Notch singalling inhibition as a multi-target therapy to overcome bone marrow microenvironment-mediated drug resistance in AML

Background: Several pro-survival proteins promoting resistance to chemother- apy, such as BCl-2, STAT3,NFκB and AKT,are over-expressed in AML cells, thus representing the basis for targeted therapies. However, only multi-target drug strategy may lead to the modulation of the pro-survival protein network, due to the simultaneous activation of alternative pathways. Notch signalling is a master developmental pathway that controls tumour cell survival by interacting with pro-survival proteins, such as β-catenin, BCL-2, STAT3, NFκB, and AKT, thus representing an ideal target to interfere with all these pathways in different cancer systems. We recently showed that Notch inhibition was capable of abrogating microen- vironment-mediated AML cell chemo-resistance (P550, EHA20); however, little is known about the mechanism involved. Aims: We studied the mechanisms underlying microenvironmental, Notch- mediated AML chemo-resistance by investigating the contribution of BCl-2, STAT3, NFκB and AKT. Using in silico and in vitro approaches we analyzed the expression changes of these proteins in ex-vivo AML cell samples in condition of pharmacological or genetical Notch down-regulation, as well as in AML cells either cultured alone or co-cultured with human bone marrow mesenchymal stromal cells (hBM-MSCs) in presence of chemoterapeutic agents, such as cytarabine (Ara-C) and Idarabucin. Methods: Cells were obtained from bone marrow (33) and peripheral blood (22) samples of AML patients. hBM-MSCs were expanded from bone marrow of 20 healthy donors (BM-MSCs) and 20 AML patients (BM-MSCs*). Gene set enrich- ment analysis (GSEA) were perform using GEOR tools on AML expression array of 304 patients previously deposited in Gene Expression Omnibus (GSE10358). Genetic inhibition of Notch signalling was achieved in AML cell lines (HL-60 and THP1) by infecting cells with lentiviral particles carrying shRNA for either RBP- jK or MALM1, two mediators of Notch signaling. Pharmacological Inhibition of Notch in AML was achieved by using Gamma secretase Inhibitors (GSIs), Notch transcription factors Inhibitor SAHM1, and combination of Notch blocking anti- bodies.Ara-C, and idarubicin were added to culture supernatants at different concentrations. Cell viability was evaluated by Annexin-V/Propidium Iodide (PI). Protein levels were analyzed by intracellular staining with corresponding fluo- rophore conjugated antibodies, followed by flow cytometry analysis. Results: In silico Gene set enrichment analysis and flow cytometry analysis showed that AML samples highly expressed Notch1, Jagged1, STAT3, NFκ-B and AKT genes and proteins. Notably, higher levels of Notch1 were found in patients with poor cytogenetic prognosis, while STAT3, NF-κB and AKT were uniformly expressed by AML patients. Protein analysis revealed low levels of pro-survival proteins AKT, STAT3 and NF-κB in RBP-jk and MALM1 knock- down cells, as compared to control cells infected with non specific shRNA. We then verified that genetic (shRNA) and pharmacological inhibition of Notch, by using either GSIs or Notch receptor blocking antibodies, was capable of sen- sitizing AML cells, either cultured alone or in presence of hBM-MSCs, to ARA- C or idarubucine. Additionally, we found that hBM-MSC-dependent induction of AML chemoresistance was associated to increase of AKT, NF-κB and STAT3 protein levels in AML cells. Similarly, Notch inhibition with GSIs prevented hBM- MSC-mediated increase of AKT, NF-κB and STAT3, thus restoring sensibility of AML cells to Idarubucin treatment. Summary/Conclusions: These results suggest that inhibition of Notch sig- nalling is sufficient to reduce protein levels of AKT, STAT3 and NF-κB proteins involved in AML chemoresistance, thus making the pro-survival core network controlled by Notch a potential target for specific Notch targeted therapies.