Characterization of a new B-ALL cell line with constitutional defect of the Notch signaling pathway

Abstract

Notch signaling contribution to B-cell acute lymphoblastic leukemia (B-ALL) development is still under investigation. The serendipitous onset of B-ALL in a patient affected by the germinal Notch mutation-dependent Alagille syndrome allowed us to establish a B-ALL cell line (VR-ALL) bearing a genetic loss of function in components of Notch signaling. VR-ALL is a common-type B-ALL cell line, grows in conventional culture medium supplemented with 10% serum, and gives rise, once injected into immunodeficient NOG mice, to a mouse xenograft model of B-ALL. Exome sequencing revealed deleterious mutations in some components of Notch signaling, including Jagged1, Notch1, and Notch2. In addition, VR-ALL is sensitive both in vitro and in vivo to γ-secretase inhibitors (GSIs) as well as conventional anti-leukemic drugs. For all these reasons, VR-ALL may help to gain more insights into the role of Notch signaling in B-ALL.

Inhibition of Notch Signaling Enhances Chemosensitivity in B-cell Precursor Acute Lymphoblastic Leukemia

“…B-ALL cell lines including VR-ALL, RS4;11, and SUP-B15 were cultured in complete RPMI (RPMI supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin). VR-ALL cells were isolated, characterized, and validated in our laboratory, as previously described (15), whereas RS4;11 and SUP-B15 cell lines were acquired from ATCC. Stability and identity of all B-ALL cell lines were controlled during and at the end of the current study using flow cytometry of membrane marker, morphologic analysis after Giemsa staining (15), and short tandem repeat (STR).…”

“…VR-ALL cells were isolated, characterized, and validated in our laboratory, as previously described (15), whereas RS4;11 and SUP-B15 cell lines were acquired from ATCC. Stability and identity of all B-ALL cell lines were controlled during and at the end of the current study using flow cytometry of membrane marker, morphologic analysis after Giemsa staining (15), and short tandem repeat (STR). The stability and identity of all other cell lines (including HEK 293, HEK 293T, CEM, and Ramos) used as control in Western immunoblot were validated only through flow cytometry of membrane marker and morphologic analysis after Giemsa staining.…”

“…All samples subjected to immunoblotting contained more than 80% of leukemia cells. The specificity of all Notch antibodies was validated on three control lysates, as previously described (5,14,15): HEK 293, HS27A stromal cell line, and CEM T-ALL cell line.…”

“…To study the specific relative basal sensitivity of B-ALL cells to different agents, cells were seeded in 96-well plates and cultured for 48 hours in presence of increasing concentrations of each modulator or drug. Then, the colorimetric 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay was performed, as previously described (5,15).…”

Bone Marrow-Derived Mesenchymal Stromal Cells: A Novel Target to Optimize Hematopoietic Stem Cell Transplantation Protocols in Hematological Malignancies and Rare Genetic Disorders

“…In particular, NOTCH/JAG1 signaling resulted in playing a fundamental role in MSC-dependent control of tumor initiation, growth, and chemoresistance. JAG1 overexpression has been shown to induce AML in mice [100], whereas NOTCH1, JAG1, and the main Notch target gene HES1 are overexpressed in AML-MSCs thus promoting survival of tumor cells exposed to chemotherapeutic agents [101] (Figure 2). Several studies have demonstrated that the tumor BM milieu is sufficient to convert a healthy BM stroma into a tumor supporting microenvironment.…”