Figure 4.

Low-dose volasertib causes selective toxicity in primary AMLs with predicted pathogenic FA pathway mutations. A, Graph represents viability of primary patient CD34⁺ AML cells treated with 10 nmol/L volasertib for 5 days. P values were calculated by two‐way ANOVA. B, Panel below bar graph indicates predicted pathogenic FA pathway mutations in patient AML samples identified through WES. C, Graph represents Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway functional pathways that were significantly enriched among identified gene mutations (P value < 0.05). D, Top depicts the position of FANCA residue L1260 within the predicted H12A helix of the C-terminal domain. Protein structure was predicted using AlphaFold Protein Structure Database (version 2022-11-01, RRID: SCR_023662). Bottom shows conservation of L1260 residue (highlighted in yellow) across multiple species. E, Representative Western blot showing FANCA expression in HeLa FANCA-KO cell line transfected with empty vector, FANCA-WT, or FANCA-Mut(L1260P). Representative graph shows the mean ± SEM cell viability (%) of empty (FANCA-KO), FANCA-WT, and FANCA-Mut(L1260P) HeLa cells treated with MMC for 4 days. P values were calculated by two‐way ANOVA. F, Representative graph shows the mean ± SEM cell viability (%) of empty (FANCA-KO) and FANCA-WT vs. FANCA-Mut(L1260P) HeLa cells treated with volasertib for 3 days. P values were calculated by two‐way ANOVA. G, Representative Western blot of FAN1 expression in control cells (Scr.Ctrl) vs. FAN1-KD (shFAN1) HeLa cells. Representative graph shows the mean ± SEM cell viability (%) of shFAN1 cells vs. Scr.Ctrl. HeLa cells treated with volasertib for 3 days. For all cell lines, viability was measured by CellTiter-Glo assay. P values were calculated by two‐way ANOVA. All experiments were repeated at least 3 times. ns, not significant.