Histone modification in TP53-mutated AML upon PLK4 inhibition. (A) Volcano plots indicating differentially expressed genes (DEG: log2FC > 1 or < −1, adjusted P value < .05) for each sample pair on day 2 post CFI-400945 treatment (20 nM). 524/413 and 158/406 DEGs were found upregulated/downregulated upon CFI-400945 treatment at T2N vs C2N and T4N vs C4N comparison, respectively. (B) GSEA showed negative enrichment of H3K27me3 and PRC2 targets upon CFI-400945 treatment (20 nM) compared with vehicle control on day 2, when 2N populations were analyzed. (C) Schematic diagram on the interaction between PLK4 and PRMT5 and the subsequent histone methylation via EZH2. (D) CFI-400945 (CFI, 10 nM) and centrinone-B (Cen, 10 nM) increased global H3K27me3 normalized to total histone H3 on day 2, compared with vehicle control (supplemental Figure 3E) (n = 3). (E) CFI-400945 (10 nM) increased global H3K27me3 normalized to total histone H3 in T2N, T4N, and polyploidy subpopulation of K052 on day 2, compared with vehicle control (supplemental Figure 3F) (n = 3). (F-G) Representative immunoblot images (F) and statistics (G) showing the coimmunoprecipitation of PLK4 and PRMT5. CFI-400945 (10-20 nM) (C10 and C20) suppressed the phosphorylation of both PLK4 and PRMT5, whereas the PRMT5 inhibitor, GSK591 (5-10 μM) (G5 and G10), only suppressed the phosphorylation of PRMT5, compared with vehicle control (n = 3). (H) Increase in global H3K27me3 upon CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment was rescued by PRC2 inhibitors, A395 (200 nM) or EED226 (500 nM) (n = 3). (I) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment increased O-GlcNAcylation in K052 on day 2, compared with vehicle control. CFI-400945 and GSK591 treatment increased O-GlcNAcylation in ML2 on day 1 and day 4, respectively. The increase was rescued by OGT inhibitor, OSMI-1 (10 μM) (OGTi) (n = 4). (J) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment increased the protein expression of EZH2, normalized by GAPDH. Increases in EZH2 and H3K27me3 were rescued by OSMI-1 (OGTi, 10 μM) (supplemental Figure 3H) (n = 3). (K) Heat map summarizing the RT-QPCR analysis upon CFI-400945 treatment in K052. It showed downregulation of gene expression in K052 upon CFI-400945 treatment (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) on day 2, compared with vehicle control. The downregulation of gene expression was rescued by EED226 (PRC2i, 500 nM) or OSMI-1 (OGTi, 10 μM) (supplemental Figure 3I) (n = 5). (L) Heat map summarizing the H3K27me3 ChIP-PCR analysis upon CFI-400945 treatment (PLK4i, 10 nM) in K052. It showed increased H3K27me3 enrichment of the promoter regions of the downregulated genes on day 2, normalized to vehicle control. The increase in H3K27me3 could be rescued by EED226 (PRC2i, 500 nM) or OSMI-1 (OGTi, 10 μM) (supplemental Figure 3J) (n = 3). (M) CFI-400945 (PLK4i, 10 nM) treatment increased DNA damage in K052 that could be rescued by EED226 (PRC2i, 500 nM) (supplemental Figure 3K) (n = 3). (N) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment induced apoptosis in K052 that could be rescued by EED226 (PRC2i, 500 nM) (n = 3). (O) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment suppressed proliferation in K052 that could be rescued by EED226 (PRC2i, 500 nM) (n = 3). D259N: knockin of TP53-D259N mutant. R280K: knockin of TP53-R280K mutant. ∗P <.05; ∗∗P < .01; ∗∗∗P < .001.

Histone modification in TP53-mutated AML upon PLK4 inhibition. (A) Volcano plots indicating differentially expressed genes (DEG: log2FC > 1 or < −1, adjusted P value < .05) for each sample pair on day 2 post CFI-400945 treatment (20 nM). 524/413 and 158/406 DEGs were found upregulated/downregulated upon CFI-400945 treatment at T2N vs C2N and T4N vs C4N comparison, respectively. (B) GSEA showed negative enrichment of H3K27me3 and PRC2 targets upon CFI-400945 treatment (20 nM) compared with vehicle control on day 2, when 2N populations were analyzed. (C) Schematic diagram on the interaction between PLK4 and PRMT5 and the subsequent histone methylation via EZH2. (D) CFI-400945 (CFI, 10 nM) and centrinone-B (Cen, 10 nM) increased global H3K27me3 normalized to total histone H3 on day 2, compared with vehicle control (supplemental Figure 3E) (n = 3). (E) CFI-400945 (10 nM) increased global H3K27me3 normalized to total histone H3 in T2N, T4N, and polyploidy subpopulation of K052 on day 2, compared with vehicle control (supplemental Figure 3F) (n = 3). (F-G) Representative immunoblot images (F) and statistics (G) showing the coimmunoprecipitation of PLK4 and PRMT5. CFI-400945 (10-20 nM) (C10 and C20) suppressed the phosphorylation of both PLK4 and PRMT5, whereas the PRMT5 inhibitor, GSK591 (5-10 μM) (G5 and G10), only suppressed the phosphorylation of PRMT5, compared with vehicle control (n = 3). (H) Increase in global H3K27me3 upon CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment was rescued by PRC2 inhibitors, A395 (200 nM) or EED226 (500 nM) (n = 3). (I) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment increased O-GlcNAcylation in K052 on day 2, compared with vehicle control. CFI-400945 and GSK591 treatment increased O-GlcNAcylation in ML2 on day 1 and day 4, respectively. The increase was rescued by OGT inhibitor, OSMI-1 (10 μM) (OGTi) (n = 4). (J) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment increased the protein expression of EZH2, normalized by GAPDH. Increases in EZH2 and H3K27me3 were rescued by OSMI-1 (OGTi, 10 μM) (supplemental Figure 3H) (n = 3). (K) Heat map summarizing the RT-QPCR analysis upon CFI-400945 treatment in K052. It showed downregulation of gene expression in K052 upon CFI-400945 treatment (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) on day 2, compared with vehicle control. The downregulation of gene expression was rescued by EED226 (PRC2i, 500 nM) or OSMI-1 (OGTi, 10 μM) (supplemental Figure 3I) (n = 5). (L) Heat map summarizing the H3K27me3 ChIP-PCR analysis upon CFI-400945 treatment (PLK4i, 10 nM) in K052. It showed increased H3K27me3 enrichment of the promoter regions of the downregulated genes on day 2, normalized to vehicle control. The increase in H3K27me3 could be rescued by EED226 (PRC2i, 500 nM) or OSMI-1 (OGTi, 10 μM) (supplemental Figure 3J) (n = 3). (M) CFI-400945 (PLK4i, 10 nM) treatment increased DNA damage in K052 that could be rescued by EED226 (PRC2i, 500 nM) (supplemental Figure 3K) (n = 3). (N) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment induced apoptosis in K052 that could be rescued by EED226 (PRC2i, 500 nM) (n = 3). (O) CFI-400945 (PLK4i, 10 nM) or GSK591 (PRMT5i, 5 μM) treatment suppressed proliferation in K052 that could be rescued by EED226 (PRC2i, 500 nM) (n = 3). D259N: knockin of TP53-D259N mutant. R280K: knockin of TP53-R280K mutant. ∗P <.05; ∗∗P < .01; ∗∗∗P < .001.

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