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I. Spinello et al.
and primary AML blasts were initially treated for 24 h with a low concentration (2.5 mM) of AC-73 and then co- treated for 48 h with Ara-C or ATO at different concentra- tions (0.01-1 mM). Cell viability assays were performed to assess leukemic cell survival and sensitivity to AC-73 treatment used in combination with Ara-C or ATO, as compared to single AC-73, Ara-C, or ATO treatment (Figure 5A and B and Online Supplementary Figure S4). We observed a significant decrease in leukemic cell viability of all leukemic cell lines after AC-73 treatment in combina- tion with Ara-C or ATO, as compared to AC-73, Ara-C or ATO used alone (Figure 5A and B and Online Supplementary Figure S4). Furthermore, we identified three subgroups of leukemic cells according to their Ara-C drug- sensitivity: high-sensitivity lines (U937 and HL-60); inter- mediate-sensitivity lines (NB4 and NB4-R4); low-sensitiv- ity lines (MV4-11 and Kasumi-1). AC-73 potentiates the sensitivity to Ara-C treatment of all leukemic cells, even
those belonging to the low-sensitivity subgroup (Figure 5A and B and Online Supplementary Figure S4). The anti- proliferative effect of AC-73 enhances sensitivity to ATO treatment of both M3 leukemic cells, such as NB4 and NB4-R4 (Figure 5B and Online Supplementary Figure S4B), and non-M3 leukemic cells, such as U937, HL-60, Kasumi- 1 and MV4-11 cells (Figure 5A and Online Supplementary Figure S4A, C and D).
Then, we showed that when used in combination with Ara-C and ATO, AC-73 treatment inhibits ERK and STAT3 activation in leukemic cells (Figure 5C).
Because ATO is also an inducer of autophagy,25 we ana- lyzed the autophagy flux in both M3 and non-M3 leukemic cells treated by ATO or AC-73 used alone or in combination, as compared to control cells. Our data showed that AC-73 and ATO are both inducers of autophagy and the autophagy flux is significantly increased in M3 (NB4) and non-M3 (U937) leukemic cells
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Figure 3. AC-73 has no significant effect on cell growth, differentiation and cell cycle progression in normal granulocytic (G) and monocytic (Mo) differ- entiating hematopoietic progenitor cells (HPCs). (A) Cell growth analysis during G and Mo differentiation of HPCs, in presence of AC-73 used at 5 mM and added every 2 days in cultures, as compared to con- trol (C) cells. (B) Cell cycle analysis at day 7 of G and Mo differentiation of HPCs treated with AC-73 (5 μM), as compared to control HPCs. (C) Phenotype analysis performed by analyzing CD11b, CD15 and CD14 expression levels in G and Mo differentiating HPCs at day 15, in the presence or not (C) of AC-73. (A-C) Mean±Standard Error of Mean (SEM) of three independent experiments is shown. *P<0.05; ns: not significant. (D) Morphological analysis at day 7 of the differentiation and maturation of G and Mo dif- ferentiating HPCs treated with AC-73, as compared to control (C) HPCs, and stained with May-Grünwald- Giemsa. (D) One representative experiment out of three is shown.
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haematologica | 2019; 104(5)