THE PIVOTAL ROLE OF NOTCH SIGNALING IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL) CHEMOSENSITIVITY

Growing evidence suggests that Notch signaling pathway can modulate drug response in hematological malignancies including T-ALL, B-CLL and AML. In B-ALL we have previously demonstrated that Notch3 and Notch4 support survival of primary B-ALL cells, suggesting a role for Notch signaling in drug response. Here, we used in vitro, in silico, and in vivo approaches to comprehensively the role of Notch pathway in B-ALL pathogenesis in terms of prognosis, proliferation, survival and drug response. B-ALL cell lines were obtained from ATCC, while B-ALL primary cells were isolated from bone marrow or peripheral blood of 51 B-ALL patients. Flow cytometry and western immunoblotting analyses showed that primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy, suggesting that Notch signaling may be critical to drug response in B-ALL. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents (Cytarabine, Ara-C; Dexamethasone, Dexa; Doxorubicin, Doxo) alone or in combination with Notch signaling modulators, including anti-Notch blocking antibodies, gamma secretase inhibitors (GSIs), and Notch transcription factor inhibitor (SAHM1). GSIs and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells, up-regulating intracellular levels of reactive oxygen species (ROS) that were then capable to modulate pro-survival protein levels such as mTor, Akt, NFκ-B and Erk. In vitro observations were successfully translated in mouse-based xenograft models of B-ALL, obtained by injecting the B-ALL line RS4;11 in NOG mice. The in vivo co-administration of Notch inhibitor GSI-XII or anti-Notch4 with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden, thus prolonging mouse survival, compared with DMSO or Ara-C alone. Overall, our results 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 inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, improving Ara-C-mediated reduction of blast cells in bone marrow, suggesting that Notch signaling is a possible therapeutic strategy to eradicate the minimal residual disease in B-ALL.