Figure 1.
Differential effects of PLK4 inhibition on TP53 wild-type and mutated AML. (A) The expression (Beat AML and Leucegene databases) and dependency (Depmap database) profile of 310 druggable genes (DGIdb), which were differentially expressed in TP53-mutated AML when compared with those with normal karyotype. Each dot represents 1 gene. Red color indicates genes that were upregulated and essential in TP53-mutated AML, and PLK4 was 1 of them. (B) In humans, public database (Bloodspot) showed the lowest PLK4 messenger RNA (mRNA) expression in mature cells compared with hematopoietic stem and intermediate cells, whereas other PLK proteins showed fluctuating expression during hematopoietic differentiation. (C) Reverse transcription QPCR (RT-QPCR) analysis showed upregulated PLK4 expression in human AML (n = 24) compared with normal peripheral blood mononuclear cells (n = 5). (D-E) Higher PLK4 mRNA expression in TP53-mutated human AML cell line (D) (n = 9) and primary AML cell line (E) (n = 24). (F-G) On day 4, PLK4-knockout by CRISPR/Cas9 (F) or PLK4 inhibitor (CFI-400945) (G) significantly suppressed leukemia growth in TP53 wild-type AML cell line ML2 but less so in TP53-mutated AML cell line K052 and THP1 (n = 3). (H) On day 4 of treatment, CFI-400945 (10 nM) treatment induced more suppression in wild-type (WT) than the TP53 mutant (Mut) AML cell lines (n = 9) as shown by a small area under curve (AUC). (I) CFI-400945 treatment induced more suppression in ML-2 than the TP53-mutant derivatives. (J) CFI-400945 treatment induced more apoptosis in ML-2 compared with K052, THP1, and TP53-mutant knockin ML2 on day 1 (n = 3). (K) CFI-400945 treatment (20 nM) induced higher level of apoptosis in TP53 WT primary AML compared with TP53 Mut primary AML on day 1 (n = 8). (L) DNA damage, as enumerated by γH2AX based on western blot and ImageJ analysis, was observed in both TP53 wild-type and mutated ML2 cell line (n = 3). (M) Representative western blot showing CFI-400945 (10-20 nM) activated p53 signaling in ML2 but not in K052. The number above the blots indicate the quantification of p53 and MDM2 normalized by GAPDH protein. Ø indicates a nonspecific band of MDM2. HSC, hematopoietic stem cell.

Differential effects of PLK4 inhibition on TP53 wild-type and mutated AML. (A) The expression (Beat AML and Leucegene databases) and dependency (Depmap database) profile of 310 druggable genes (DGIdb), which were differentially expressed in TP53-mutated AML when compared with those with normal karyotype. Each dot represents 1 gene. Red color indicates genes that were upregulated and essential in TP53-mutated AML, and PLK4 was 1 of them. (B) In humans, public database (Bloodspot) showed the lowest PLK4 messenger RNA (mRNA) expression in mature cells compared with hematopoietic stem and intermediate cells, whereas other PLK proteins showed fluctuating expression during hematopoietic differentiation. (C) Reverse transcription QPCR (RT-QPCR) analysis showed upregulated PLK4 expression in human AML (n = 24) compared with normal peripheral blood mononuclear cells (n = 5). (D-E) Higher PLK4 mRNA expression in TP53-mutated human AML cell line (D) (n = 9) and primary AML cell line (E) (n = 24). (F-G) On day 4, PLK4-knockout by CRISPR/Cas9 (F) or PLK4 inhibitor (CFI-400945) (G) significantly suppressed leukemia growth in TP53 wild-type AML cell line ML2 but less so in TP53-mutated AML cell line K052 and THP1 (n = 3). (H) On day 4 of treatment, CFI-400945 (10 nM) treatment induced more suppression in wild-type (WT) than the TP53 mutant (Mut) AML cell lines (n = 9) as shown by a small area under curve (AUC). (I) CFI-400945 treatment induced more suppression in ML-2 than the TP53-mutant derivatives. (J) CFI-400945 treatment induced more apoptosis in ML-2 compared with K052, THP1, and TP53-mutant knockin ML2 on day 1 (n = 3). (K) CFI-400945 treatment (20 nM) induced higher level of apoptosis in TP53 WT primary AML compared with TP53 Mut primary AML on day 1 (n = 8). (L) DNA damage, as enumerated by γH2AX based on western blot and ImageJ analysis, was observed in both TP53 wild-type and mutated ML2 cell line (n = 3). (M) Representative western blot showing CFI-400945 (10-20 nM) activated p53 signaling in ML2 but not in K052. The number above the blots indicate the quantification of p53 and MDM2 normalized by GAPDH protein. Ø indicates a nonspecific band of MDM2. HSC, hematopoietic stem cell.

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