Pb1827: two-level inhibition of hedgehog signalling pathway to exit quiescent state in acute myeloid leukaemia

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: One of the possible explanations for relapse in patients with acute myeloid leukaemia is the chemoresistance of cells in a quiescent state. This quiescent phase is regulated by several signalling pathways. One of the most important players in this setting is the Hedgehog pathway. The Hedgehog pathway is activated canonically by PTCH and SMO, terminating the activation cascade at the GLI transcription factors, but several authors have stressed the importance of non-canonical activations of the pathway, activating GLI transcription factors without the need for upstream proteins from the Hedhehog pathway. Therefore, dual inhibition of the pathway could bring leukaemic cells out of the quiescent phase, regardless of the type of pathway activation, and help to eradicate them with conventional chemotherapy. Aims: Decrease of the quiescent state by inhibiting Hedgehog in acute myeloid leukemia after targeting SMO membrane protein and GLI transcription factor. Methods: Three acute myeloid leukaemia cell lines were used: HL60, OCI-AML3 and KASUMI1. They were treated with the drugs GANT61 and Glasdegib, GLI and SMO inhibitors of the Hedgehog pathway, respectively. The concentration and exposure to the drugs varied depending on the cell line (see Figure). Subsequently, cell cycle analysis was performed by flow cytometry (9 replicates for each treatment) through KI-67 protein expression and 7-aminoactinomycin D (7-AAD) fluorescent staining. In addition, the viability of the cell lines after Hedgehog inhibition and cytarabine combination was studied by absorbance with the compound WST8. Absorbance analyses were performed using WST8 to study the cytotoxicity of the treatments (12 replicates for each treatment). Results: A statistically significant decrease in quiescent phase is observed in all cell lines. Glasdegib treatment decreased quiescent phase by approximately 40% in all cell lines (p < 0.01; p < 0.001) compared to DMSO control. On the other hand, treatment with GANT61 significantly reduced quiescence only in OCI-AML3 and KASUMI1, reducing quiescence by 60% compared to the control (p < 0.0001). Furthermore, the combination of these treatments reduced the quiescent phase by 40% (p < 0.001) in the latter two cell lines. Finally, in the viability assays, no significant results were obtained with the proposed treatments in combination with cytarabine. Summary/Conclusion: Inhibition of the Hedgehog pathway helps to bring acute myeloid leukaemia cells out of quiescence. No differences were observed between two-level inhibition and monotherapy inhibition, which may suggest that activation of the Hedgehog pathway in acute myeloid leukaemia is by canonical activation via the SMO protein.Figure. Cell cycle in HL60 (A), OCI-AML3 (B) and KASUMI1 (C) after inhibition of Hedgehog with SMO and GLI inhibitors. P-values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0,0001 Keywords: Cell cycle progression, Cell cycle, Chemotherapy, Acute myeloid leukemia