Abstract
Background: Growing evidences suggest that Notch signaling can modulate drug response in hematological malignancies including T-ALL, B-CLL and AML. In B-cell acute lymphoblastic leukemia (B-ALL) we have previously demonstrated that Notch3 and Notch4 support survival of primary B-ALL cells, precluding a role of Notch signaling in drug response.
Aims: In this study we used in vitro, in silico and in vivo experiments to comprehensively define the role of Notch signaling in B-ALL pathogenesis in terms of prognosis, proliferation survival and drug response.
Methods: B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were obtained from bone marrow or peripheral blood of 45 B-ALL patients. Flow cytometry and western immunoblotting were used to study the expression of Notch receptors and ligands. Drugs used were Cytarabine (Ara-C), Dexamethasone (Dexa) and Doxorubicin (Doxo) alone or in combination with Notch modulators including anti-Notch blocking antibodies, gamma secretase inhibitors (GSIs), and Notch transcription factor inhibitor (SAHM1). 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: Our data revealed a robust expression of Notch receptors and ligands in B-ALL primary cells and B-ALL cell lines. The expression pattern consisted in high expression levels of Notch1, Notch3, Notch4, Jagged2, DLL3 and DLL4. Notably, in primary blast cells deriving from patients, the expression of Notch3, Notch4 and Jagged2, were significantly higher in the cases refractory to treatment as compared to patients achieving complete remission. Having also observed in samples derived from patients after chemotherapy a reduction in expression levels of Notch1-4 compared to samples collected prior the treatment, we hypothesized that Notch signaling was critical to drug response in B-ALL. Analysis of B-ALL cells treated with conventional chemo-agents (Ara-C, Doxo and Dexa) alone or in combination with Notch signaling inhibitors, showed that Notch signaling inhibitors including GSIs and anti-Notch4 were all able to significantly potentiate drug-induced cell death in B-ALL cells, up-regulating intracellular levels of reactive oxygen species (ROS). These high intracellular levels or ROS were then capable to modulate prosurvival-protein levels such as mTor, Akt, NFκ-B and Erk. In vitro observations were successfully translated in NOG mice, where GSI-XII in association with Ara-C significantly lowered leukemic burden in bone marrow of xenograft models of B-ALL, prolonging survival of mice compared to DMSO or Ara-C alone.
Conclusion: In this study, we highlighted the prognostic value of Notch expression in B-ALL as well as its critical role in B-ALL cell survival and response to chemotherapy in vitro and in vivo ; we demonstrated that Notch inhibitors can improve Ara-C-mediated reduction of blast cells in bone marrow, revealing that Notch signaling is a possible therapeutic strategy to eradicate minimal residual disease in B-ALL. Overall, our results support further investigations of Notch inhibitors in clinical trials of B-ALL.
Bonifacio: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.
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