Direct isolation of human central nervous system stem cells (CNS‐SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS‐SC were isolated from fetal brain tissue using the cell surface markers CD133+, CD34–, CD45–, and CD24–/lo (CD133+ cells). Fluorescence‐activated cell sorted (FACS) CD133+ cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133–, CD34–, and CD45– sorted cells (∼95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD‐SCID mice proliferated, migrated, and differentiated in a site‐specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (β‐tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133+‐derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP+ cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD‐SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP+ cells were present in the subventricular zone‐rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS‐SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model. © 2002 Wiley‐Liss, Inc.
Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 ؋ 10 5 infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.
Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the United States (US). DLBCL is a heterogeneous lymphoma, including the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which have different gene expression profiles, oncogenic aberrations, and clinical outcomes (Alizadeh, Nature 2000; Staudt, Adv Immunol 2005). ABC-DLBCL is characterized by chronic active B-cell receptor (BCR) signaling (Davis, Nature 2010), which is required for cell survival. Thus, the BCR signaling pathway is an attractive therapeutic target in this type of B-cell malignancy. Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling, is covalently bound with high affinity by ibrutinib, a first-in-class BTK inhibitor approved in the US for mantle cell lymphoma and chronic lymphocytic leukemia (CLL) patients (pts) who have received at least one prior treatment, CLL with del17p, and WaldenstršmÕs macroglobulinemia. A recent phase 2 clinical trial of single-agent ibrutinib in DLBCL pts revealed an overall response rate of 40% for ABC-DLBCL (Wilson, Nat. Med 2015); however, responses to single kinase-targeted cancer therapies are often limited by the cellÕs ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway (Nardi, Curr Opin Hematol 2004; Gazdar, Oncogene 2009). The serine/threonine-protein kinase PIM1 is one of several genes exhibiting differential expression in ibrutinib-resistant ABC-DLBCL cells compared with wild-type (WT) cells. We identified and report herein the role of PIM1 in ABC-DLBCL ibrutinib-resistant cells. Methods: PIM1 gene expression was analyzed by RT-qPCR. In vitro, cell viability was assessed in the human ABC-DLBCL cell line HBL-1 after treatment with ibrutinib and/or a pan-PIM inhibitor for 3 days, and the effect on colony formation was determined 7 days post-treatment. PIM1 mutational analysis was performed with clinical tumor biopsy samples from 2 studies, PCYC-04753 (NCT00849654) and PCYC-1106-CA (NCT01325701). PIM1 protein stability was analyzed by treating cells with cycloheximide and examining protein levels at different time points up to 8 hours. Results: Gene expression profiling of ibrutinib-resistant ABC-DLBCL cells revealed an upregulation of PIM1 (15-fold increase compared with WT cells) as well as PIM2 and PIM3. We also found that, compared with single-drug treatment, in vitro cell growth could be synergistically suppressed with a combination of ibrutinib and a pan-PIM inhibitor. This effect was observed in both WT (combination index (C.I.) = 0.25; synergy score = 3.18) and ibrutinib-resistant HBL-1 cells (C.I. = 0.18; synergy score = 4.98). In HBL-1 cells, this drug combination reduced colony formation and suppressed tumor growth in a xenograft model (Figure 1). In 48 DLBCL patient samples with available genomic profiling, PIM1 mutations appeared more frequently in pts diagnosed with ABC-DLBCL compared with GCB-DLBCL (5 out of 6 DLBCL pts with PIM1 mutations were ABC-subtype). 4 of these 5 pts exhibited a poor clinical response to ibrutinib, ie, 80% of ABC-DLBCL pts with PIM1 mutations had progressive disease, compared with only 13 of 26 (ie, 50%) ABC-DLBCL pts without PIM1 mutations. Subsequent characterization of the mutant PIM1 proteins (L2V, P81S, and S97N) confirmed that they were more stable than WT PIM1, suggesting increased protein levels by 2 potential mechanisms (WT PIM1 gene up-regulation or increased mutant PIM1 protein half-life). The impact of these mutations on PIM1 function and ibrutinib sensitivity is under investigation. Conclusions: Ibrutinib-resistant ABC-DLBCL cells have increased PIM1 expression, and synergistic growth suppression was observed when ibrutinib was combined with a pan-PIM inhibitor. PIM1 mutations identified in ABC-DLBCL pts with poor responses to ibrutinib contributed to increased PIM1 protein stability. A better understanding of the role of PIM1 in ibrutinib-resistant ABC-DLBCL tumors could provide a rationale for the design of combination therapies. Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Figure 1. Combination of ibrutinib and a pan-PIM inhibitor in the HBL-1 xenograft model. Ibrutinib and PIM inhibitor treatment suppressed tumor growth by 62% compared with the vehicle-treated group (*p < 0.01, repeated measures MANOVA adjusted univariate F-test). Disclosures Kuo: Pharmacyclics LLC, an AbbVie Company: Employment. Hsieh:pharmacyclics LLC, an AbbVie Company: Employment. Schweighofer:Pharmacyclics LLC, an AbbVie Company: Employment. Cheung:Pharmacyclics LLC, an AbbVie Company: Employment. Wu:Pharmacyclics LLC, an AbbVie Company: Employment. Apatira:Pharmacyclics LLC, an AbbVie Company: Employment. Sirisawad:Pharmacyclics LLC, an AbbVie Company: Employment. Eckert:Pharmacyclics LLC, an AbbVie Company: Employment. Liang:Pharmacyclics LLC, an AbbVie Company: Employment. Hsu:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.
Ibrutinib led to significant improvements in hematologic function and disease symptomatology versus ofatumumab, and can restore quality of life while prolonging survival in relapsed/refractory CLL/SLL.
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