scholarly journals Acute Myeloid Leukemia (AML) in a 3D Bone Marrow Niche Showed High Performance for in Vitro and In Vivo Drug Screenings

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 544-544
Author(s):  
Giulia Borella ◽  
Ambra Da Ros ◽  
Elena Porcù ◽  
Claudia Tregnago ◽  
Maddalena Benetton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Board Of Directors ◽  
Advisory Committees ◽  
In Vivo Models ◽  
3D Cultures ◽  
Nsg Mice

Chemotherapy still remains the pillar of treatment of children with AML, a disease in which refinements in diagnostic approaches, minimal residual disease monitoring, and patient stratification have resulted into remarkable progresses during the past decade. However, most of the recently tested, novel anti-leukemia agents failed during pre-clinical and clinical validation phases, and one main limit in AML field is the inappropriateness of current preclinical models used to study drug efficacy, this jeopardizing the advance of phase II and III clinical trials, especially for children. In light of this consideration, we aimed at creating novel robust in vitro and in vivo approaches to discover or to re-assess alternative treatments to improve the portfolio of agents active in childhood AML. For this purpose, we developed new protocols for long-term 3D-AML cultures to perform more predictable high throughput drug screening in vitro, and, once identified the best compounds, to create new pre-clinical in vivo models. We set up the bone marrow (BM) endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, where we seeded mesenchymal stromal cells derived either from AML patients (AML-MSCs) or from healthy BM donors (h-MSCs), together with osteoblasts, endothelial cells and finally AML blasts. We studied AML cell proliferation and clonogenicity cultured in 3D. We obtained results from twenty 3D long-term cultures of different primary AML, confirming blast proliferation up to 21 days. Clonogenic potential and immunophenotype preservation of the original AML blasts was also documented. At the same time, we compared AML-MSCs with h-MSCs, finding that AML-MSCs exhibited a higher proliferation rate (40% increase proliferation at 72 and 96 hours, p<0.001), and commitment to osteogenic differentiation, this latter occurring after 7 days with respect to 21 days of h-MSCs (p<0.01). To better characterize AML-MSCs features supporting AML cell growth, we cultured AML-MSCs and h-MSCs in an inflammatory environment (IL1β, IL6 and TNFα), observing that AML-MSCs did not exert anti-inflammatory activity by HUVEC tube formation assay (n=6, p<0.001). This latter finding was supported by a peculiar secretome profile defined by mass-spectrometry revealing factors as SERPINE1, CHI3L1, TIMP1, and PTX3 being differentially secreted. Thus, a drug targeting of AML-MSCs would be desirable, and we performed a screening of 480 compounds. This screening identified 17/480 active compounds capable of reducing AML-MSCs proliferation without toxicity over h-MSCs and AML blasts. We identified one main compound able to selectively reduce AML-MSCs proliferation, that, when combined to novel therapeutic agents for AML, such as the Quizartinib, I-BET inhibitor and Dasatinib in the 3D cultures, showed a synergistic effect (CI=0.5, p<0.05) towards FLT3-ITD, MLL-rearranged or c-KIT mutated AML. We then proceeded with two pilot studies in order to define the use of this 3D-AML cultures in in vivo setting. We implanted these organoids in the back of NSG mice and monitored leukemia engraftment in the scaffolds, as well as AML dissemination in peripheral blood. We documented the niche being vascularized and well organized by immunohistochemical staining for mCD31, hCD45 and hOPN, whereas we had a low rate of success in AML dissemination out of the niche (1 AML out of 14 implanted in 15 months). On the contrary, we observed that AML cells proliferated in the 3D up to 9 months after implantation, suggesting that this system is more suitable for an easy and quick in loco drug testing. Thus, we implanted AML, after being transduced with luciferase, in the 3D niche in NSG mice and monitored luciferase activity during intra-peritoneal drug treatments. We evaluated some new and old compounds known to target leukemia cells and documented a significant reduction of luciferase in the 3D when a drug was active, supporting our 3D scaffolds as a novel useful in vivo system to screen selected drugs before the prioritization of the best one to be used in a pre-clinical setting in PDXs. In conclusion, our data support the possibility to work with long-term 3D cultures of AML in vitro to identify new drugs, and we attribute to AML-MSCs a crucial supportive role to be further considered in in vivo settings for novel combo strategies. Finally, these findings could help test new compounds to be validated in future clinical trials. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy.

LNA Anti-MicroRNA-155: A Novel Therapeutic Strategy in Waldenstrom Macroglobulinemia and Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2728-2728
Author(s):  
Yong Zhang ◽  
Christopher P. Rombaoa ◽  
Aldo M Roccaro ◽  
Susanna Obad ◽  
Oliver Broom ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Mrna Levels ◽  
Advisory Committees ◽  
Qrt Pcr

Abstract Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 672-672
Author(s):  
Elizabeth R Macari ◽  
Alison Taylor ◽  
David Raiser ◽  
Kavitha Siva ◽  
Katherine McGrath ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Board Of Directors ◽  
Transcriptional Activity ◽  
Employment Equity ◽  
Advisory Committees ◽  
In Vivo Models ◽  
Diamond Blackfan Anemia ◽  
Equity Ownership

Abstract Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP. Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

scholarly journals Spatial Transcriptomics Reveals DPP4 As Novel Marker of a More Proliferative Phenotype in Early AML Progression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3310-3310
Author(s):  
Christa Haase ◽  
Karin Gustafsson ◽  
Shenglin Mei ◽  
Jelena Milosevic ◽  
Shu-Chi Yeh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Board Of Directors ◽  
Hematologic Malignancy ◽  
New Technology ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Privately Held

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis for which the standard-of-care chemotherapy treatment regimen has remained virtually unchanged over the past 40 years. We have employed "Image-Seq", a new technology that was developed in our laboratory, to study spatial variations in early leukemia progression in a mouse model of HoxA9-Meis1 AML. We visualized leukemia cells with differing proliferative phenotype using intravital microscopy, captured these cells under image guidance from individual bone marrow microenvironments and studied their differential expression by single-cell RNA sequencing. This analysis identified DPP4 as a key upregulated gene in AML cells from more proliferative bone marrow compartments and associated DPP4 expression with a cell cluster enriched in progenitor cell markers for HoxA9-Meis1 AML, including Flt3, Itgb7 and Ddx4. Strikingly, DPP4 is not expressed in vitro, and its expression in vivo (as quantitated by FACS analysis) correlated with disease progression and marked a more proliferative phenotype both at the 1-week and 2-week time-points during disease progression. Disclosures Sykes: Clear Creek Bio: Current equity holder in publicly-traded company; SAFI Biosolutions: Consultancy, Current equity holder in publicly-traded company; Keros Therapeutics: Consultancy. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; FOG Pharma: Consultancy; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Editas Medicines: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: sponsored research; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2160-2160
Author(s):  
Gabriel Pabst ◽  
Johannes Foßelteder ◽  
Angelika Schlacher ◽  
Lisa Auinger ◽  
Daniel Martinez-Krams ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Genome Engineering ◽  
Specific Effect ◽  
Monocytic Differentiation ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays. Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition. Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed "Acute Myeloid Leukemia" among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells. Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. Figure 1 Figure 1. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

scholarly journals A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2069-2069
Author(s):  
Biagio De Angelis ◽  
Marika Guercio ◽  
Domenico Orlando ◽  
Stefano Di Cecca ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Short Term ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 887-887
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Phong Quang ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Confocal Microscopy ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vivo Confocal Microscopy ◽  
Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

scholarly journals Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4274-4274 ◽  
Author(s):  
Thomas Balligand ◽  
Younes Achouri ◽  
Ilyas Chachoua ◽  
Christian Pecquet ◽  
Jean-Philippe Defour ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Mutational Status ◽  
Thrombopoietin Receptor ◽  
Exon 9 ◽  
Calr Mutations

Abstract In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation. References 1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. N Engl J Med. 2013 Dec 10;369(25):2379-90. 2. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. N Engl J Med. 2013 Dec 10;369(25):2391-405. 3. Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Blood. 2015 Dec 14;10.1182/blood-2015-11-681932. 4. Marty C, Pecquet C, Nivarthi H, Elkhoury M, Chachoua I, Tulliez M, et al. Blood. 2015 Nov 25;10.1182/blood-2015-11-679571. 5. Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C, et al. Leukemia. 2016 Feb 29;10.1038/leu.2016.47. Disclosures Constantinescu: Teva: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Selectin Inhibition Disrupts Multiple Myeloma Cells Interaction with the Bone Marrow Microenvironment and Sensitizes Them to Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 453-453
Author(s):  
Abdel Kareem A. Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
John Magnani ◽  
John Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Bioluminescence Imaging ◽  
Plasma Cells ◽  
Tumor Burden ◽  
Advisory Committees ◽  
Normal Plasma

Abstract Abstract 453 Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications. Methods AND RESULTS: Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 < MGUS, n=20 < smoldering MM, n=23 < MM, n=68 p<0.01). Moreover, it was shown that both BMSCs (isolated from MM patients and HS5 cell line) and ECs (isolated from MM patients and HUVECs) had high expression of P-selectin. SDF1 treatment increased the expression of P-selectin on ECs but it had no effect on the expression of PSGL on MM cells. The interaction of PSGL and P-selectin played a major role in the adhesion of MM cell to both BMSCs and ECs, and the inhibition of this interaction either by the pan-selectin inhibitor GMI-1070 (500uM, 3hrs) or by knock-down of P-selectin expression significantly decreased (50-60%) the adhesion of MM cells to BMSCs and ECs. The CXCR4 inhibitor AMD3100 (25uM, 3hrs) similarly induced similar inhibition of adhesion, and the combination of AMD3100 and GM1070 had more profound inhibition of MM adhesion to BMSCs and ECs (p = 0.006). Both AMD3100 and GMI1070 induced MM cell de-adhesion from BMSCs and ECs, but the combination of both drugs was not additive. AMD3100, GMI1070 or their combination prevented BMSCs or ECs mediated induction of proliferation of MM cells. Moreover, it was shown that the co-culture of MM cells with BMSCs and ECs reduced their sensitivity to bortezomib (5nM, 24hrs) and dexamethasone (25nm, 24hrs) compared to MM cells cultured alone. Importantly, GMI1070 restored the sensitivity of MM cells to bortezomib and dexamethasone to the level observed without co-culture with BMSCs or ECs. These effects were next tested in vivo using an orthotopic xenograft model of MM. SCID-beige mice were injected with luciferase-expressing MM1S cells, and tumor burden was assessed bioluminescence imaging. Mice with established disease were divided into treatment groups (n=10 per group) treated with vehicle, GMI1070 by osmotic pump, velcade at 1.5 mg/kg IP weekly, or a combination of GMI1070 and bortezomib. Tumor burden was determined by bioluminescence imaging. Treatment with GMI1070 alone was not different from vehicle treated control mice. While treatment with bortezomib alone had a minimal delay in tumor progression, the combined treatment of bortezomib and GMI1070 resulted in synergistic anti-tumor efficacy (p=0.0017). Conclusion: We have shown that PSGL-1 is highly expressed in MM cells as compared to normal plasma cells, and that it plays a major role in the interaction of MM cells with the BM microenvironment in relation with the SDF1/CXCR4 axis in vitro, an effect which was inhibited by the pan-selectin inhibitor GMI1070. We also demonstrated that selectin inhibition by GMI1070 reduced MM cell proliferation induced by BMSCs and ECs sensitized MM cells to bortezomib and dexamethasone in vitro, and significantly increased the sensitivity of MM tumors to bortezomib in vivo. This information provides the rationale for future clinical trials for increasing efficacy of existing therapies through a combination with selectin inhibitors for the treatment of myeloma. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

CS1-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells and T Cells Enhance In Vitro and In Vivo Anti-Tumor Activity Against Human Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 14-14
Author(s):  
Jianhong Chu ◽  
Youcai Deng ◽  
Don M. Benson ◽  
Shun He ◽  
Tiffany L. Hughes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Trials ◽  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Variable Region ◽  
Advisory Committees ◽  
Empty Vector

Abstract Multiple myeloma (MM) is a B-cell malignancy characterized by the aberrant clonal expansion of plasma cells (PCs) within the bone marrow (BM). Despite the use of proteasome inhibitors and immune-modulating drugs, which have improved overall survival, MM remains an incurable malignancy for which novel therapeutic approaches are urgently needed. Immunotherapy that specifically targets antigens expressed by MM would be a promising approach to treat MM patients refractory to any current treatments. Chimeric antigen receptors (CARs) are engineered fusion proteins containing tumor antigen-recognition moieties and immune cell activation domains. CAR-expressing T cells have been demonstrated successful in the clinic to treat chronic lymphocytic leukemia (CLL) and acute lymphoid leukemia (ALL). However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells or T cells to target MM-expressed CS1 to treat MM has not been previously explored, and is the focus of our study. CS1 is a surface glycoprotein and represents an ideal target for the treatment of MM. CS1 is highly, and nearly ubiquitously, expressed on MM cells, while expression remains very low on NK cells, some T-cell subsets, and normal B cells, and also it is almost undetectable on myeloid cells. In addition, monoclonal antibody directed against CS1, elotuzumab, has already been proven safe in phase 1 and 2 clinical trials, and phase 3 trials are ongoing. Therefore, it should be safe to target CS1 for the treatment of MM. We successfully generated a specific CS1-CAR construct with a lentiviral vector backbone, sequentially containing a signal peptide (SP), a heavy chain variable region (VH), a linker, a light chain variable region (VL), a hinge, CD28 and CD3ζ. Flow cytometry analysis with an antibody against VH and VL regions indicated that CS1-CAR was successfully expressed on the surface of NK cells and T cells transduced with the CAR construct. In vitro, CS1-CAR NK cells and T cells displayed enhanced MM cytolysis (detected by Cr51 release assay) and augmented production of cytokines (determined by enzyme-linked immunosorbent assay, ELISA), such as IFN-g for NK and T cells and IL-2 for T cells, when co-cultured with CS1-expressing MM cell lines. These effects relied on CS1-dependent recognition of MM cells because CS1-CAR NK or T cells possessed higher activity when they were co-cultured with CS1-expressing cells, but remained much lower activity when they were co-cultured with CS1-negative cells. However, CS1-CAR NK or T cells launched significantly higher killing of MM cells and secrete more abundant cytokines when the CS1-negative MM cells ectopically overexpressed CS1 and were co-cultured with the CAR cells. Ex vivo, compared to NK or T cells transduced with the empty vector, NK or T cells transduced with CS1-CAR also showed significantly enhanced effector functions when responding to purified primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, when compared to untreated mice or mice treated with empty vector-tranduced NK or T cells, adoptive transfer of 5 × 106 NK or T cells expressing CS1-CAR once every five days efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged survival of mice bearing IM9 MM cells. Our efforts to translate these findings into clinical trials are ongoing. In summary, CS1 is a promising target for using CAR NK or T cells for MM treatment, and we have generated a CAR that recognizes CS1. We demonstrate that NK cells or T cells armed with this CS1-CAR can recognize and eradicate myeloma cells in vitro and in vivo. Autologous or allogeneic transplantation of these CS1-specific CAR NK cells or CAR T cells may be a promising strategy to treat MM. Disclosures: Caligiuri: Innate Pharma: Membership on an entity’s Board of Directors or advisory committees. Hofmeister:Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Mouse Versus Human Extrinsic Cues Dictate Transformation Potential In BCR-ABL/BMI1-Induced Leukemia In Humanized Xenograft Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 515-515
Author(s):  
Pallavi Sontakke ◽  
Richard W.J. Groen ◽  
Jennifer Jaques ◽  
Huipin Yuan ◽  
Anton Martens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blast Crisis ◽  
Human Cells ◽  
Bone Marrow Niche ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Transformation Potential

Abstract The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document