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Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.

ABSTRACT: Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

SUBMITTER: Pearce L

PROVIDER: S-EPMC2833085 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.

Pearce Laurence L Morgan Liam L Lin Thet Thet TT Hewamana Saman S Matthews R James RJ Deaglio Silvia S Rowntree Clare C Fegan Christopher C Pepper Christopher C Brennan Paul P

Haematologica 20100301 3

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. T ...[more]

PMID: 20207849

Similar Datasets

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells.

Project description:BACKGROUND: VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions. METHODS: VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined. RESULTS: About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro. CONCLUSIONS: Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration, but only a limited role in their protection by stromal cells.

| S-EPMC3158106 | biostudies-literature

CD38/CD31 interactions activate genetic pathways leading to proliferation and migration in chronic lymphocytic leukemia cells.

Project description:Human CD38 is a pleiotropic glycoprotein belonging to a family of enzymes/receptors involved in the catabolism of extracellular nucleotides. CD38-receptor activities are regulated through binding to the nonsubstrate ligand CD31. CD38 expression above a critical threshold is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. Activation of CD38 by means of agonistic monoclonal antibodies or the CD31 ligand induces proliferation and immunoblast differentiation of CLL cells. Here we define the genetic signature that follows long-term in vitro interactions between CD38(+) CLL lymphocytes and CD31(+) cells. The emerging profile confirms that the CD31/CD38 axis activates genetic programs relevant for proliferative responses. It also indicates a contribution of this pathway to the processes mediating migration and homing. These results further support the notion that the CD31/CD38 axis is part of a network of accessory signals that modify the microenvironment, favoring localization of leukemic cells to growth-permissive sites.

| S-EPMC2785473 | biostudies-literature

CD38 and chronic lymphocytic leukemia: a decade later.

Project description:This review highlights a decade of investigations into the role of CD38 in CLL. CD38 is accepted as a dependable marker of unfavorable prognosis and as an indicator of activation and proliferation of cells when tested. Leukemic clones with higher numbers of CD38(+) cells are more responsive to BCR signaling and are characterized by enhanced migration. In vitro activation through CD38 drives CLL proliferation and chemotaxis via a signaling pathway that includes ZAP-70 and ERK1/2. Finally, CD38 is under a polymorphic transcriptional control after external signals. Consequently, CD38 appears to be a global molecular bridge to the environment, promoting survival/proliferation over apoptosis. Together, this evidence contributes to the current view of CLL as a chronic disease in which the host's microenvironment promotes leukemic cell growth and also controls the sequential acquisition and accumulation of genetic alterations. This view relies on the existence of a set of surface molecules, including CD38, which support proliferation and survival of B cells on their way to and after neoplastic transformation. The second decade of studies on CD38 in CLL will tell if the molecule is an effective target for antibody-mediated therapy in this currently incurable leukemia.

| S-EPMC3574275 | biostudies-literature

Calcium-RasGRP2-Rap1 signaling mediates CD38-induced migration of chronic lymphocytic leukemia cells.

Project description:CD38 is a transmembrane exoenzyme that is associated with poor prognosis in chronic lymphocytic leukemia (CLL). High CD38 levels in CLL cells are linked to increased cell migration, but the molecular basis is unknown. CD38 produces nicotinic acid adenine dinucleotide phosphate and adenosine 5'-diphosphate-ribose, both of which can act to increase intracellular Ca2+ levels. Here we show that CD38 expression increases basal intracellular Ca2+ levels and stimulates CLL cell migration both with and without chemokine stimulation. We find that CD38 acts via intracellular Ca2+ to increase the activity of the Ras family GTPase Rap1, which is in turn regulated by the Ca2+-sensitive Rap1 guanine-nucleotide exchange factor RasGRP2. Both Rap1 and RasGRP2 are required for CLL cell migration, and RasGRP2 is polarized in primary CLL cells with high CD38 levels. These results indicate that CD38 promotes RasGRP2/Rap1-mediated CLL cell adhesion and migration by increasing intracellular Ca2+ levels.

| S-EPMC6039665 | biostudies-literature

Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways.

Project description:Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

| S-EPMC10968363 | biostudies-literature

Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia.

Project description:PurposeCD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied.Experimental designWe investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT.ResultsIn addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model.ConclusionsOverall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

| S-EPMC6744942 | biostudies-literature

Genetic susceptibility to chronic lymphocytic leukemia.

Project description:Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the West and is an incurable malignancy. No firmly established evidence exists for environmental risk factors in the etiology of CLL. However, CLL is estimated to have one of the highest familial risks for a hematologic malignancy; this along with other evidence strongly supports an inherited genetic component. In the past 5 years, genome-wide association studies (GWAS) have provided the foundation for new avenues in the investigation of pathogenesis of this disease with 22 susceptibility loci currently identified. We review here the advances made in identifying these loci, the potential to translate these findings into clinical practice, and future directions needed to advance our understanding of the genetic susceptibility of CLL.

| S-EPMC3834539 | biostudies-literature

CD38 modulates CXCR4 signals in chronic lymphocytic leukemia cells: potential therapeutic implications

Project description:Recent data indicate that CD38, a cell surface enzyme and receptor, is part of the complex network of signals underlying the development, maintenance and progression of chronic lymphocytic leukemia (CLL). Here we show that CD38 signals directly facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by CXCL12 exposure. Use of i) a specific enzyme inhibitor, ii) an agonistic anti-CD38 monoclonal antibody (mAb) and iii) microarray studies led to the conclusion that the receptor-like functions of CD38 are responsible for the observed effects. Interactions between CD38 and its non substrate ligand CD31 define a genetic signature characterized by modulation of a significant number of genetic pathways, involved in proliferation and migration. Blocking CD38 with domain-specific mAbs significantly lowers CXCL12 effects, in terms of ERK1/2 phosphorylation and chemotaxis. The molecular explanation behind the blocking activity of these mAbs lies in physical proximity between CD38 and the CXCL12 receptor CXCR4, as demonstrated by i) co-immunoprecipitation, ii) confocal microscopy and iii) direct measurement of CXCL12 binding to its receptor. Lastly, blocking anti-CD38 mAbs were successfully used in vivo to interfere with CLL cell recirculation from blood to the lymphoid organs, an event critically controlled by CXCL12. These results represent a first step towards the validation of anti-CD38 mAbs as potential therapeutics for certain CLL patients. Keywords: gene expression profiling by microarray, CLL, CD31, CD38

2009-12-22 | GSE14063 | GEO

Genetic susceptibility variants for chronic lymphocytic leukemia.

Project description:BackgroundThere is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association study of CLL identified seven genetic variants that increased the risk of CLL within a European population.MethodsWe evaluated the association of these variants, or variants in linkage disequilibrium with these variants, with CLL risk in an independent sample of 438 CLL cases and 328 controls.ResultsOf these seven single nucleotide polymorphisms (SNP), six had P trend < 0.05 and had estimated odds ratios (OR) that were strikingly comparable to those of the previous study. Associations were seen for rs9378805 [OR, 1.47; 95% confidence intervals (CI), 1.19-1.80; P trend = 0.0003] near IRF4 and rs735665 near GRAMD1B (OR, 1.47; 95% CI, 1.14-1.89; P trend = 0.003). However, no associations (P > 0.05) were found for rs11083846, nor were any found for any SNP in linkage disequilibrium with rs11083846.ConclusionsOur results confirm the previous findings and further support the role of a genetic basis in the etiology of CLL; however, more research is needed to elucidate the causal SNP(s) and the potential manner in which these SNPs or linked SNPs function in CLL pathogenesis.

| S-EPMC2852480 | biostudies-literature

The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia.

Project description:The interactions of chronic lymphocytic leukemia cells with the microenvironment in secondary lymphoid tissues and the bone marrow are known to promote CLL cell survival and proliferation. CD38 and CD49d are both independent prognostic risk parameters in CLL with important roles in shaping these interactions. Both are reported to influence CLL cell trafficking between blood and lymphoid organs as well as their survival and proliferation within the lymphoid organs, thereby impacting the pathophysiology of the disease. The expression of CD38 and CD49d is associated in the majority of cases, and they exist as part of macromolecular complexes. Here, we review the current evidence for the individual and associated contributions of these molecules to CLL pathophysiology.

| S-EPMC4032465 | biostudies-literature

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