Figure 5. SMYD2-mediated methylation of KMT2D affects the therapeutic response of breast cancer to PI3K/AKT inhibitors.

(A) PrestoBlue assays with K1330A CRISPR KI clones compared to unmodified MCF7 cells using a dose response of the PI3K inhibitor alpelisib for a treatment period of 5 days. Significance at individual doses was assessed using an unpaired, two-tailed Student’s t test. p values are indicated as follows: *p < 0.05,**p < 0.01,***p < 0.001, and ****p < 0.0001 (n = 3 biological replicates, mean, SEM).

(B) PrestoBlue assays with K1330A CRISPR KI clones compared to unmodified MCF7 cells using a dose response of the AKT inhibitor capivasertib for a treatment period of 5 days (n = 3 biological replicates, mean, SEM).

(C) PrestoBlue assays using K1330A CRISPR KI clones compared to unmodified MCF7 cells to assess the effect of fulvestrant, alpelisib, and the combination. All drugs were used at 1 μM concentration. Treatments were conducted for 7 days and statistical analysis was conducted using a two-tailed unpaired Student’s t test (n = 3 biological replicates, mean, SEM).

(D) Xenograft study using unmodified MCF7 and the K1330A CRISPR KI Clone-2. Tumor volume was measured for the indicated time course. p values were calculated using a two-sided Mann-Whitney U test (n = 15 per group, mean, SEM).

(E) Heatmap of RNA-seq conducted in the K1330A CRISPR KI clones with and without alpelisib treatment. Genes were filtered using an adjusted p value <0.1 and the heatmap was generated using k-means clustering. The overlapped differential genes for the two K1330A clones were used for the analysis. The number of genes in each cluster (1–5) is indicated on the left (n = 3 technical replicates).

(F) Heatmap showing the differential expression of genes regulated by alpelisib treatment in the K1330A CRISPR KI clones compared to the unmodified MCF7 control. (total of 436 genes, 137 differentially expressed with log₂fold change >0.5 and adjusted p value <0.1) Cells were treated with 1 μM alpelisib for 24 h before collection.

(G) RT-qPCR analysis of ER target genes in MCF7 K1330A CRISPR KI clones compared to wild type, treated with ethanol or 100 nM estradiol (E2) for 24 h. p values were generated using a two-tailed Student’s t test (n = 3 biological replicates, representative shown, mean, SEM).

(H) Venn diagrams indicating the overlapped genes from the MCF7 shSMYD2 RNA-seq and the MCF7 K1330A CRISPR KI RNA-seq in each treatment condition (DMSO or alpelisib). Differential genes from each RNA-seq dataset were used for the knockdown (shSMYD2 versus control) and for the KI (overlap between K1330A clones versus wild type), filtered by adjusted p value <0.1.