26 results on '"Bernhard, Gentner"'
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2. TEM-GBM: An Open-Label, Phase I/IIa Dose-Escalation Study Evaluating the Safety and Efficacy of Genetically Modified Tie-2 Expressing Monocytes to Deliver IFN-α within Glioblastoma Tumor Microenvironment
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Bernhard Gentner, Filippo Gagliardi, Gabriele Antonarelli, Bianca Pollo, Maria Grazia Bruzzone, Monica Patanè, Elena Anghileri, Stefania Mazzoleni, Fabio Ciceri, Marica Eoli, Valentina Brambilla, Capotondo Alessia, Silvia Snider, Carlo Russo, Matteo Maria Naldini, Valeria Ferla, Matteo Carrabba, Rosina Paterra, Giorgio D'Alessandris, Alessandro Olivi, Zahid Bashir, Matteo Barcella, Luigi Naldini, Valeria Cuccarini, Marco Saini, Roberto Pallini, Francesco Di Meco, Francesca Farina, Paolo Ferroli, Federico G. Legnani, Mariagrazia Garramone, G. Finocchiaro, Tiziana Magnani, and Pietro Mortini more...
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Tumor microenvironment ,Chemistry ,Immunology ,Cancer research ,Dose escalation ,medicine ,Cell Biology ,Hematology ,Open label ,medicine.disease ,Biochemistry ,Genetically modified organism ,Glioblastoma - Abstract
Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs. Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan). The primary endpoints for this study are: Engraftment of Temferon over the first 90 DaysThe proportion of patients achieving hematologic recovery by Day +30 from ASCTShort-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent Lentivirus The figure below reports the TEM-GBM study design. Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing. Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. Figure 1 Figure 1. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder. more...
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- 2021
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Catalog
3. Longitudinal Tracking of Acute Myeloid Leukemia Stem Cells in Xenografts and Patients By microRNA Reporters and Single Cell RNA Sequencing
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Stefano Beretta, Bernhard Gentner, Matteo Barcella, Giacomo Desantis, Matteo Maria Naldini, Carolina Caserta, Ivan Merelli, Francesca Pavesi, Fabio Ciceri, and Gabriele Casirati
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education.field_of_study ,NPM1 ,Myeloid ,Immunology ,Population ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Gene signature ,Biology ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,medicine.anatomical_structure ,Cancer research ,medicine ,education - Abstract
Current understanding of acute myeloid leukemia (AML) assumes a developmental hierarchy, in which a minor fraction of primitive and quiescent leukemia stem cells (LSC) sustain clonal propagation of disease. These therapy-resistant LSC may be the basis of relapse, as supported by data correlating the presence of LSC gene expression signatures at diagnosis with poor prognosis (Ng et al, Nature 2016). Novel approaches tracing LSC fates at single cell level before, during and after chemotherapy (CTX) are needed to confirm their biological relevance and derive new, LSC-focused diagnostic and therapeutic strategies. We and others have previously linked key LSC properties, such as quiescence and therapy resistance, to complex transcriptional regulation orchestrated by miR-126 (Lechman et al, Cancer Cell 2016). Furthermore, we provided proof of concept that LSCs could be prospectively isolated as miR-126(high) cells exploiting a lentiviral reporter vector capturing miR-126 bioactivity in live cells with single cell resolution. To extend these studies, we transduced primary blasts from n=3 AML patients (pts) carrying NPM1 and FLT3-ITD mutations with the miR-126 reporter, followed by xenografting (PDX). Blasts showed intra-tumor heterogeneity (ITH) in terms of miR-126 activity, with a minor fraction identified as miR-126(high). Limiting dilution secondary transplantation of FACS sorted miR-126(high) and -(low) blasts proved strong enrichment of repopulating activity within the miR-126(high) compartment in all 3 pts. On the contrary, no LSC enrichment could be verified in the CD34+CD38- fraction in 1 patient, suggesting that high miR-126 activity represents a more robust LSC identifier than commonly used surface markers. Next, we investigated the impact of daunorubicin and cytarabine CTX on miR-126-reporter+ blasts (n=3 AML) in PDX. Surprisingly, overall miR-126 activity diminished in post CTX residual AML compared to controls, compatible with a loss of blast quiescence. CTX accentuated ITH by uncovering a subset of blasts with very high levels of miR-126, distinct from the bulk population, which may correspond to residual quiescent LSC (Fig A). We then performed bulk RNA sequencing on miR-126(high) and miR-126(low) subsets from CTX and control PDX. While miR-126(low) blasts from both groups expressed markers of myeloid differentiation, miR-126(high) blasts were enriched for published hematopoietic stem cell hallmark signatures. Integrating differentially expressed genes between miR-126(high) and-(low) subsets at steady state and post CTX, we extrapolated a novel 8 gene signature associated with miR-126(high) blasts. Of note, patients from the AML TCGA PanCancer Atlas Cohort (n=161) harboring overexpression in one or more of these 8 genes had significantly decreased overall survival (10 vs 19 months, Logrank test p-value = 0.018). To further test whether our 8-gene miR-126(high) signature reveals ITH in patients, we performed single cell RNA sequencing (scRNAseq) of AML patient BM aspirates at diagnosis (n= 6). Blasts were identified based on the detection of mutated NPM1 transcripts in single cells. In 5 out of 7 patients expression of the miR-126 signature mapped to specific clusters of blasts identified by unsupervised shared nearest neighbor algorithm, confirming that it identifies ITH in patient samples. Interestingly, we detected miR-126 signature(high) blasts in pts with poor prognosis (n=4) and not in those with favorable outcome (n=2) (Fig B). To investigate ITH across longitudinal samples, we next performed scRNAseq of residual AML from a representative patient assessed early after CTX. In line with our PDX CTX model displaying increased miR-126 ITH, blasts on day14 of induction CTX segregated into 2 different clusters: cluster 1 containing LSC-like cells with miR-126(high) signature and similar transcriptional profile to the diagnosis counterpart; cluster 2, instead, was composed of actively cycling blasts. Residual blasts at day30, in addition to uniformly expressing the miR-126(high) signature, differed from diagnosis and day14 blasts by displaying cell cycle quiescence and induction of oxidative phosphorylation genes (Fig C). In summary, we have set up and applied PDX LSC modeling to clinically relevant patient samples to address AML intra-tumor heterogeneity and to pinpoint novel relevant transcriptomic features of LSC at diagnosis and after chemotherapy. Figure Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. more...
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- 2019
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4. Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial
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Maria Ester Bernardo, Serena Acquati, Attilio Rovelli, Silvia Gregori, Bernhard Gentner, Alessandro Aiuti, Francesca Tucci, Giancarlo la Marca, Luigi Naldini, Silvia Pontesilli, Paolo Silvani, Francesca Fumagalli, Fabio Ciceri, Rossella Parini, Eugenio Montini, and Erika Zonari more...
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Oncology ,medicine.medical_specialty ,business.industry ,Plerixafor ,Mucopolysaccharidosis ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Enzyme replacement therapy ,Leukapheresis ,Hematopoietic stem cell transplantation ,Neutropenia ,medicine.disease ,Biochemistry ,Internal medicine ,medicine ,Cognitive decline ,business ,Busulfan ,medicine.drug - Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) performed early in life is the current standard of care for patients with severe type 1 mucopolysaccharidosis (Hurler disease), a metabolic disorder caused by mutations in the alpha-L-iduronidase (IDUA) gene, leading to impaired breakdown of glycosaminoglycans (GAG). Secretion of IDUA by donor-derived hematopoietic cells may cross-correct non-hematopoietic cells, slowing progression of tissue damage and cognitive decline. Nevertheless, Hurler patients undergoing HSCT manifest substantial residual disease burden, e.g. on the skeleton and central nervous system (CNS). We conducted a phase I/II clinical study (NCT03488394) to test whether infusion of autologous CD34+ hematopoietic stem and progenitor cells (HSPC) transduced ex vivo with a lentiviral vector coding for the IDUA gene was feasible, safe and capable of restoring enzymatic activity in the patients' blood and tissues, up to supraphysiologic levels. The trial originally planned to enroll 6 Hurler patients with preserved neurocognitive function (DQ/IQ>70) that had no access to a suitable allogeneic donor. Sample size has recently been increased to 8 patients. By July 2019, six patients have been treated at a median age of 24 months (range: 14-34), with a median follow up of 4 months (range: 1-13). In all patients, we collected a high number of autologous HSPC by leukapheresis following mobilization with lenograstim and plerixafor, resulting in drug products with a median of 21 million CD34+ cells/kg (range: 13-29). Transduction efficiency was high with a median above 80% and a vector copy number (VCN) of 1.7 (range: 1.0-5.2), employing a shortened, 2 day transduction protocol that included prostaglandin E2. All patients showed rapid hematopoietic recovery following myeloablative conditioning with busulfan (targeted to an AUC of 80mg*h/L), fludarabine (160mg/sqm) and rituximab (375mg/sqm). Median duration of grade 4 neutropenia associated with conditioning was 15.5 days (range: 13-19). Also associated with conditioning, Grade 3 thrombocytopenia lasted 4 days, while only 2 out of 6 patients experienced a platelet drop below 20,000/mcL on a single day, in the absence of transfusion support. Adverse events were mild and compatible with myeloablative conditioning, with the exception of patient 3 who experienced an anaphylactic reaction on day+12, which promptly responded to antihistamines, IV fluids and steroids. All evaluable patients showed sustained, supraphysiologic blood IDUA activity (dried blood spot), which was on average 3 fold above the upper limit of normal (evaluable patients: n=5 at 1 month, n=4 at 2 months, n=3 at 3 months). Notably, in n=4 Hurler patients treated with allogeneic HSCT, we detected IDUA activity that ranged within the lowest quartile of normal in spite of full donor chimerism, suggesting substantial gain achieved by overexpressing IDUA in ex vivo genetically-modified autologous HSPC. Urinary GAG excretion fell to normal levels within 3-6 months. IDUA activity was also detected in the cerebrospinal fluid (CSF) of treated patients, accompanied by a logfold reduction in CSF GAGs in the 2 patients with longest follow up. This suggests that gene therapy accomplishes full metabolic correction of tissues, including the CNS. Gene therapy did not induce antibodies against the IDUA protein, while pre-existing antibodies induced by enzyme replacement therapy before gene therapy rapidly disappeared. Patient 1 who reached the 1-year follow-up demonstrated a stable cognitive score, improved findings on brain and spine MRI, resumed growth velocity and an improvement of his skeletal phenotype. The preliminary results from our phase I/II study compare favorably with the standard of care in terms of safety and efficacy, and highlight the potential of genetic engineering of HSPC grafts for therapeutic gain-of-function. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Parini:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; BioMarin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Ultragenyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Orphan Europe: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support. Naldini:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI. more...
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- 2019
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5. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma
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Bernhard Gentner, Fabio Ciceri, Chiara Bonini, Margherita Norelli, Claudio Bordignon, Maurilio Ponzoni, Barbara Camisa, Attilio Bondanza, Laura Falcone, Monica Casucci, Luigi Naldini, Gianpietro Dotti, Fabiana Gullotta, Aurore Saudemont, Benedetta Nicolis di Robilant, Magda Marcatti, Barbara Savoldo, Pietro Genovese, Massimo Bernardi, Casucci, M, Nicolis di Robilant, B, Falcone, L, Camisa, B, Norelli, M, Genovese, P, Gentner, B, Gullotta, F, Ponzoni, Maurilio, Bernardi, M, Marcatti, M, Saudemont, A, Bordignon, Claudio, Savoldo, B, Ciceri, Fabio, Naldini, Luigi, Dotti, G, Bonini, MARIA CHIARA, and Bondanza, Attilio more...
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Myeloid ,business.industry ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,CD28 ,Cell Biology ,Hematology ,Immunotherapy ,Suicide gene ,Biochemistry ,Chimeric antigen receptor ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Stem cell ,business - Abstract
Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM. ""Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem\\\/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3\\\/CD28 beads and interleukin (IL)-7\\\/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM."" more...
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- 2013
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6. Microrna-126 Levels Modulate B-ALL Aggressiveness but Require Tight Regulation to Guarantee Disease Maintenance
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Davide Cittaro, Silvia Nucera, Carolina Caserta, Matteo Maria Naldini, Giacomo Desantis, Bernhard Gentner, Nadia Foligno, and Fabio Ciceri
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Cell cycle regulator ,Immunology ,microRNA ,Cell Biology ,Hematology ,Disease ,Biology ,Biochemistry ,Transcription factor ,Cell biology - Abstract
We have previously identified microRNA-126 as a central modulator of B-ALL biology (Nucera et al, Cancer Cell). Ectopic miR-126 expression in murine stem and progenitor cells is sufficient and necessary to induce and maintain a B-ALL with a Philadelphia(Ph)-like gene expression profile. Importantly, miR-126 is expressed in the majority of human Ph+ B-ALL. Interestingly, there is marked intra-tumoral heterogeneity with regards to miR-126 levels, defining a new layer of subclonal architecture different from genetic subclones. By prospectively isolating miR-126(high) and miR-126(low) subpopulations exploiting a lentiviral miRNA reporter vector, we consistently found a more aggressive behavior of the miR-126(high) subset in xenograft models. To detect miR-126-dependent pathways that may explain this more aggressive behavior, we engineered 2 primary human B-ALLs with lentiviral vector cassettes containing (1) a reverse tetracycline transactivator (rTTA) and (2) a Tet-operon driving overexpression of miR-126 (miR-126-OE), a miR-126-3p seed mutant (miR-126-OE-SM) or a miR-126 sponge (miR-126-KD) performing serial passages in mice following enrichment for transduced cells. Vector cassettes were induced in vivo by doxycycline administration to mice that have developed full blown leukemia, and blasts were collected within 72hr. In patient 1, acute miR-126-OE induced apoptosis, while it was well tolerated in the disease from patient 2 that we hence studied in more detail. RNA sequencing highlighted more than 2,000 deregulated genes between miR-126-OE and miR-126-OE-SM or miR-126-KD blasts (qValue 0.2). Genes down-regulated upon miR-126-OE included transcription factors associated with B cell differentiation (PAX5, IKZ1, TCF3, BCL6), cell cycle regulators (CCND2, RBL1, E2F7, EP300) and ribosomal genes. Moreover, pathway analysis revealed significant de-regulation in JAK-STAT signaling, cell proliferation, adhesion/migration, BCR signaling, FOXO signaling and cell metabolism. Strikingly, there was a strong overlap (221 genes) with a miR-126 switch-off signature obtained from the Ph-like B-ALL mouse model. These shared genes were enriched in the categories "cell proliferation", "cancer-related signaling", "FOXO signaling" and "chromatin assembly during cell cycle", pointing to a miR-126-dependent core circuit shared between human Ph+ B-ALL and the mouse model. We started to validate the transcriptomic data at the protein level measuring phosphorylation status and subcellular localization of signaling hubs. Primary B-ALL from patient 2 showed increased phospho-STAT5 shortly after miR-126-OE, which returned to baseline within 6 days, suggesting adaptive responses counteracting a potentially deleterious excess of signaling in this disease. We hypothesize that a surge in signaling strength upon acute induction of miR-126-OE may have contributed to the apoptotic phenotype observed in patient 1. In keeping with this, constitutive miR-126-OE induced apoptosis in an additional 5 Ph+ B-ALL and, consequently, reduced engraftment in NSG mice, similar to knockdown of miR-126. Our results further consolidate an important role of miR-126 in human B-ALL, whereby a tight regulation of this miRNA appears critical for leukemia maintenance. Disclosures No relevant conflicts of interest to declare. more...
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- 2018
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7. Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing
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Stefano Beretta, Gabriele Casirati, Ivan Merelli, Erika Zonari, Bernhard Gentner, Giacomo Desantis, Andrea Cammarata, Fabio Ciceri, and Matteo Maria Naldini
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Severe combined immunodeficiency ,Genetic enhancement ,Immunology ,Cell ,RNA ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Cell biology ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Male-pattern baldness ,Stem cell ,Progenitor cell - Abstract
Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy. We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture. By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between. After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC. Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO). To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status. As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. Disclosures No relevant conflicts of interest to declare. more...
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- 2018
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8. Human CD34+ Cells from Different Sources Disclose a Specific Stemness Signature
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Maria Rosa Lidonnici, Marta Claudia Frittoli, Matilde Zambelli, Giuliana Ferrari, Giacomo Mandelli, Annamaria Aprile, Bernhard Gentner, Fabio Ciceri, Maria Domenica Cappellini, Ylenia Paleari, Sarah Marktel, Antonello E. Spinelli, Laura Zanaboni, Elena Cassinerio, Cristina Parisi, and Laura Bellio more...
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0301 basic medicine ,biology ,Plerixafor ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biochemistry ,CXCR4 ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,medicine ,biology.protein ,Stromal cell-derived factor 1 ,Bone marrow ,Progenitor cell ,Stem cell ,Homing (hematopoietic) ,medicine.drug - Abstract
Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis. In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment. These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients. Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications. Disclosures Ciceri: MolMed SpA: Consultancy. more...
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- 2016
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9. microRNA-126 Orchestrates a Stem Cell-like Program in AML and B-ALL and Can be Exploited to Dissect Disease Heterogeneity
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Massimo Bernardi, Luca Vago, Jose Manuel Garcia-Manteiga, Bernhard Gentner, Andrea Cosentino, Matteo Carrabba, Giacomo Desantis, Carolina Caserta, Silvia Nucera, Matteo Maria Naldini, Francesca Pavesi, and Fabio Ciceri more...
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Acute leukemia ,Immunology ,CD34 ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biology ,CD38 ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Cancer cell ,medicine ,Cancer research ,Progenitor cell ,Stem cell - Abstract
MicroRNA-126 reinforces hematopoietic stem cell (HSC) quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in human AML, it was required to maintain leukemic stem cell (LSC) quiescence (Lechman et al, Cancer Cell 2016), while its ectopic expression in mouse HSC induced leukemia (75% B-ALL, 25% AML) that fully regressed when switching a tetracycline-repressible miR-126 cassette off (Nucera et al, Cancer Cell 2016). RNA sequencing showed that miR-126 targeted cell cycle, apoptosis and p53 response genes, prevented differentiation and sustained oncogenic/pro-survival pathways typically associated with stem and progenitor cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We quantified miR-126 expression levels in a cohort of 45 newly diagnosed AML patients (n=38 de novo, of which 37% favorable, 34% int-1, 16% int-2, 13% adverse according to the ELN classification) presenting at the San Raffaele Hospital between 2010 and 2015 using a robust digital droplet PCR assay. In addition to core-binding factor (CBF) mutated AML, we found that the group of AML with chromosomal aneuploidy showed significantly elevated miR-126 levels, suggesting that this subgroup is characterized by high LSC frequencies and/or a specific need to suppress p53 responses, a hypothesis supported by the data obtained in our mouse model. To measure miR-126 at single cell resolution, we stably transduced primary AML blasts (n=5 diseases) with a lentiviral miR-126 reporter vector, xenotransplanted them into NSG mice and quantified miR-126 activity by FACS in the engrafted cells recovered from the mice's bone marrow. Across different genetic subgroups including CBF leukemia, cells with the highest miR-126 activity were enriched in the CD34+ or CD34+CD38- fractions, consistent with LSC. To facilitate prospective LSC isolation based on miRNA activity, we screened a series of combination miRNA reporters incorporating response elements for LSC-enriched and LSC-depleted miRNAs and have now identified an optimized construct that highlights easily sortable, distinct subpopulations that are currently undergoing functional validation. Applying this tool to AML with complex and monosomal karyotype, we are addressing whether high miR-126 expression refers to elevated LSC frequency or is a specific feature of this clinically relevant AML entity. We next applied the miR-126 reporter to n=15 primary, human B-ALL measuring miR-126 activity in the xenograft. Surprisingly, we identified well-separated blast subpopulations that differed in miR-126 activity within single diseases. Heterogeneity for miR-126 appeared to be a general feature of B-ALL as we detected 2-3 subpopulations in most of the diseases studied. We purified miR-126(high) and miR-126(low) B-ALL subpopulations from the primografts and verified up to 1log differences in miR-126 expression, while we detected equal levels of the BCR-ABL fusion transcript in all subpopulations from Philadelphia+ B-ALL confirming their neoplastic nature. When transplanting miR-126(high) and miR-126(low) subpopulations into secondary or tertiary recipients, post-sorting miR-126 levels were tightly maintained indicating that miR-126 levels were static rather than dynamically regulated in distinct B-ALL subpopulations, as expected from a subclonal architecture. RNAseq performed on miR-126(high) and miR-126(low) human B-ALL fractions evidenced a miR-126 signature reminiscent of the one obtained in the B-ALL mouse model and cord blood CD34+ cells, uncovering physiological miR-126 activity in human primary B-ALL. Taken together, these data support a broad, pathogenetically important role for miR-126 in human AML and B-ALL that goes beyond LSC and open up opportunities to better understand leukemia disease biology, dissect intratumoral heterogeneity and therapeutically target resistant and refractory disease, considering that miR-126 knockdown expands normal HSC while depleting the leukemia. Disclosures Ciceri: MolMed SpA: Consultancy. more...
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- 2016
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10. Incremental Innovation of Ex Vivo Hematopoietic Stem Cell Engineering to Expand Clinical Gene Therapy Applications
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Giuliana Ferrari, Carolina Petrillo, Maria Rosa Lidonnici, Sarah Marktel, Fabio Ciceri, Anna Kajaste-Rudnitski, Oriana Meo, Samantha Scaramuzza, Erika Zonari, Luigi Naldini, Bernhard Gentner, Giacomo Desantis, and Alessandro Aiuti more...
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Immunology ,CD34 ,Hematopoietic stem cell ,Cell Biology ,Hematology ,CD38 ,Biology ,Biochemistry ,Haematopoiesis ,medicine.anatomical_structure ,NSG mouse ,medicine ,Cancer research ,Stem cell ,Progenitor cell ,Ex vivo - Abstract
Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. Disclosures Ciceri: MolMed SpA: Consultancy. more...
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- 2016
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11. Intrinsic Molecular Features of Human Hematopoietic Stem Cells from Different Sources Define Their Specific Functional Properties
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Bernhard Gentner, Annamaria Aprile, Sarah Marktel, Laura Bellio, Ylenia Paleari, Marta Claudia Frittoli, Elena Cassinerio, Fabio Ciceri, Giacomo Mandelli, Laura Zanaboni, Maria Domenica Cappellini, Giuliana Ferrari, and Maria Rosa Lidonnici more...
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Plerixafor ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biology ,Biochemistry ,CXCR4 ,medicine.anatomical_structure ,medicine ,Cancer research ,Autologous transplantation ,Bone marrow ,Stem cell ,Progenitor cell ,Homing (hematopoietic) ,medicine.drug - Abstract
Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs. So far, the intrinsic molecular features of human primitive HSCs from different sources have not been investigated in comparative studies to unravel their variable reconstitution potential. Diverse strategies are currently used to disengage HSCs from the niche, promoting egress from BM to PB. Traditionally the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, many other compounds have been tested to this regard. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the PB. This drug, which was shown in preclinical combination studies with G-CSF to enhance mobilization compared to G-CSF alone, is currently approved by FDA and EMA "in combination with G-CSF to enhance the mobilization of HSCs into the peripheral blood for collection and autologous transplantation of patients affected by lymphoma or multiple myeloma whose cells mobilize poorly" We investigated functional and molecular hallmarks of human HSCs from different sources, i.e. BM and PB following mobilization by G-CSF and/or Plerixafor. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+ CD38/low CD90+ CD45RA- CD49f+ cells and primitive populations of HSCs. These cells are able to provide stable long-term hematopoietic engraftment in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The quiescence status of these cells correlates with the enriched scid-repopulating cell frequency. Noteworthy, the combined use of G-CSF and Plerixafor mobilizes a CD34+ population enriched in immature cells and with a lower engraftment capacity respect to cells mobilized by Plerixafor alone. Since the signaling provided by the interaction of SDF-1 with CXCR4, plays an essential role in maintaining HSC quiescence and regulating homing, we analyzed the CXCR4 expression. Interestingly, this analysis reveals that the proportion of CXCR4+ primitive cells was lower when using G-CSF combined to Plerixafor in respect to Plerixafor alone. These data indicate that the combination of the two mobilizing agents induce a higher amount of circulating CD34+ cells but containing a lower proportion of cells capable of homing to BM in NSG mice. . As a result, at a defined dose of transplanted CD34+ cells, less SRCs are observed when G-CSF is added to Plerixafor. Indeed, it is expected to observe also a rapid rescue of hematopoiesis in myeloablated subjects conferred by high amount of short-term progenitors. Insights into the transcriptional program reveal the molecular machinery underlying stemness features of cells derived from different sources, defining their specific functional properties. Noteworthy, CD34+ cells exposed to Plerixafor but still resident in the BM acquire an intermediate signature between steady-state and circulating cells, suggesting an effect of this agent on HSC function. From preliminary data, genes of Prostaglandin signaling are up-regulated in HSCs mobilized by Plerixafor, suggesting a role of this pathway. These data uncover unique HSCs properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need. Disclosures No relevant conflicts of interest to declare. more...
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- 2015
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12. Hoxa9/Meis1 Mediate Leukemic Programming through Microrna-155
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Hartmut Döhner, Arefeh Rouhi, Bernhard Gentner, Lars Palmqvist, Michael Heuser, Florian Kuchenbauer, Milijana Mirkovic-Hoesle, Christian Buske, R. Keith Humphries, Edith Schneider, Jens Ruschmann, Anna Staffas, and Konstanze Döhner more...
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Myeloid ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Leukemogenic ,Transplantation ,Leukemia ,medicine.anatomical_structure ,Downregulation and upregulation ,In vivo ,microRNA ,medicine ,Cancer research - Abstract
Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood. In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR. Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p Therefore, to dissect the leukemic potential of miR-155 to program mbm, 5-FU-stimulated mbm cells were retrovirally transduced with miR-155, leading to significantly increased proliferation in vitro (p The leukemic potency of Hoxa9/miR-155 was further investigated in a murine transplantation model in vivo. Transplantation of mbm co-overexpressing Hoxa9/miR-155 led to significantly increased engraftment levels already after four weeks (wks) (57.8%±31.3, n=16) compared to Hoxa9/ctrl (11.7%±19.3%, p Considering the central role of the Hoxa9/Meis1 in both myeloid and lymphoid acute leukemias, we demonstrate for the first time the leukemogenic relevance of a miRNA within this transcriptional axis. Disclosures No relevant conflicts of interest to declare. more...
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- 2014
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13. Off-Tumor Target Expression Levels Do Not Predict CAR-T Cell Killing: A Foundation For The Safety Of CD44v6-Targeted T Cells
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Luigi Naldini, Maurilio Ponzoni, Fabio Ciceri, Bernhard Gentner, Chiara Bonini, Claudio Bordignon, Gianpietro Dotti, Pietro Genovese, Margherita Norelli, Barbara Camisa, Aurore Saudemont, Benedetta Nicolis di Robilant, Laura Falcone, Attilio Bondanza, and Monica Casucci more...
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CD40 ,biology ,Immunology ,Cell Biology ,Hematology ,Natural killer T cell ,Biochemistry ,Interleukin 21 ,Antigen ,biology.protein ,Cancer research ,Cytotoxic T cell ,IL-2 receptor ,Antigen-presenting cell ,Interleukin 3 - Abstract
Introduction Off-tumor expression of the target antigen raises justified safety concerns about newly designed chimeric antigen receptors (CARs). We have recently developed a CAR targeting the tumor-promoting antigen CD44v6 and demonstrated potent antitumor effects against acute myeloid leukemia (AML) and multiple myeloma (MM) both in vitro and in vivo. Despite promising activity against epithelial tumors, the administration of the CD44v6-specific mAb used for deriving our CAR (bivatuzumab) showed reversible myelosuppression and mucositis when conjugated with radioisotopes, and severe skin toxicity when conjugated with the potent cytotoxic drug mertansine. Preclinically evaluating the potential off-tumor toxicities of CD44v6-targeted T cells is therefore crucial before they can be safely translated to the clinic. Aim To profile the off-tumor expression of CD44v6 and to verify the susceptibility of expressing cells to CAR-T cell killing. Results Quantitative RT-PCR analysis on a wide panel of cDNA from normal tissues revealed restricted CD44v6 expression on flat stratified epithelia, like the skin, albeit at considerably lower levels compared with primary leukemic blasts. We therefore addressed the issue of keratinocyte recognition in co-culture experiments. Strikingly, at the E:T ratios allowing the potent antitumor effects of CD44v6-targeted T cells, keratinocytes were not killed and there was no cytokine production. Interestingly, comparative analysis of accessory molecules showed that, differently from leukemic blasts, keratinocytes expressed significant lower levels of adhesion/costimulatory molecules, including (ICAM-1, LFA-3 and B7.2), but higher levels of the critical checkpoint molecule PD-L1. Of the different cells of the hematopoietic system analyzed, only circulating CD14+ monocytes expressed CD44v6 and were killed by CD44v6-targeted T cells. Interestingly, by immunohistochemistry, we found no CD44v6 expression on bone-marrow monocytes, lymph-node macrophages, brain microglia, liver Kuppfer cells and dermal macrophages, suggesting a low risk for by-stander toxicity against these tissues. Moreover, CD44v6-targeted T cells did not interfere with the generation of virus-specific CTLs by antigen-specific stimulation in vitro. Importantly, both RT-qPCR and FACS demonstrated lack of CD44v6 expression on hematopoietic stem cells (HSCs) and progenitors. Accordingly, CD44v6-targeted T cells did not interfere with their clonogenic potential in vitro and, in co-culture experiments with whole bone marrow from MM patients, were able to selectively eliminate tumor cells, while sparing HSCs and progenitors. Finally, we tested the potential hematological toxicities of CD44v6-targeted T cells in NSG mice transgenic for human IL-3, SCF and GM-CSF (NSG-3GS). NSG-3GS mice transplanted with human CD34-selected cord blood cells showed enhanced myeloid reconstitution compared to NSG mice, including CD44v6+ monocytes. The infusion of CD44v6-targeted T cells in reconstituted NSG-3G mice resulted in the selective elimination of monocytes, but in the preservation of other cell subsets. Importantly, after in vivo exhaustion of CD44v6-targeted T cells, NSG-3G mice reconstituted monocytes de novo, indicating preservation of the HSC pool. For enabling rapid and conditional ablation of CD44v6-targeted T cells, we have finally co-expressed the CD44v6-CAR with TK or the inducible caspase-9 and validated the suicide gene approach in hyperacute xenogeneic GVHD surrogating maximal toxicity. Conclusions Our results indicate that off-tumor target expression levels do not automatically predict the susceptibility to CAR T-cell killing. Moreover they suggest that, differently from mAb-derived pharmaceuticals, therapeutic doses of suicidal CD44v6-targeted T cells might associate with acceptable and/or reversible toxicities. Disclosures: Bordignon: MolMed SpA: Employment. more...
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- 2013
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14. A Mechanistic Role For Mir-126, a Hematopoietic Stem Cell Microrna, In Acute Leukemias
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Eric R. Lechman, Luigi Naldini, Bernhard Gentner, Francesco Boccalatte, Tiziana Plati, Alice Giustacchini, Anna Ranghetti, John E. Dick, Maurilio Ponzoni, Cristiana Fanciullo, Massimo Bernardi, Luca Vago, and Silvia Nucera more...
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Acute leukemia ,biology ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,CD38 ,medicine.disease ,Biochemistry ,CD19 ,medicine.anatomical_structure ,Acute lymphocytic leukemia ,medicine ,Cancer research ,biology.protein ,Stem cell ,Progenitor cell ,B cell - Abstract
We have recently shown that miR-126 expression faithfully identifies the engrafting fraction of bone marrow (BM) and cord blood (Gentner et al, Sci Transl Med 2010), in which it regulates hematopoietic stem cell (HSC) pool size by modulating cell cycle progression (Lechman et al., Cell Stem Cell 2012). Antagonizing miR-126 expands HSC by enhancing PI3K/AKT signaling, without causing their exhaustion or transformation. By reconstituting mice with lentivirally transduced BM cells ectopically expressing miR-126, we noted differential effects in HSC and progenitors: while miR-126 overexpressing (126OE) HSC were more quiescent and outcompeted by HSC with physiologic miR-126 expression, progenitor subsets were increased in number and proliferated more upon 126OE. In particular, B lymphopoiesis was enhanced, with the appearance of 126OE-vector marked, CD45(low)CD19+Ig(-) B cell progenitors in the peripheral blood. This abnormal population was transiently observed in all 126OE (n=45), but in none of the control mice (n=30), and BM B cell precursors and progenitors from 126OE mice showed significantly reduced levels of apoptosis, as well as a differentiation block at the immature B cell stage. At least part of this effect was due to interference of miR-126 with p53 activation. Consequentially, 20-30% of 126OE mice (as compared to 0% of control mice) followed over 1 year developed vector-marked, high-grade B-cell neoplasms which resembled diffuse large B-cell lymphoma, lymphoblastic lymphoma or acute lymphoblastic leukemia (ALL). Strikingly, by using a tetracycline-regulated, conditional miR-126OE vector, we observed full regression of the expanded abnormal B cell population after switching miR-126 expression off by doxycycline administration, demonstrating that miR-126 is an oncogenic driver in hematologic malignancies. To establish the relevance of miR-126 in human disease, we studied primary samples from patients with acute leukemia. We measured miR-126 expression in blasts from 12 adult patients with acute lymphoblastic leukemia (ALL). miR-126 was highly expressed in all studied cases of ALL (Phil+: n=7, Phil-: n=5), often surpassing the levels found in normal HSC. Unlike in HSC, miR-126 expression was independent from expression of its host gene EGFL7, suggesting an ALL-specific regulation of the miR-126 locus. By exploiting a lentiviral miR-126 reporter vector, we quantified miR-126 activity with single cell resolution after xenotransplantation of miR-126 reporter vector transduced ALL blasts into NSG mice. In 7 out of 12 ALL cases, we identified distinct subpopulations exhibiting different levels of miR-126 activity, and miR-126 (hi) ALL cells were more frequently contained in the CD34+ cell fraction. Moreover, high miR-126 expression correlated with faster and higher ALL engraftment in NSG mice. In acute myeloid leukemia (AML), miR-126 is significantly enriched in leukemic stem cells (LSC) where it governs quiescence and chemotherapy resistance (see abstract by Lechman et al), and analogous studies in ALL are ongoing. To further substantiate the role of miR-126 in AML, we studied miR-126 activity in 11 primary AML cases (4 AML with CBF, core binding factor, mutations; 7 AML with normal karyotype) using the reporter vector technology and the NSG xenotransplantation model. Globally, we found highest miR-126 activity in CBF-mutated AML. However, there was substantial heterogeneity in subpopulations of each single disease. Significantly higher miR-126 levels were found in CD34+, and particularly in CD34+CD38- AML cells, a fraction that is enriched for LSC. To see whether there is a correlation between miR-126 expression and disease progression, we measured miR-126 levels in sorted blasts from paired diagnosis/relapse (n=6) and diagnosis/chemotherapy-refractory (n=8) patient samples. Strikingly, we found a significant up-regulation of miR-126 in the disease progression sample, suggesting a mechanistic role for miR-126 in AML persistence after chemotherapy. In summary, these data support a clinically relevant role of miR-126 in the pathogenesis, treatment response and progession of acute leukemias. Authors AG and SN contributed equally, and LN and BG contributed equally. Disclosures: No relevant conflicts of interest to declare. more...
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- 2013
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15. CD44v6 Is Required For In Vivo Tumorigenesis Of Human AML and MM Cells: Role Of Microenvironmental Signals and Therapeutic Implications
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Laura Falcone, Claudio Bordignon, Monica Casucci, Bernhard Gentner, Attilio Bondanza, Luigi Naldini, Benedetta Nicolis di Robilant, Chiara Bonini, Barbara Camisa, and Fabio Ciceri
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biology ,Daunorubicin ,Immunology ,Mesenchymal stem cell ,CD44 ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease_cause ,Biochemistry ,medicine.anatomical_structure ,medicine ,biology.protein ,Cancer research ,Bone marrow ,Osteopontin ,Carcinogenesis ,medicine.drug ,Homing (hematopoietic) - Abstract
Background Targeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. Aim To preclinically validate CD44v6 as a therapeutic target in AML and MM Results By FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P Conclusions These results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures: No relevant conflicts of interest to declare. more...
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- 2013
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16. Mir-126 Governs Human Leukemia Stem Cell Quiescence and Chemotherapy Resistance
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Eric R. Lechman, Bernhard Gentner, Peter van Galen, Stanley Wai-Kwong Ng, Kolja Eppert, Rene Marke, Liran Slush, James A. Kennedy, Amanda Mitchell, Weihsu Claire Chen, Veronique Voisin, Gary Bader, Mark D. Minden, Jun Lu, Jean C.Y. Wang, Luigi Naldini, and John E. Dick more...
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Immunology ,Hematopoietic stem cell ,Myeloid leukemia ,Combination chemotherapy ,Cell Biology ,Hematology ,Cell cycle ,Biology ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,medicine.anatomical_structure ,microRNA ,medicine ,Cancer research ,Stem cell - Abstract
miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. more...
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- 2013
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17. The Challenge Of HSCs Procurement For Gene Therapy: Exploring Plerixafor As Mobilization Agent
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Maria Rosa Lidonnici, Annamaria Aprile, Marta Claudia Frittoli, Giacomo Mandelli, Bernhard Gentner, Laura Bellio, Elena Cassinerio, Laura Zanaboni, Maria Domenica Cappellini, Silvano Rossini, Fabio Ciceri, Sarah Marktel, and Giuliana Ferrari more...
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business.industry ,Plerixafor ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,CXCR4 ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,Medicine ,Bone marrow ,Stem cell ,Progenitor cell ,business ,medicine.drug ,Homing (hematopoietic) - Abstract
Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of autologous genetically engineered hematopoietic stem/progenitor cells (HSPCs) in myeloablated patients. Hematopoietic reconstitution and clinical benefit are related to cell dose, although single disease features might play a role favoring selection of relevant progenitor populations. Gene therapy trials in young pediatric patients are performed isolating CD34+ cells from bone marrow (BM), while in adults mobilized peripheral blood stem cells (PBSC) should represent the favorite target. In the context of gene therapy for thalassemia, the choice of HSPC source is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilization agent and prompt investigation of new agents. Moreover, adult thalassemic patients may possibly have a decreased BM stem cell reservoir, due to the BM suppression in response to multiple transfusions. A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected by transfusion dependent beta-thalassemia (EudraCT 2011-000973-30) started in 2012 at our hospital. Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood. The availability of a new source of HSPCs, potentially superior in terms of CD34+ cell yield, transduction efficiency and biological features to steady-state BM, would have a significant impact on the feasibility and efficacy of gene therapy. Four subjects were enrolled and treated by subcutaneously administration of Plerixafor at the single dose of 0.24 mg/kg followed by leukoapheresis. Mobilization of CD34+ cells occurred very rapidly with a peak between 7 to 9 hrs. Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse event occurred. To the aim of engineering Plerixafor-mobilized CD34+ cells for gene therapy, we performed a comprehensive characterization of their biological, molecular and functional properties. In vivo reconstitution potential and lympho-myeloid differentiation were tested following transplantation in NSG mice and compared to those of PBSCs mobilized by G-CSF. Percentages of engrafted human cells in NSG mice transplanted with Plerixafor -PBSCs were about 2- to 5-fold higher than those found in mice transplanted with G-CSF PBSCs. On the same line, the SRC frequency, obtained by pooled engraftment data, was significantly higher (1 SRC out of 47.875 CD34+ cells vs.1 SRC out of 141.203 CD34+ cells). The phenotypic analysis of the frequency of primitive hematopoietic sub-populations revealed that Plerixafor mobilizes preferentially HSPCs and LT-HSPCs, with a percentage of CD34+ CD38-/low CD90+ CD45RA- CD49f+ cells higher than that found in G-CSF PBSCs. This result mirrors the enhanced number of SRCs found in the CD34+ cell population mobilized by Plerixafor. In order to further define the molecular features of HSPCs from different sources, we are studying signalling networks in response to specific cytokines by phospho-proteins analysis and gene expression by microarrays analysis. Our studies are focused on self-renewal, homing, engraftment and multilineage differentiation processes and bioinformatic analysis will reveal the molecular machinery underlying 'stemness' properties of Plerixafor mobilized cells. Disclosures: No relevant conflicts of interest to declare. more...
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- 2013
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18. Hematopoietic Stem Cell Expansion, without Exhaustion or Transformation, by Stable Microrna Antagonism in Vivo
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John E. Dick, Luigi Naldini, Alice Giustacchini, Massimo Saini, Bernhard Gentner, Peter van Galen, Eric R. Lechman, and Francesco Boccalatte
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Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Lymphopoiesis ,Progenitor cell ,Stem cell ,Protein kinase B ,PI3K/AKT/mTOR pathway - Abstract
Abstract 30 The precise mechanisms controlling hematopoietic stem cell (HSC) quiescence, self renewal and differentiation remain incompletely understood, and HSC expansion remains a long sought after goal for cell- and gene therapy. Genetic manipulation of single transcription factors and signal transduction pathways has so far failed to provide sustained HSC expansion, often resulting in stem cell exhaustion or the emergence of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate multiple genes in a coordinated fashion, making them attractive targets to manipulate signaling networks and complex cellular functions. We have systematically manipulated miR-126, a miRNA enriched in the primitive hematopoietic compartment, in mouse and human HSC by stably knocking down its activity with a sponge lentiviral vector (126/KD LV) or forcing its overexpression by a 126/OE LV. Hematochimeric mice were generated by transplanting 126/OE LV- or 126/KD LV- transduced progenitors into congenic mice (murine HSC) or NSG mice (human HSC), and the effects of altered miR-126 levels on hematopoiesis were investigated in vivo. Steady state hematopoiesis was comparatively normal, with no alterations in complete blood cell counts except a minor skewing towards or against lymphopoiesis upon 126/OE or 126/KD, respectively. Strikingly, under stress conditions such as 5-fluorouracil exposure, secondary transplantation or the NSG xenograft environment, 126/OE resulted in a progressive loss of HSC, while 126/KD increased HSC numbers as measured by immunophenotype and functional output upon competitive transplantation into secondary recipients. Limiting dilution analysis confirmed a 3–4 fold expansion of mouse and human HSC upon miR-126/KD, and the competitive advantage of 126/KD HSC was maintained long-term including post tertiary transplantation. To study the mechanism by which miR-126 regulates HSC numbers, cell cycle analysis was performed on 126/KD- or 126/OE progenitors isolated from steady state bone marrow. Human CD34+CD38-CD90+ and murine Lineage-Kit+Sca+CD150+ HSC were shifted towards the S/G2/M or the G0 phase upon 126/KD or 126/OE, respectively, indicating that miR-126 antagonism increased cycling while excessive levels of miR-126 favoured HSC quiescence within the niche microenvironment. Interestingly, this effect was highly specific for HSC and the most primitive progenitors. An in vitro culture model of HSC-enriched cord blood cells was set up to study the molecular basis of miR-126 mediated control of HSC cycling. This model reproduced key aspects of the HSC phenotype observed in vivo, i.e. increased or decreased proliferation of primitive progenitors upon 126/KD or 126/OE, respectively. Transcriptome analysis indicated that miR-126 manipulation altered several cellular functions and pathways, most significantly the PI3K/AKT/GSK3β signalling axis. We confirmed PIK3R2 and CRKII as direct miR-126 targets in CB CD34+38- cells and show enhanced PI3K pathway activation as reflected by increased AKT and GSK3β phosphorylation upon 126/KD, while 126/OE caused opposite effects. Pharmacologic inhibition of the beta subunit of PI3K abrogated the 126/KD phenotype, indicating that miR-126 mainly acts through the PI3K pathway. In contrast to published studies showing HSC expansion upon hyperactivation of the PI3K pathway using various genetic approaches, miR-126/KD did not cause HSC exhaustion or leukemogenesis. On the contrary, evidence will be provided that 126/OE is oncogenic in hematopoietic cells. Thus, miR-126 represents a principle target for HSC expansion for cell and gene therapy applications. In summary, we propose that in vivo HSC pool size is influenced by endogenous levels of miR-126 through modulation of the HSC quiescence/proliferation equilibrium. Disclosures: No relevant conflicts of interest to declare. more...
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- 2012
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19. Co-Expression of a Suicide Gene in CAR-Redirected T Cells Enables the Safe Targeting of CD44v6 for Leukemia and Myeloma Eradication
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Fabio Ciceri, Pietro Genovese, Barbara Camisa, Monica Casucci, Gianpietro Dotti, Claudio Bordignon, Benedetta Nicolis di Robilant, Laura Falcone, Luigi Naldini, Attilio Bondanza, Barbara Savoldo, Bernhard Gentner, and Chiara Bonini more...
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biology ,medicine.medical_treatment ,Immunology ,CD44 ,Myeloid leukemia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Biochemistry ,Chimeric antigen receptor ,Leukemia ,medicine.anatomical_structure ,Antigen ,biology.protein ,medicine ,Bone marrow - Abstract
Abstract 949 Background: Acute myeloid leukemia (AML) and multiple myeloma (MM) share common features, such as preferential homing to the bone marrow (BM) and the tendency to relapse after chemotherapy. These phenomena are possibly linked, as signals provided within the BM niche induce chemoresistance. Relapsing AML and MM can however be cured by allogeneic hematopoietic stem cell transplantation (HSCT), suggesting that allospecific T may specifically sterilize tumor cells that survive chemotherapy. Unfortunaley, the universal application of HSCT is severely limited by the occurrence of life-threatening GVHD and by the fact that leukemia often escapes the alloimmune pressure by losing specific HLAs, as we have recently demonstrated (Vago et al., NEJM 2009). The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and MM, and is crucially involved in BM homing. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Thanks to the chimeric antigen receptor (CAR) technology it is now possible to efficiently redirect T cells against virtually any antigen for which there is availability of a specific mAb. Our experience with suicide gene therapy in the context of HSCT (Ciceri et al., Lancet Oncol 2009) clearly indicates that genetic switches are efficient tools for controlling the off-tumor toxicities of T cells. Aim: To develop a strategy for the safe targeting of CD44v6 through the co-expression of a suicide gene in CAR-modified T cells for AML and MM treatment Results: CD44v6 surface expression was observed in 11/14 (79%) AML cases. Co-culturing primary leukemic cells with BM-derived mesenchymal stromal cells (MSCs) ex vivo caused the significant up-regulation of CD44v6 expression (P Conclusions: CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM and can be ablated at will. We are currently designing a phase I/II clinical trial where CD44v6 CAR-redirected suicidal T cells will be applied soon after autologous HSCT for sterilizing residual tumor, and routinely ablated thereafter not to interfere will full hematological reconstitution. The co-expression of a suicide gene will also allow to control any potential toxicity deriving from background expression of CD44v6 on healthy tissues. Disclosures: Bordignon: Molmed SpA: Employment. more...
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- 2012
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20. Plerixafor Single Agent for Autologous Stem Cells Mobilization and Collection in Adult Thalassemic Patients: Towards the Assessment of the Suitable Hematopoietic Stem Cell Source for Gene Therapy of Beta-Thalassemia
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Matilde Zambelli, Salvatore Gattillo, Maria Rosa Lidonnici, Laura Zanaboni, Bernhard Gentner, Elena Cassinerio, Raffaella Milani, Marta Claudia Frittoli, Fabio Ciceri, Giuliana Ferrari, Silvano Rossini, Annamaria Aprile, Sarah Marktel, Maria Domenica Cappellini, and Laura Bellio more...
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business.industry ,Plerixafor ,Immunology ,CD34 ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Pharmacology ,Biochemistry ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Autologous transplantation ,Progenitor cell ,Stem cell ,business ,medicine.drug - Abstract
Abstract 586 Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34+ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent. In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34+ cells at a level comparable to that obtained using steady state BM. Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34+ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34+ cells/kg: 1.84 × 106 (01) and 4.43 × 106 (02) with a unique leukoapheretic procedure, and 3.57 × 106 (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34+ cells/mcl in peripheral blood was not reached. CD34+ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 106 CD34+ cells/Kg, 65% recovery (01), 2.66 × 106 CD34+ cells/Kg, 60% recovery (02), 1.78 × 106 CD34+ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 103/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34+ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM. Purified CD34+ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34+ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy. Disclosures: No relevant conflicts of interest to declare. more...
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- 2012
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21. Intensive Treatment in Elderly AML Patients Induce Prolonged Survival and Disease Remission If Early Mortality Is Prevented: The Case Record of the San Raffaele Scientific Institute
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Massimo Bernardi, Alessandro Vignati, Luca Vago, Matteo Carrabba, Alessandro Crotta, Bernhard Gentner, Consuelo Corti, Carlo Messina, Elisa Sala, Raffaella Greco, Andrea Assanelli, Jacopo Peccatori, Simona Piemontese, Elena Guggiari, Michela Tassara, and Fabio Ciceri more...
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medicine.medical_specialty ,Chemotherapy ,business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Biochemistry ,Chemotherapy regimen ,Surgery ,Fludarabine ,Transplantation ,Internal medicine ,medicine ,Cytarabine ,Idarubicin ,Autologous transplantation ,business ,medicine.drug - Abstract
Abstract 4583 Background: the outcome of pts older than 65 with AML is dismal because of the unfavourable characteristics of the disease and the frequent co-morbidities. Intensive chemotherapeutic programs, with or without haematopoietic stem cells transplantation (HSCT), are not usually offered to these pts since they are considered too toxic. If treated with intensive therapeutic programs these pts show initial complete remission (CR) rates around 45% to 55% and 2 years survival of 10–20%. Since 2001 in our institution we have systematically screened AML elderly patients older than 65 yrs for comorbidites and offered to “fit” AML elderly pts an intensive approach aimed at curing the disease. In this single-center retrospective study we describe the outcome of this court of pts. Aim: to evaluate toxicity and survival after intensive treatments in an homogeneous court of AML pts older than 65. Methods: from 10/2001 to 3/2011 we treated 60 pts older than 65, median age 70 (66–77), 30 pts ≤70 yrs (median 68, 66–70), and 30 pts >70 (median 73, 71–77). Among AML pts older than 65 years old we considered eligible for intensive chemotherapy those with PS (ECOG) ≤ 2, renal, hepatic and cardiac function parameters within normal ranges. All pts signed informed consent for intensive chemotherapy. Diagnosis (WHO): 33 AML with MDS related changes, 4 AML with minimal differentiation, 6 AML w/o maturation, 8 AML with maturation, 5 AMML, 1 erythrolaeukemia, 1 AML with mutated NPM1, 2 t-AML. Cytogenetics: 43 intermediate risk (33 with normal karytotype), 10 high risk (7 complex, 3 with Monosomal Karyotype), 2 good risk, 5 not evaluable. Induction with one or two of the following chemo cycles was administered to each patient: FLAG-IDA (fludarabine + cytarabine + idarubicin) or 3–7 (cytarabine + daunorubicin, idarubicin or mitoxantrone), high-dose cytarabine (HiDAC). Post-remission treatment: chemotherapy (CHT) 1 patient, HiDAC 12 pts, autologous transplantation (AUTO) 14 pts, allogeneic transplantation (ALLO) 2 pts. Results: overall induction related mortality was 18.3%, 7% in pts ≤70 yrs, 30% in 30 pts >70 p=0.041, CR rate was 57% (34 pts), 73% in pts ≤70 yrs, 40% in pts >70 p=0.018. Relapse rate was 73.5% (25 pts), 68% pts ≤70 yrs, 83% in pts >70. Median OS was 386 days, 2 and 5 yrs OS was 38 and 19%, respectively, median DFS was 397 days and 2 yrs DFS was 22%. In pts ≤70 and >70 yrs, median OS and DFS were 574 and 313, 397 and 425 days, respectively, p=ns, 2 and 5 yrs OS was 41% and 27%, 30% and 8%, 2 yrs DFS 27% and 17%, respectively, p=ns. OS of pts who obtained the CR after induction: median 1155 and 790 days for pts ≤70 and >70 yrs, respectively, p=ns, 2 yrs OS 51% and 58%, respectively, p=ns. Conclusions: our data suggest that prolonged survival can be achieved in elderly AML who are fit to receive intensive chemotherapy. Data on mortality and remission induction are very promising in pts 66–70 yrs old, whereas early death is an important cause of treatment failure in older pts (>70), together with disease relapse. However, also older pts, with a median age of 73, who obtain the CR can reach a survival plateau at 2 yrs. Overall, these results i) indicate that selection of elderly AML pts for treatment assignment should be refined by the development of new tools for oncologic-geriatric assessment rather than by PS and vital organ function evaluation ii) prompt to design of prospective trials addressing the role of intensive regimens and transplantation programs in “fit” elderly AML pts. Disclosures: No relevant conflicts of interest to declare. more...
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- 2011
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22. Dual Transgenesis of T Cells with a Novel CD44v6-Specific Chimeric Antigen Receptor and a Suicide Gene for Safe and Effective Targeting of Chemoresistance in Hematopoietic Tumors
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Gianpietro Dotti, Barbara Camisa, Claudio Bordignon, Bernhard Gentner, Chiara Bonini, Attilio Bondanza, Benedetta Nicolis di Robilant, Monica Casucci, Pietro Genovese, Fabio Ciceri, Luigi Naldini, Laura Falcone, and Barbara Savoldo more...
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biology ,medicine.medical_treatment ,Immunology ,CD44 ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Biochemistry ,Chimeric antigen receptor ,Leukemia ,Haematopoiesis ,medicine.anatomical_structure ,Antigen ,biology.protein ,medicine ,Bone marrow - Abstract
Abstract 3125 Background: We have previously demonstrated that the genetic induction of a conditional suicidal phenotype in donor T cells allows for an operational dissociation of the GVL effect from GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, leukemia often escapes the immunological pressure of alloreactive donor T cells by losing “passenger” mismatched HLAs. Conversely, redirecting T cells against a non HLA-restricted antigen critically involved in the neoplastic phenotype may circumvent tumor escape due to the emergence of antigen-loss variants. The isoform variant 6 of CD44 is expressed by different epithelial and hematological cancers, and is possibly involved in tumor-cell survival and proliferation. Clinical experience with chemo-conjugated CD44v6-specific mAbs in epithelial tumors showed substantial efficacy, which was however limited by skin toxicity due to background expression of CD44v6 on keratinocytes Aim: By analogy with our experience in HSCT, we reasoned that CD44v6 targeting with suicide gene-modified T cells would provide a major therapeutic effect against hematological tumors, while granting a safety switch in case of toxicity. To this aim, we designed a novel CD44v6-specific chimeric antigen receptor (CAR) and developed a strategy for its co-expression with a suicide gene Results: CD44v6 expression by FACS was observed at high levels in 6/17 (37%) cases of acute myeloid leukemia (AML), but did not associate with enhanced leukemia initiation after infusion into NSG mice (83% vs 88%). In all cases, however, AML cells isolated from the bone marrow (BM) of engrafting mice were brightly positive for CD44v6, suggesting in vivo regulation by microenvironmental factors. In vitro, co-culturing primary AML cells with human BM-derived mesenchymal stromal cells (MSCs) caused a selective up-regulation of CD44v6 (P90% elimination: 18 hrs vs 112 hrs average, P Conclusions: We demonstrated that LV-mediated dual transgenesis of primary human T cells with a novel CD44v6-specific CAR and a suicide gene is feasible, results into a powerful antitumor effect against chemoresistant AML and MM cells, and enables effective T-cell ablation in case of toxicity. The premise that suicide gene-modified CAR-redirected T cells can widen the therapeutic index of CD44v6 targeting awaits clinical confirmation Disclosures: Bordignon: Molmed SpA: Employment. Bonini:MolMed: Consultancy. more...
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- 2011
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23. Identification and Function of Hematopoietic Stem and Progenitor Cell Specific Micrornas
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Massimo Saini, Bernhard Gentner, Alice Giustacchini, Eric R. Lechman, Francesco Boccalatte, Luigi Naldini, Hidefumi Hiramatsu, Giulia Schira, and John E. Dick
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Myeloid ,Cell growth ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Cell biology ,Haematopoiesis ,medicine.anatomical_structure ,Cord blood ,medicine ,Bone marrow ,Progenitor cell ,Stem cell ,Clonogenic assay - Abstract
Abstract 2631 Little is known about microRNA function in hematopoietic stem and progenitor cells (HSPC). Using a lentivector genetic reporter strategy to functionally detect miRNA activity in hematopoietic cells at single cell resolution, we identified several miRNAs which were specifically expressed in mouse and human HSC and early progenitors, defined according to cell surface phenotype and functional repopulation assays. One of these HSPC-specific miRNAs, miR-126, was further studied. We generated a stable miR-126 knockdown (kd) or forced its expression (“knock-in”, ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for >1 year in both primary and secondary recipients. miR-126 kd HSPC displayed enhanced myeloid and/or lymphoid contribution during the early phases of reconstitution, while they subsequently contributed similarly as the control cells. When this steady state bone marrow (BM) was transplanted into secondary recipients, we noted an even more pronounced over-contribution of miR-126 kd cells to hematopoiesis. In the long run, however, some secondary mice showed signs of exhaustion of miR-126 kd cells. These data suggest that miR-126 kd enhances hematopoiesis, likely at the stem/early progenitor level and in particular under stress conditions. On the other hand, forced expression of miR-126 (ki) resulted in an early competitive disadvantage in vivo, with progressively decreasing contribution to all hematopoietic lineages, paralleled by a nearly complete depletion of Kit+Sca+Lin- (KSL) miR-126 ki cells in the BM at 6 weeks after transplant. At 3 weeks post-transplant, when miR-126 ki KSL cells could still be detected, we found an increased proliferative index in these cells as judged by EdU incorporation in vivo, paralleled by a higher hematopoietic output respect to control cells at week 2–4 after transplant. These data suggest that miR-126 ki might favor HSC commitment at the cost of self-renewal. This phenotype was specific for miR-126 and not due to vector toxicity, as we demonstrate stable, long term overexpression of several control miRNAs in vivo. Moreover, miR-126 ki cells showed normal clonogenic activity in vitro. We then optimized a protocol to stably knock down miR-126 in human cord blood (huCB) HSPC, and validated this approach by demonstrating upregulation of previously described miR-126 targets including the beta subunit of phosphoinositide-3-kinase. Manipulation of miR-126 activity changed cell growth and differentiation of huCB, and we show altered activation of key signal transduction pathways upon miR-126 kd. Identification of additional miR-126 targets is ongoing using unbiased proteomic and transcriptomic approaches. In summary, these data suggest that a narrow range of miR-126 activity is required for robust and sustained HSC function, and that its manipulation may provide novel insights into stem cell biology. Disclosures: No relevant conflicts of interest to declare. more...
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- 2010
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24. Enriched MicroRNA-126 Bioactivity Marks the Primitive Compartment In AML and Regulates LSC Numbers
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Bernhard Gentner, Mark D. Minden, John E. Dick, Eric R. Lechman, Hidefumi Hiramatsu, Kristin J Hope, Kolja Eppert, Luigi Naldini, and Katsuto Takenaka
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education.field_of_study ,Immunology ,Population ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,CD38 ,Biology ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,hemic and lymphatic diseases ,Cancer research ,medicine ,Progenitor cell ,Stem cell ,education - Abstract
Abstract 94 Previous work has shown miRNAs to be dysregulated in acute myeloid leukemia (AML), however, there is little known regarding miRNA expression and function in leukemia stem cells (LSC). In order to elucidate the role of miRNA in LSC, we performed miRNA profiling on fractionated subpopulations of primary AML patient samples. Supervised analysis guided by the in vivo SCID leukemia initiating capacity (SL-IC) of each sub-population generated a unique miRNA signature associated with LSC enriched fractions. An in vitro antagomir-based functional miRNA knockdown screen identified miR-126, our top array candidate, for further study. After RT-PCR validation, the biological activity of miR-126 was confirmed at single cell resolution by using a novel bidirectional lentivirus miRNA reporter system in the 8227 cell line in vitro and within primary AML patient samples xenografted into immune-deficient NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast populations. To test the hypothesis that AML stem cells are marked by high miR-126 bioactivity, we FACS sorted miR-126 genetic reporter vector transduced primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these proof-of-concept experiments demonstrates our ability to prospectively isolate LSC enriched fractions in all 4 AML patient samples tested using only a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive AML cells, stable enforced expression and knockdown of miR-126 was achieved using lentiviral vectors. Enforced expression of miR-126 in CD34+CD38- 8227 cells resulted in reduced AML blast colony formation, an increase/maintenance of CD34+ cells and a decrease in differentiation marker positive (CD14, CD15) AML blasts. Similarly, enforced miR-126 expression in 4 primary AML xenografts resulted in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells after 12 weeks. In addition, the miR-126 cells showed reduced differentiation marker expression (CD14, CD15) with no significant differences in AML graft size. To determine if the expanded population had SL-IC activity or was a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary recipient mice for 12 weeks. A 13 fold increase in LSC activity was observed with miR-126 forced expression compared to control vector expressing cells. These data suggest that high levels of miR-126 bioactivity may support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Conversely, in vitro knockdown of miR-126 in CD34+CD38- 8227 cells increased AML blast colony formation, while no phenotype was observed in xenotransplanted primary AML, with secondary LDA transplant experiments ongoing. Target prediction algorithms and previously described target genes were used to ascertain the principal signaling pathway(s) under direct control of miR-126 in primitive AML cells. In summary, these experiments demonstrate that miR-126 is more abundantly expressed and biologically active within the leukemia stem/progenitor cell compartment of the AML functional hierarchy and serves to regulate AML stem cell numbers. Disclosures: No relevant conflicts of interest to declare. more...
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- 2010
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25. Bone-to-Bone Stem Cell Infusion In Graft Failure After Haploidentical Hematopoietic Stem Cell Transplantation: Safety and Feasibility. Three Case Report
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Bernhard Gentner, Fabio Ciceri, Carlo Messina, Fabio Giglio, Jacopo Peccatori, Matilde Zambelli, Vincenzo Matozzo, Simone Claudiani, Simona Malato, Sara Mastaglio, Consuelo Corti, Massimo Bernardi, Andrea Assanelli, and Daniela Clerici more...
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Oncology ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Immunology ,Immunosuppression ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Pancytopenia ,Surgery ,Granulocyte colony-stimulating factor ,Transplantation ,Leukemia ,medicine.anatomical_structure ,Graft-versus-host disease ,Internal medicine ,medicine ,Bone marrow ,business - Abstract
Abstract 4438 BACKGROUND: Graft Failure (GF) occurs in 5–27% of patients (pts) after allogeneic hamatopoietic stem cell transplant (HSCT) and is associated with high morbidity and mortality related to infections and hemorrhagic events. Graft function may be poor as result of graft rejection, primary disease relapse or Poor Graft Function (PGF). The incomplete recovery of blood counts is defined primary PGF and the decreasing blood counts after successful engraftment secondary PGF. Several factors may determine GF: disease risk and status, conditioning regimen, HSC source, HLA compatibility, T cell content, immunosuppression, GvHD, viral infections, drugs. GCSF and Rhu-EPO are readily available and effective in PGF but with no effects on platelets. Second transplantation from the same donor, with or without conditioning therapy, can boost the haematopoietic recovery in pts with GF. Unfortunately, both a second peripheral CD34+ mononuclear cells (MNC) mobilization and a marrow harvest in the operating room may be contraindicated early after the first donation as not safe for donors. Intrabone SCT can overcome the risk of graft failure even with a low number of CD34+ MNC, as it has been demonstrated in cord blood transplant. Here we investigate in three adult pts with GF a bone-to-bone boost (BBB) with a small marrow harvest from respective donors, unfit for a second conventional donation. AIM: to evaluate the feasibility of the BBB technique in 3 pts with graft failure. METHODS: pts were 2 males (57, 53 y) with PGF with a diagnosis of AML and CMML, respectively, and a female (44 y) with graft rejection and AML relapse. In the first two patients prolonged pancytopenia and hypoplastic marrow were documented, with diagnosis of primary PGF and secondary PGF, respectively, donor chimerism ranging from 80–100% (STR and HLA), without evidence of leukemia. In the third patient, after prolonged pancytopenia an AML relapse was documented with 89% blasts on bone marrow aspirate. In PGF patients no conditioning regimen was administered before the boost at day 30 and 72 after SCT, respectively. In the patient with AML relapse Melphalan 200 mg/mq was given 48 h before the infusion, at day 35 after SCT. The 3 donors were related, haploidentical. For the BBB procedure small quantities of bone marrow (< 200ml) were collected from the posterior iliac crest bilaterally of the donors, at the bedside, during deep sedation and analgesya. Shortly after the unmanipulated marrow harvested was infused in superior-posterior iliac crest mono- or bilaterally, depending on the volume, during deep sedation and analgesya. In pt 1 Mononucleated cell (MNC) dose was 0.9 × 10^8/Kg for a volume of 166 ml. In pt 2 MNC dose was 0.4 × 10^8/Kg for a volume of 88 ml. In pt 3 MNC dose was 0.3 × 10^8/Kg for a volume of 140 ml. RESULTS AND CONCLUSION: In this cases the BBB technique proved feasible and safe for both the donor and the patient. Patient 1 received a second PBSC boost, without conditioning, 3 months after the BBB, and he's now alive, in CR, 13 months after the first transplant. Patient 2 died 3 months after the first transplant for pneumonia and sepsis. Patient 3 is alive, in CR, 4 months after the first HSCT. This practice can give the chance of HSC boost to patients with GF without the need of a GCSF mobilization for donors, with a minimal invasive operation. This can give the option to overcome and resolve infectious and hemorrhagic complications, bridging patients to further therapies and procedures. The intra-bone SCT may be a facilitating tool for microenvironment reconstitution, seeding and subsequent differentiation and may as well have a tolerogenic effect, through the mesenchymal stromal cells infused with the harvest. Further studies are necessary to assess the efficacy of this procedure. Disclosures: No relevant conflicts of interest to declare. more...
- Published
- 2010
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26. High Levels of MicroRNA-126 Bioactivity Specify the LSC Compartment in AML
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Bernhard Gentner, Kristin J Hope, Luigi Naldini, Eric R. Lechman, Mark D. Minden, Hidefumi Hiramatsu, Katsuto Takenaka, Kolja Eppert, and John E. Dick
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Regulation of gene expression ,education.field_of_study ,Gene knockdown ,Immunology ,Cell ,Population ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Cell biology ,Leukemia ,medicine.anatomical_structure ,microRNA ,medicine ,Stem cell ,education - Abstract
Leukemia stem cells (LSCs) are a biologically distinct blast population positioned at the apex of the acute myeloid leukemia (AML) developmental hierarchy. A more complete understanding of the unique properties of LSCs is crucial for the identification of novel AML regulatory pathways and the subsequent development of innovative therapies that effectively target these cells in leukemia patients. However, most studies overlook the heterogeneity of AML and the existence of LSC, potentially masking important molecular pathways. MicroRNAs (miRNAs) are an emerging class of non-coding small RNAs that negatively regulate the expression of protein-encoding genes. Normal miRNA expression is tissue and developmental stage restricted, suggesting important roles in tissue specification and/or cell lineage determination. Several studies have already demonstrated that miRNA expression levels are dysregulated in AML. However, little is known of the contribution of miRNAs to the regulation of gene expression and maintenance of LSCs. In order to elucidate the role of miRNAs in the regulation and maintenance of the leukemic stem cell state and the leukemogenic process, primary AML patient samples were fractionated into sub-populations based on the expression of CD34 and CD38 cell surface expression and purified RNA was extracted for miRNA array profiling. Supervised analysis guided by the in vivo SCID leukemia initiating capacity of each sub-population identified a unique 2 miRNA signature associated with leukemia stem cell enriched fractions. Validation of top miRNA candidates was confirmed by standard RT-PCR. However, to confirm that our miRNA candidates were biologically active within the AML developmental hierarchy at a single cell level, we utilized bidirectional miRNA sensor lentivectors (Bd.LV). Bd.LVs co-express truncated NGFR and enhanced GFP (eGFP), and permit post-transcriptional regulation of eGFP when miRNA target sequences (miRT) are placed downstream. One week post-transduction of a unique primary AML patient sample (8227), which can maintain a phenotypic and functional hierarchy for over 200 days in serum-free in vitro culture conditions, multicolor flow cytometry analysis revealed that miR-126 strongly suppressed (17 fold eGFP repression vs. control vector) eGFP expression in CD34+CD38-cells, the sub-fraction of AML that is highly enriched in LSC, while miR-126 activity declined along a gradient of further “differentiation” of the cells. Knockdown of miR-126 activity in 8227 cells with antagomirs designed to target miR-126 resulted in an increase in eGFP expression within the CD34+CD38-population, confirming the specificity of the biosensor lentivector. Furthermore, this expression gradient of miR-126 was conserved in 2 primary patient AML samples after 10 weeks post-transplant into pre-conditioned NOD/SCID mice. These experiments demonstrate that mir-126, a miRNA candidate identified by miRNA array, is more abundantly expressed and biologically active within the leukemia stem cell compartment of the AML hierarchy. Moreover, these data demonstrate the utility of biosensor vector technology in discerning miRNA expression patterns at the single cell level and will be useful for the further refinement of LSC purification. more...
- Published
- 2008
- Full Text
- View/download PDF
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