Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: One of the main obstacles in the treatment of acute myeloid leukaemia is the relapse of patients after treatment with conventional chemotherapy, whose main target is proliferative cells. A possible explanation may be the existence of leukaemic stem cells in a reversible state of quiescence (outside the cell cycle) that confer chemoresistance. The activation of the Hedgehog, Notch and Wnt/β-catenin pathways seems to favour the maintenance of quiescence, so their inhibition could induce the entry of these cells into the cell cycle and improve the efficacy of conventional chemotherapy such as cytarabine, previously studied by the group, or azacitidine and idarubicin, presented in this study. Aims: Improve conventional chemotherapy cell death after inhibiting quiescence state by targeting Hedgehog, Notch and Wnt/β-catenin signalling pathways. Methods: Three human AML cell lines were used: HL-60, OCI-AML3 and KASUMI-1. The cells were treated for 48h and 72h with the IC50 concentration of Idarrubicin or Azacitidine, previously determined by the group, and different combinations of the drugs Glasdegib, Nirogacestat and PRI-724, inhibitors of the Hedgehog, Notch and Wnt/β-catenin pathways, respectively. To determine the cytotoxicity of the treatments (24 replicates for each treatment), the WST8 agent was used to obtain a measure of absorbance proportional to the number of viable cells. Results: Inhibiting the Notch pathway in combination with idarrubicin decreased the viability of HL60 cell line in 14% compared to control (p < 0.05), in 30% when Hedgehog and Notch pathways were inhibited together (p < 0.001), and in 16% when all three pathways were inhibited (p < 0.01). Combined treatments of azacytidine and signalling pathway inhibitors revealed a reduction in cell viability relative to the monotherapy control of 20% when inhibiting the Notch pathway (p < 0.01) and 18% when inhibiting the Wnt pathway (p < 0.05) in OCI-AML3, and between 12%-24% when all three signalling pathways were inhibited together in both OCI-AML3 and KASUMI1 (p < 0.001) during 72h. Summary/Conclusion: Combinations of chemotherapy with signalling pathway inhibitors during 72h showed a greater reduction in cell viability compared to the monotherapy control. Also, HL60 showed a more sensitive pattern to idarrubicin, while OCI-AML3 and KASUMI1 were more sensitive to azacitidine. The results obtained show a greater efficacy of conventional chemotherapy when Hedgehog, Notch and Wnt/β-catenin pathways are inhibited in acute myeloid leukaemia cell lines.Figure. Cell viability in HL60, OCI-AML3 and KASUMI1 after inhibition of Hedgehog, Notch and Wnt/Beta-catenin signalling pathways in combination with (A-C) idarrubicin and (D-F) azacytidine. P-values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0,0001 Keywords: Acute myeloid leukemia, Cell cycle, Chemotherapy