Fig. 2: AUR augments VEN activity against MLLr BCP-ALL cell lines and PDXs in vitro and in vivo.

A SEM and RS4;11 MLLr BCP-ALL cell lines were treated with EC20 and EC50 concentration of VEN or AUR (calculated by nonlinear regression dose-response analysis using GraphPad7), as well as with a combination of both drugs for 48 h. Subsequently, cells were subjected to MTT assay. The viable cells are presented as the percentage of untreated control (DMSO). Bars represent mean + SD, n = 8. Based on the drug response, the combination index (CI) was calculated using CompuSyn software employing Chou-Talalay algorithm [57]. Right panel shows CI matrices representing particular VEN+AUR CI values. CI = 1 shows the additive effect, CI < 1 indicates the synergistic effect of the drugs. B CI matrices presenting antileukemic activity of VEN+AUR tested in MLLr BCP-ALL PDXs (n = 8). Pediatric (marked in red color) and adult (marked in green color) MLLr PDXs derived at diagnosis (n = 5) and relapse (n = 3) were co-cultured with primary BM-MSC cells and treated with VEN, AUR or a combination of both for 4–5 days. After treatment, the total amount of viable cells was determined by flow cytometry using anti-CD19 antibody, 7AAD dye and counting beads. CI value for each drug combination was calculated with CompuSyn. C In vivo experimental scheme. PDX#1 cells were injected into NSG mice and disease progression was determined weekly based on peripheral blood staining. When the percentage of human leukemic cells (hCD45⁺, hCD19⁺) reached 1% of murine CD45⁺ cells (day 18), VEN and AUR administration was initiated. Randomly selected mice were treated through o.g. with 100 mg/kg of VEN, intraperitoneally (i.p.) with 10 mg/kg of AUR, a combination of both drugs or control (DMSO – i.p., 60% Phosal 50PG; 30% PEG400; 10% ethanol – o.g.) for 21 days (3 cycles of 5 days treatment/2 days off). D Median fold of human leukemic cells over murine CD45⁺ cells calculated at day 41 post engraftment, pooled from two independent experiments, n = 6 (control), n = 8 (AUR), n = 7 (VEN, VEN+AUR), each dot represents a separate mouse. P values were calculated with the Mann–Whitney U test. E Event-free survival depicted on Kaplan–Meier survival plot from two independent experiments. Curve comparison was performed by log-rank (Mantel–Cox) test. F Experimental scheme for PDX#2. PDX#2 cells were injected into NSG mice and disease progression was determined based on peripheral blood staining. After 1 week, once leukemia cells propagation was confirmed in murine blood (0.2–1% of human leukemic cells among murine CD45⁺ cells), VEN and AUR administration was initiated. Randomly selected mice were treated with VEN, AUR, and a combination of both drugs as described in (C), daily, for 28 days. G Median fold of human leukemic cells over murine CD45⁺ cells calculated at day 25 post engraftment, n = 7 (control, AUR), n = 8 (VEN, VEN+AUR), each dot represents a separate mouse. P values were calculated with the Mann–Whitney U test. H Event-free survival for PDX#2 depicted on Kaplan–Meier survival plot. Curve comparison was performed by log-rank (Mantel–Cox) test; numbers of mice were as reported in (G).