Fig. 7

Potential enhancement of selective inhibitory effects on KRAS (G13C) HPCs by combination treatment. a, b Comparison of growth inhibitory effects between single- and combined treatments. Shown are the dose–response plots assessing treatment effects on either control (C2-1, blue) or KRAS-mutant (R2-1, orange) HPCs (technical replicates: n = 3). (a) The IC₅₀ value for R2-1 samples gets lower (1.6 nM) in the presence of 0.5 nM Palbociclib (PAL) compared with that obtained with Trametinib alone (3.6 nM). In contrast, the values stay similar for C2-1 samples (6.5 nM with TRA alone and 7.3 nM in combination). (b) Lowered IC₅₀ values observed for Navitoclax treatment with the addition of Trametinib (+ 0.4 nM TRA) in R2-1 samples (2.7 nM vs. 0.7 nM). The two values do not differ significantly for C2-1 (16.5 nM with NCX alone and 23.0 nM in combination). c, d Two-way analysis of variance (ANOVA) tests assessing the combination effects on iPSC-HPCs with the fixed concentration for each drug (TRA, 0.4 nM; NCX, 5 nM). Shown is the mean ± SD (technical replicates: n = 3) with the mean values provided on top of each histogram. Three P values are shown for each graph with underlines placed where they reach the statistical significance (< 0.05). Overall, we detected no remarkable interaction between the two main effects (TRA and NCX) and performed no post-hoc tests. (c) Results of the isogenic pair iPSC-HPCs derived from patient 2 (C2-1 and R2-1). (d) Results of the isogenic pair iPSC-HPCs derived from patient 1 (C8, a genetically corrected clone originated from the mutant iPSC R1-2; F4, a non-edited counterpart clone). Note that the combination treatment shows marked inhibitory effects on the mutant HPCs (27.0% for R2-1 and 30.2% for F4) while allowing limited toxicity to the control cells (viability of 77.7% for C2-1 and 63.7% for C8). e BM CD34⁺ cells isolated from RALD Pt 1 were tested. The pre-culture assessment revealed that the patient HPCs consisted of a ~ 1: 1 mixture of WT and KRAS-G13C cells. Shown is a box and whiskers plot comparing the estimated KRAS-G13C/WT ratio in each group of cells following a 7-day culture (technical replicates: n = 12. Recent death of the patient limits repetitive assessment with the primary samples). The decrease in ratio values is considered consistent with the selective inhibition of mutant cells over the control. The P values are based on the 2-way ANOVA. The analysis proved no interaction between the two effects, with only the inhibitory effect of navitoclax (NCX) reaching statistical significance (underlined). Despite the lack of interaction, it seems that a sensitizing effect of trametinib (TRA) exists selectively for the primary HPCs carrying KRAS-G13C on NCX-mediated growth inhibition.