancer cells are exposed to (e.g., hypoxia, low nutrients, endoplasmic reticulum stress and chemotherapy administration). In this regard, the latest multi-omics analyses unveiled intra-tumor phenotypic heterogeneity, which determines the polyclonal nature of breast tumors and constitutes a challenge for clinicians, correlating with patient poor prognosis. The present work reviews BC classification and epidemiology, focusing on the impact of metastatic disease on patient prognosis and survival, while describing general principles and current in vitro/in vivo models of the BC metastatic cascade. The authors address here both genetic and phenotypic intrinsic heterogeneity of breast tumors, reporting the latest studies that support the role of the latter in metastatic spreading. Finally, the review illustrates the mechanisms underlying adaptive stress responses during BC metastatic progression.
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- 2022
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43. Quantifying geographical accessibility to cancer clinical trials in different income landscapes.
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Tini G, Trapani D, Duso BA, Beria P, Curigliano G, Pelicci PG, and Mazzarella L
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- Geography, Humans, Incidence, Income, Italy epidemiology, Registries, Clinical Trials as Topic, Health Services Accessibility, Neoplasms epidemiology, Neoplasms therapy
- Abstract
Background: Clinical trials are increasingly perceived as a therapeutic opportunity for cancer patients. Favoring their concentration in few high-expertise academic centers maximizes quality of data collection but poses an issue of access equality. Analytical tools to quantify trial accessibility are needed to rationalize resources., Materials and Methods: We constructed a distance-based accessibility index (dAI) using publicly available data on demographics, cancer incidence and trials. Multiple strategies were applied to mitigate or quantify clear sources of bias: reporting biases by text mining multiple registries; reliability of simple geographical distance by comparison with high-quality travel cost data for Italy; index inflation due to highly heterogeneous cancer incidence by log-transformation. We studied inequalities by Gini index and time trend significance by Mann-Kendall test. We simulated different resource allocation models in representative countries and identified locations where new studies would maximally improve the national index., Results: The dAI approximated well a more realistic but not widely applicable travel cost-based index. Accessibility was unevenly distributed across and within countries (Gini index ∼0.75), with maximal inequalities in high- and upper-middle-income countries (China, United States, Russian Federation). Over time, accessibility increased but less than the total number of trials, most evidently in upper-middle-income countries. Simulations in representative countries (Italy and Serbia) identified ideal locations able to maximally raise the national index., Conclusions: Access to clinical trials is highly uneven across and within countries and is not mitigated by simple increase in the number of trials; a rational algorithmic approach can be used to mitigate inequalities., Competing Interests: Disclosure LM is member of the scientific board of Tethis S.p.A.; GC reports grants from Roche and Pfizer and personal fees from Daichii Sankyo, MSD and AstraZeneca outside the submitted work. All other authors have declared no conflicts of interest. Data sharing The data underlying this article were derived from sources in the public domain: ClinicalTrials.gov (https://clinicaltrials.gov) via the Clinical Trials Transformation Initiative database (http://www.ctti-clinicaltrials.org); Gridded Population of the World v4 (geoquery.org); GLOBOCAN 2018 (https://gco.iarc.fr/); World Bank Open Data (https://data.worldbank.org/). A GitHub repository with the data and code used for accessibility computation and analysis is available at https://github.com/translational-oncology-lab/CTAccessibilityTool., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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44. TMBleR: a bioinformatic tool to optimize TMB estimation and predictive power.
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Fancello L, Guida A, Frige G, Ceol AGM, Babini G, Scaglione GL, Zanfardino M, Mazza T, Ferrando L, Pelicci PG, and Mazzarella L
- Subjects
- Humans, Mutation, Immunotherapy, Biomarkers, Tumor genetics, Computational Biology, Neoplasms pathology
- Abstract
Motivation: Tumor mutational burden (TMB) has been proposed as a predictive biomarker for immunotherapy response in cancer patients, as it is thought to enrich for tumors with high neoantigen load. TMB assessed by whole-exome sequencing is considered the gold standard but remains confined to research settings. In the clinical setting, targeted gene panels sampling various genomic sizes along with diverse strategies to estimate TMB were proposed and no real standard has emerged yet., Results: We provide the community with TMBleR, a tool to measure the clinical impact of various strategies of panel-based TMB measurement., Availability and Implementation: R package and docker container (GPL-3 Open Source license): https://acc-bioinfo.github.io/TMBleR/. Graphical-user interface website: https://bioserver.ieo.it/shiny/app/tmbler., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2022
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45. Novel Tools to Measure Single Molecules Colocalization in Fluorescence Nanoscopy by Image Cross Correlation Spectroscopy.
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Pelicci S, Furia L, Scanarini M, Pelicci PG, Lanzanò L, and Faretta M
- Abstract
Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM. We propose new tools based on Image Cross Correlation Spectroscopy (ICCS) to quantify the colocalization of fluorescent signals at single molecule level. These analysis procedures have been inserted into an experimental pipeline to optimize the produced results. We show that Fluorescent NanoDiamonds targeted to an intracellular compartment can be employed (i) to correct spatial drift to maximize the localization precision and (ii) to register confocal and SMLM images in correlative multiresolution, multimodal imaging. We validated the ICCS based approach on defined biological control samples and showed its ability to quantitatively map area of interactions inside the cell. The produced results show that the ICCS analysis is an efficient tool to measure relative spatial distribution of different molecular species at the nanoscale.
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- 2022
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46. Regulation of LncRNAs in Melanoma and Their Functional Roles in the Metastatic Process.
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Melixetian M, Pelicci PG, and Lanfrancone L
- Subjects
- Carcinogenesis genetics, Gene Expression Regulation, Neoplastic, Humans, Oncogenes, Melanoma genetics, Melanoma pathology, RNA, Long Noncoding genetics
- Abstract
Long non-coding RNAs (lncRNAs) are key regulators of numerous intracellular processes leading to tumorigenesis. They are frequently deregulated in cancer, functioning as oncogenes or tumor suppressors. As they act through multiple mechanisms, it is not surprising that they may exert dual functions in the same tumor. In melanoma, a highly invasive and metastatic tumor with the propensity to rapidly develop drug resistance, lncRNAs play different roles in: (i) guiding the phenotype switch and leading to metastasis formation; (ii) predicting the response of melanoma patients to immunotherapy; (iii) triggering adaptive responses to therapy and acquisition of drug resistance phenotypes. In this review we summarize the most recent findings on the lncRNAs involved in melanoma growth and spreading to distant sites, focusing on their role as biomarkers for disease diagnosis and patient prognosis, or targets for novel therapeutic approaches.
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- 2022
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47. Single-Cell Technologies to Decipher the Immune Microenvironment in Myeloid Neoplasms: Perspectives and Opportunities.
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Caprioli C, Nazari I, Milovanovic S, and Pelicci PG
- Abstract
Myeloid neoplasms (MN) are heterogeneous clonal disorders arising from the expansion of hematopoietic stem and progenitor cells. In parallel with genetic and epigenetic dynamics, the immune system plays a critical role in modulating tumorigenesis, evolution and therapeutic resistance at the various stages of disease progression. Single-cell technologies represent powerful tools to assess the cellular composition of the complex tumor ecosystem and its immune environment, to dissect interactions between neoplastic and non-neoplastic components, and to decipher their functional heterogeneity and plasticity. In addition, recent progress in multi-omics approaches provide an unprecedented opportunity to study multiple molecular layers (DNA, RNA, proteins) at the level of single-cell or single cellular clones during disease evolution or in response to therapy. Applying single-cell technologies to MN holds the promise to uncover novel cell subsets or phenotypic states and highlight the connections between clonal evolution and immune escape, which is crucial to fully understand disease progression and therapeutic resistance. This review provides a perspective on the various opportunities and challenges in the field, focusing on key questions in MN research and discussing their translational value, particularly for the development of more efficient immunotherapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caprioli, Nazari, Milovanovic and Pelicci.)
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- 2022
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48. Biomedical omics: first insights of a new MSc degree of the University of Milan.
- Author
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Alcalay M, Jereczek-Fossa BA, Pepa M, Volpe S, Zaffaroni M, Fiore F, Marvaso G, Pravettoni G, Curigliano G, Santaguida S, and Pelicci PG
- Subjects
- COVID-19 virology, Epigenomics education, Genomics education, Humans, Metabolomics education, Proteomics education, SARS-CoV-2 pathogenicity, Biomedical Research education, COVID-19 genetics, Pandemics prevention & control, SARS-CoV-2 genetics
- Abstract
The advent of technologies allowing the global analysis of biological phenomena, referred to as "omics" (genomics, epigenomics, proteomics, metabolomics, microbiomics, radiomics, and radiogenomics), has revolutionized the study of human diseases and traced the path for quantitative personalized medicine. The newly inaugurated Master of Science Program in Biomedical Omics of the University of Milan, Italy, aims at addressing the unmet need to create professionals with a broad understanding of omics disciplines. The course is structured over 2 years and admits students with a bachelor's degree in biotechnology, biology, chemistry, or pharmaceutical sciences. All teaching activities are fully held in English. A total of nine students enrolled in the first academic year and attended the courses of radiomics, genomics and epigenomics, proteomics, and high-throughput screenings, and their feedback was evaluated by means of an online questionnaire. Faculty with different backgrounds were recruited according to the subject. Due to restrictions imposed by the coronavirus disease 2019 (COVID-19) pandemic, laboratory activities were temporarily suspended, while lectures, journal clubs, and examinations were mainly held online. After the end of the first semester, despite the difficulties brought on by the COVID-19 pandemic, the course overall met the expectations of the students, specifically regarding teaching effectiveness, interpersonal interactions with the lecturers, and courses organization. Future efforts will be undertaken to better calibrate the overall workload of the course and to implement the most relevant suggestions from the students together with omics science evolution in order to guarantee state-of-the-art omics teaching and to prepare future omics specialists.
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- 2022
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49. Lower probability and shorter duration of infections after COVID-19 vaccine correlate with anti-SARS-CoV-2 circulating IgGs.
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Ronchini C, Gandini S, Pasqualato S, Mazzarella L, Facciotti F, Mapelli M, Frige' G, Passerini R, Pase L, Capizzi S, Mastrilli F, Orecchia R, Natoli G, and Pelicci PG
- Subjects
- Adult, Aged, Aged, 80 and over, Antibodies, Anti-Idiotypic immunology, Antibodies, Viral blood, Antibodies, Viral immunology, COVID-19 blood, COVID-19 prevention & control, COVID-19 virology, Female, Humans, Immunoglobulin G immunology, Male, Mass Vaccination, Middle Aged, SARS-CoV-2 immunology, SARS-CoV-2 pathogenicity, Time Factors, Young Adult, Antibodies, Anti-Idiotypic blood, COVID-19 immunology, COVID-19 Vaccines administration & dosage, Immunoglobulin G blood
- Abstract
The correlation between immune responses and protection from SARS-CoV-2 infections and its duration remains unclear. We performed a sanitary surveillance at the European Institute of Oncology (IEO) in Milan over a 17 months period. Pre-vaccination, in 1,493 participants, we scored 266 infections (17.8%) and 8 possible reinfections (3%). Post-vaccination, we identified 30 infections in 2,029 vaccinated individuals (1.5%). We report that the probability of infection post-vaccination is i) significantly lower compared to natural infection, ii) associated with a significantly shorter median duration of infection than that of first infection and reinfection, iii) anticorrelated with circulating antibody levels., Competing Interests: The authors have declared that no competing interests exist.
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- 2022
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50. Dual targeting of the DNA damage response pathway and BCL-2 in diffuse large B-cell lymphoma.
- Author
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Rossi A, Orecchioni S, Falvo P, Tabanelli V, Baiardi E, Agostinelli C, Melle F, Motta G, Calleri A, Fiori S, Corsini C, Casadei B, Mazzara S, Vitolo U, Bertolini F, Zinzani PL, Alcalay M, Pelicci PG, Pileri S, Tarella C, and Derenzini E
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Antineoplastic Agents pharmacology, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Drug Therapy, Combination, Female, Follow-Up Studies, Humans, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Male, Middle Aged, Prognosis, Prospective Studies, Retrospective Studies, Survival Rate, Urea pharmacology, Young Adult, Bridged Bicyclo Compounds, Heterocyclic pharmacology, DNA Repair Enzymes antagonists & inhibitors, Gene Expression Regulation, Leukemic drug effects, Lymphoma, Large B-Cell, Diffuse drug therapy, Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors, Sulfonamides pharmacology, Thiophenes pharmacology, Urea analogs & derivatives
- Abstract
Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient's population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL., (© 2021. The Author(s).)
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- 2022
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