Page 203 - Haematologica March 2020

Venetoclax response and maturation stage in AML
sitive and 1 of eight, 12.5% in resistant) and IDH1/IDH2 (10 of 25, 40% in sensitive and 1/8, 12.5% in resistant) mutations supporting the good clinical activity of veneto- clax seen in this patient group (Figure 6A, Online Supplementary Table S7).
To assess the efficacy and clinical relevancy of 27 drug combinations against blasts, we used concentrations achieved in patients’ plasma during treatment. The results demonstrated prominent inter-patient variability with the most synergistic drug combinations when blast-specific drug responses were measured by FC (Online Supplementary Figure S8). Of the 27 tested drug combina- tions, venetoclax plus kinase inhibitors showed the high- est average synergistic and blast killing effect (Table 2, higher BLISS score and lower mean % live blasts). Importantly, blasts were highly sensitive to single-agent venetoclax in 76% (25 of 33) of the samples with IC50<20nM. Thus, we did not observe synergy in the majority of the samples with a single venetoclax concen- tration of 50nM as this concentration alone was sufficient to kill the blasts (Figure 5A). To study the drug combina- tion effect in more detail, we conducted additional drug testing of venetoclax with a more detailed concentration range on four AML samples. We observed that with lower
venetoclax concentrations (10nM) a synergistic effect with MEK and/or JAK inhibitors was also detected in samples that were sensitive to single agent 50nM venetoclax treat- ment (Online Supplementary Figure S9).
Importantly, venetoclax (50nM) plus ruxolitinib (300nM) showed high efficacy (apoptosis/death>70%) and synergism in 6 of eight venetoclax resistant samples (Figure 5A-B). Strikingly, by combining venetoclax (50nM) with trametinib (25nM), all venetoclax resistant blasts were effectively targeted (Figure 6A-B). Although the combinations showed substantial toxicity to healthy CD34+ cells, they targeted most effectively leukemic blasts (Figure 6B). As a comparison, a drug combination used during induction treatment (cytarabine+idarubicin) showed remarkable inter-patient differences in blast toxi- city and it was also toxic to healthy CD34+ cells (Figure 6B). Furthermore, the broad-spectrum tyrosine kinase and FLT3 inhibitor sunitinib (100nM) or mTOR inhibitor everolimus (10nM) were not as effective when combined with venetoclax (Figure 6A, Table 2). Our data demon- strate that by simultaneously inhibiting JAK and/or MEK signaling and Bcl-2, blast cells involving chemorefractory AML cells, can be effectively targeted ex vivo in physiolog- ically relevant concentrations.
Table 2. Drug combination synergism and combination sensitivity in blasts.
Drug I
Venetoclax 50nM
Cytarabine 300nM
Trametinib 25nM
Venetoclax 50nM
Idarubicin 10nM
Trametinib 25nM
Venetoclax 50nM
Sunitinib 100nM
Venetoclax 50nM
Idarubicin 10nM
Venetoclax 50nM
Sunitinib 100nM
Sunitinib 100nM
Everolimus 10nM
Cytarabine 300nM
Idarubicin 10nM
Idarubicin 30nM
Idarubicin 10nM
Cytarabine 300nM
Everolimus 10nM
Cytarabine 300nM
Sunitinib 100nM
Idarubicin 10nM
Idarubicin 10nM
Cytarabine 300nM
Cytarabine 300nM
Idarubicin 10nM
Drug II
Trametinib 25nM
Trametinib 25nM
Everolimus 10nM
Ruxolitinib 300nM
Trametinib 25nM
Ruxolitinib 300nM
Everolimus 10nM
Trametinib 25nM
Sunitinib 100nM
Ruxolitinib 300nM
Cytarabine 300nM
Ruxolitinib 300nM
Everolimus 10nM
Ruxolitinib 300nM
Sunitinib 100nM
Cytarabine 300nM
Cytarabine 1000nM
Everolimus 10nM
Everolimus 10nM
Ruxolitinib 300nM
Ruxolitinib 300nM
Everolimus 10nM
Ruxolitinib 300nM
Sunitinib 100nM
Ruxolitinib 300nM
Ruxolitinib 300nM
Ruxolitinib 300nM
Drug III
Mean BLISS score*
0.083
0.076 0.075 0.068 0.057 0.046 0.040 0.034 0.030 0.029 0.023 -0.004 -0.001 -0.018 -0.020 -0.026 -0.029 -0.038 -0.046 -0.051 -0.058 -0.067 -0.073 -0.088 -0.123 -0.129 -0.146
Mean % of live blasts**
11.3
56.7 62.9 13.4 51.3 59.7 18.6 69.0 21.8 61.8 19.6 73.3 80.8 73.9 75.9 64.9 40.1 69.5 80.1 52.4 72.2 63.8 39.6 74.7 62.5 54.4 57.3
Trametinib 25nM
Ruxolitinib 300nM Trametinib 25nM
Everolimus 10nM Trametinib 25nM Everolimus 10nM
*Synergism calculated using BLISS score **Normalized to DMSO treated cells.
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