Inhibition of PLK4 triggered cytokinesis failure and polyploidy in TP53-mutated AML. (A) CFI-400945 treatment induced senescence (increased staining of senescence-associated [SA] β-galactosidase) in K052, THP-1, and TP53-mutant knockin ML2 but not ML2 on day 2 (n = 3). (B) CFI-400945 treatment induced senescence in primary TP53-mutated AML, compared with vehicle control on day 6 (n = 5). (C) Representative immunofluorescence images of DAPI (4′,6-diamidino-2-phenylindole; blue), α-tubulin (green), and pericentrin (purple) staining in K052 upon CFI-400945 treatment on day 2. (D) CFI-400945 treatment increased the number of centrosomes in K052, THP-1, ML-2, and TP53-mutant knockin ML2 on day 2 (n = 3). (E) Representative time-lapse microscopy images showing defective cell division of K052 upon CFI-400945 treatment on day 2. (F) CFI-400945 treatment increased the number of cells with cytokinesis failure in K052 and THP-1 on day 2 from time-lapsed microscopy for 18 hours (n = 3). (G) CFI-400945 treatment (10 nM) induced polyploidy in K052, THP-1, and TP53-mutant knockin ML2 but not in ML2 (n = 3). (H) PLK4-knockdown by shRNA resulted in polyploidy in K052 (n = 3). (I) CHI-400945 treatment (10 nM) induced the formation of cytoplasmic chromatin in K052 but not in MOLM-13 or ML2 (n = 3). (J-N) CFI-400945 treatment (10 nM) increased the mitochondrial mass (J), reactive oxygen species production (K), and apoptosis (L); reduced proliferation (M); and induced DNA damage (by western blot) (N) in both T2N and polyploidy subpopulation of K052 at day 2 (n = 3). (O-P) CFI-400945 treatment (10 nM) increased the formation of cytoplasmic chromatin (O) and senescence in polyploidy subpopulation of K052 (P) on day 2 (n = 3). (Q) T2N, T4N, and treated polyploidy cell of K052 failed to proliferate compared with control arms (n = 3). (R) On day 12, PLK4-knockout by CRISPR/Cas9 significantly suppressed leukemia growth in K052 and THP-1 (n = 3). (S) On day 12, CFI-400945 treatment significantly suppressed leukemia growth in K052, THP-1, and TP53-mutant knockin ML2 (n = 3). (T) CFI-400945 reduced clonogenicity of K052 and THP-1 on day 14 (n = 3). (U) CFI-400945 treatment reduced leukemia growth in TP53-mutated primary AML (n = 10) but not normal human cord blood CD34⁺ cells on day 6 (n = 5). (V) CFI-400945 had no effect on clonogenicity of normal human cord blood CD34⁺ cells on day 14 (n = 3). D259N: knockin of TP53-D259N mutant. R280K: knockin of TP53-R280K mutant. In panels A-I, cell numbers were obtained by manual counting based on stained images or videos or by flow cytometry. In panels J-Q, 2N, 4N, and > 4N refer to diploid (2N), tetraploid (4N), and polyploid (> 4N) cells in the control (C) or treatment (T) arm. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant.