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, 9 (26), 18341-18350

Characterization of a New B-ALL Cell Line With Constitutional Defect of the Notch Signaling Pathway

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Characterization of a New B-ALL Cell Line With Constitutional Defect of the Notch Signaling Pathway

Paul Takam Kamga et al. Oncotarget.

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.

Keywords: ALGS; Alagille syndrome; B-ALL; B-acute lymphoblastic leukemia; Notch signaling.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing financial interests. Conflict-of-interest disclosure.

Figures

Figure 1
Figure 1. Growth and morphological patterns of VR-ALL
(A) Initial proliferation rate of VR-ALL cells isolated from the ALGS patient. Blast cells derived from the patient were grown in RPMI with 10% FBS, cell count was performed routinely. (B) Proliferation rate of VR-ALL cells 3 years following isolation; cells were grown in RPMI with 10% FBS, cell count was performed every 24 hours. Data are reported as mean ± SEM of 4 independent experiments performed in duplicate. (C) Cell morphology of B-ALL cell lines stained with May Grunwald-Giemsa staining and observed using Axiovert Z1 Observer Microscope (Zeiss).
Figure 2
Figure 2. Engraftment of VR-ALL cells into NOG mice
5 × 106 VR-ALL cells were injected via the tail vein into 8 to 12 week-old mice. Mice were either (A) sacrificed (n = 4) at 8 weeks following the initial injection of VR-ALL cells to evaluate leukemic burden in organs as levels of hCD45+ cells, or (B) monitored (n = 4) for their survival. “Sham”: control or naive mice.
Figure 3
Figure 3. Notch expression and activation in B-ALL samples
Immunoblot of VR-ALL cells and B-ALL cell lines RS4;11 and SUP-B15, probed for Notch1-4, Jagged1-2, DLL4, Hes1 and GADPH. Data are representative of 6 independent experiments. “FL”: Full Length, “NTM”: Notch Transmembrane Domain, “NICD”: Notch Intracellular Domain.
Figure 4
Figure 4. GSIs reduces VR-ALL cell viability
(A) Relative proliferation of VR-ALL cells stained with CFSE and treated for 2 days with GSI-XII (10 μM) and GSI-IX (15 μM); CFSE dilution was analyzed though flow cytometry and expressed as relative proliferation. Data are reported as mean ± SEM of 4 independent experiments performed in duplicate **p < 0.01, **p < 0.001. (B) Apoptosis levels in VR-ALL cells treated for 2 days with GSI-XII (10 μM) and GSI-IX (15 μM); cells were stained with Annexin-V and propidium before analysis through flow cytometry. Data are reported as mean ± SEM of 4 independent experiments performed in duplicate **p < 0.01, ***p < 0.001. (C–D) Effect of GSI-XII administration on leukemic burden and mouse overall survival after injection of VR-ALL cell line (DMSO: solvent of GSI-XII, negative control).

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