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  • 1
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3343-3343
    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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
  • 3
    In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 8588-8589
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 916-916
    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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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    detail.hit.zdb_id: 80069-7
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  • 5
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 4600-4600
    Abstract: Introduction Allogeneic stem cell transplantation (allo-HCT) survivors are at a defined relevant risk of developing long-term complications: the prevalence of chronic health conditions approaches 75% among HCT survivors. The endocrine system is one of the most frequent targets of complications, providing justification for a long-term and continuous follow-up (LTFU) to assure a timely and appropriate treatment. The aim of our study is to evaluate the incidence of endocrinopathies in survivors in respect of sex, age, donor type, conditioning regimen and GvHD occurrence. Methods A standardized LTFU is applied at our center. We here analyze data consecutively collected in an Institutional database, starting from 2006, including 402 adult patients (pts) who underwent an allo-HCT between 1992 and 2016 at our Institution. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. We considered for the analysis pts with an overall survival 〉 /=1y. We reviewed pts chapters with a focus on occurrence, management and treatment of diabetes, thyroid disfunctions, dyslipidemia and osteopenia / osteoporosis: diagnosis and follow-up were performed according to guidelines for long-term HCT survivors. Results With a median follow-up of 7y (r 2-25y) 328 pts were evaluable; donor was a match unrelated donor in 107 cases, HLA identical sibling in 88, haploidentical relative in 129 and cord blood in 4. The 5y-incidence of diabetes type 2 was 3%, with a median time after allo-HCT of 1138 days (r 5-4181 days); 13/22 developed diabetes after diagnosis of GvHD (median time 884 days, r 30-3753 days). All pts received indication for diet modification, 11 pts were treated with insulin and 8 pts with metformin. Thyroid disfunction was documented in 38 pts (5y-incidence 8,5%): 2 pts were diagnosed with hyperthyroidism and treated with methimazole or radioiodine treatment. Hypothyroidism was documented in 36 pts (median time after allo-HSCT 799 days, r 65-5021 days). Thirteen pts developed hypothyroidism following the diagnosis of GvHD (median time 1236 days, r 166-3540 days). Only 2 pts did not receive a specific treatment, while all the others received substitutive therapy with levothyroxine. Furthermore 1 pt was diagnosed with a papillary thyroid cancer. The 5y-incidende of dyslipidemia was 30% with a median time after allo-HSCT of 1433 days (r 366-7629), 47 pts developed dyslipidemia after the diagnosis of GVHD (median time 1425 days, r 134-7403 days). Diet-therapy was recommended to all the pts, 29 pts received a statin-based pharmacological treatment, 20 pts a polyenoic-fatty-acids based treatment, while a nutraceutical compound was given in 13 pts. Osteopenia was documented in 120 pts (median time after allo-HSCT 994 days, range 31-6605 days) with a 5y-incidence of 36%. Seventy-nine pts presented osteopenia after diagnosis of GvHD (median time after GvHD diagnosis 707 days, r 13-6379 days). Eight pts did not receive a specific treatment. Two pts received treatment with biphosphonates plus oral vitamin D and calcium supplementation, the 110 remaining pts received oral vitamin D +/- calcium supplementation only. Sixty-four pts developed osteoporosis (median time after allo-HSCT 1000 days, r 60-8836 days), the 5y-incidence was 26%. Forty-four pts developed osteoporosis following the diagnosis of GvHD (median time 724 days, r 28-8280 days). Only 4 pts did not receive any specific therapy; 31 pts received therapy with bisphosphonates, 2 pts denosumab and 27 pts oral vitamin D and calcium supplementation. In univariate analysis no relationship between host sex, age at transplant, TBI exposure, donor or history of GvHD and development of diabetes, thyroid disfunction and dyslipidemia was outlined. Otherwise, osteopenia development was strongly associated with GvHD occurrence and osteoporosis was strongly associated with age, sex and GvHD occurrence (table 1). Conclusions Allo-HCT survivors are at relevant risk of endocrinopathies after transplantation, providing justification for specific monitoring to individualize treatment and follow-up. Of note, classical transplant-related variables are not enough to justify the occurrence of endocrinological disfunction: a further deeper evaluation of a misdiagnosed donor-mediated autoimmune predisposition will be essential. Disclosures Bonini: Intellia Therapeutics: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    In: Clinical Nutrition ESPEN, Elsevier BV, Vol. 57 ( 2023-10), p. 782-
    Type of Medium: Online Resource
    ISSN: 2405-4577
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
    detail.hit.zdb_id: 2816659-0
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  • 7
    In: Nature, Springer Science and Business Media LLC, Vol. 621, No. 7978 ( 2023-09-14), p. 404-414
    Abstract: Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia 1,2 , the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens 3–5 . Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34 + HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.
    Type of Medium: Online Resource
    ISSN: 0028-0836 , 1476-4687
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    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 120714-3
    detail.hit.zdb_id: 1413423-8
    SSG: 11
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  • 8
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 14, No. 1 ( 2023-03-08)
    Abstract: Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 ( NPM1 ) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2553671-0
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  • 9
    In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 303-304
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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