Figure 6.

Defactinib in combination with anti-CD44 is additive/synergistic in preventing AML cell adhesion. (a) FC in non-adhered KG1a AML cells (mean ± SD) when treated with increasing doses of anti-CD44 alone, increasing doses of defactinib alone or in combination. Synergy plot (right) was generated using SynergyFinder software (version 3.0, https://synergyfinder.fimm.fi (accessed on 15 February 2023)), showing an additive mean Bliss score of 1.627 (>1 = additive) and maximum of 4.39 (5 µg/mL + 5 µM). Results are compared to the no-treatment control, which is equal to 1. (b) Representative scatter plots of no drug, anti-CD44, defactinib and the combination of both, showing the proportions of total and CD34⁺ non-adhered primary AML cells after 2 min of acquisition on a Cytoflex S flow cytometer. This shows substantially more viable non-adhered CD34⁺ and CD34⁻ AML cells in the presence of both anti-CD44 and defactinib than no drug or either alone. (c) Individual FC (compared to no drug) in viable non-adhered primary AML cell numbers (mean ± SD) when treated with increasing doses of anti-CD44, defactinib or both and co-cultured for 3 h with a confluent layer of autologous stromal cells. Different concentrations of anti-CD44 alone and defactinib alone versus the combination of both were determined using a one-way ANOVA, and the results are tabulated in Supplementary Table S5; all comparisons were significant. (d) Combined FC in non-adhered primary AML cells (n = 3, mean ± SD) when treated with increasing doses of anti-CD44 alone, increasing doses of defactinib alone or in combination. A representative synergy contour plot for patient AML13 (right) was generated using SynergyFinder and shows a mean Bliss score of 8.12 (>1 = additive) and a synergistic maximum of 18.74 (2.5 µg/mL + 2.5 µM; >10 = synergistic).