Contributions
Abstract: PB1597
Type: Publication Only
Background
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.
Aims
We used the VR-ALL cell line in comparison with two other B-ALL lines (RS4;11 and SUB-P15) and primary samples from B-ALL patients to analyze the contribution of defective notch signaling in B-ALL cell lines as well as cell line based-xenograft models of B-ALL.
Methods
B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were obtained from bone marrow or peripheral blood of 30 B-ALL patients. Flow cytometry and western immunoblotting were used to study the expression of Notch receptors and ligands. Drugs used were Cytarabine, Dexamethasone and Doxorubicin alone or in combination with gamma secretase inhibitors (GSIs). Mice xenograft model of B-ALL were obtained by injecting the B-ALL line RS4;11 in NOD/Shi-scid/IL-2Rγnull mice (NOG). Cell viability was evaluated by Annexin-V/PI and MTT assay; proliferation was assessed through CFSE dilution.
Results
Flow cytometry analysis and May-Grünwald Giemsa staining revealed that the VR-ALL was a Pre-B-ALL cell line. Similarly, to RS4;11 and SUP-B15 cell lines, VR-ALL cells grew easily in RPMI or IMDM supplemented with 10% FBS. Western blot and flow cytometry analysis showed that VR-ALL cells as well as primary blast cells displayed a Notch expression pattern consisting in lower expression levels of Notch2 and Jagged1, high expression levels of Notch1, Notch3, Notch4, Jagged2, DLL3 and DLL4. But in contrary to SUP-B15 and RS4;11, the Notch target Hes1 was absent in VR-ALL cells. However, VR-ALL cells were still highly sensitive in vitro to GSI XII as shown by MTS and Annexin assays. Then, we successfully obtained a mice xenograft model of B-ALL with high bone marrow leukemic burden, by injecting the VR-ALL cell line in NOD/Shi-scid/IL-2Rγnull mice. Concordantly with in vitro observation, administration of GSI-XII to mice, significantly lowered the leukemic burden in the bone marrow. These activities of GSI-XII in cells with a defective Notch activity suggested a Notch-independent role of gamma secretase inhibitors. Seeking the influence of Notch status on drug response, we observed that the treatment of the cell lines in vitro with Cytarabine, Dexamethasone or Doxorubicin induced a dose-dependent decrease in VR-ALL cell viability. Noteworthy, VR-ALL cells were less sensitive to the treatment with glucocorticoids than the two other B-ALL cell lines. This reduction was abrogated when the glucocorticoid was associated to GSI-XII.
Conclusion
Through this study we propose a new cell line that has allowed us to understand B-ALL pathogenesis, representing an appropriate tool to better unravel the mechanistic role of Notch signaling in B-ALL. Moreover, VR-ALL could be a valuable tool to investigate the mechanisms of B-ALL relapse determined by glucocorticoid refractoriness.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): B cell acute lymphoblastic leukemia, Notch signaling
Abstract: PB1597
Type: Publication Only
Background
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.
Aims
We used the VR-ALL cell line in comparison with two other B-ALL lines (RS4;11 and SUB-P15) and primary samples from B-ALL patients to analyze the contribution of defective notch signaling in B-ALL cell lines as well as cell line based-xenograft models of B-ALL.
Methods
B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were obtained from bone marrow or peripheral blood of 30 B-ALL patients. Flow cytometry and western immunoblotting were used to study the expression of Notch receptors and ligands. Drugs used were Cytarabine, Dexamethasone and Doxorubicin alone or in combination with gamma secretase inhibitors (GSIs). Mice xenograft model of B-ALL were obtained by injecting the B-ALL line RS4;11 in NOD/Shi-scid/IL-2Rγnull mice (NOG). Cell viability was evaluated by Annexin-V/PI and MTT assay; proliferation was assessed through CFSE dilution.
Results
Flow cytometry analysis and May-Grünwald Giemsa staining revealed that the VR-ALL was a Pre-B-ALL cell line. Similarly, to RS4;11 and SUP-B15 cell lines, VR-ALL cells grew easily in RPMI or IMDM supplemented with 10% FBS. Western blot and flow cytometry analysis showed that VR-ALL cells as well as primary blast cells displayed a Notch expression pattern consisting in lower expression levels of Notch2 and Jagged1, high expression levels of Notch1, Notch3, Notch4, Jagged2, DLL3 and DLL4. But in contrary to SUP-B15 and RS4;11, the Notch target Hes1 was absent in VR-ALL cells. However, VR-ALL cells were still highly sensitive in vitro to GSI XII as shown by MTS and Annexin assays. Then, we successfully obtained a mice xenograft model of B-ALL with high bone marrow leukemic burden, by injecting the VR-ALL cell line in NOD/Shi-scid/IL-2Rγnull mice. Concordantly with in vitro observation, administration of GSI-XII to mice, significantly lowered the leukemic burden in the bone marrow. These activities of GSI-XII in cells with a defective Notch activity suggested a Notch-independent role of gamma secretase inhibitors. Seeking the influence of Notch status on drug response, we observed that the treatment of the cell lines in vitro with Cytarabine, Dexamethasone or Doxorubicin induced a dose-dependent decrease in VR-ALL cell viability. Noteworthy, VR-ALL cells were less sensitive to the treatment with glucocorticoids than the two other B-ALL cell lines. This reduction was abrogated when the glucocorticoid was associated to GSI-XII.
Conclusion
Through this study we propose a new cell line that has allowed us to understand B-ALL pathogenesis, representing an appropriate tool to better unravel the mechanistic role of Notch signaling in B-ALL. Moreover, VR-ALL could be a valuable tool to investigate the mechanisms of B-ALL relapse determined by glucocorticoid refractoriness.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): B cell acute lymphoblastic leukemia, Notch signaling