Figure 4. SMYD2 inhibition improves the therapeutic response of breast cancer to PI3K/AKT inhibitors in vitro and in vivo.

(A) MTT proliferation assays with doxycycline-inducible SMYD2-knockdown MCF7 cell lines using a dose response of 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, representative shown, mean, SD).

(B) MTT proliferation assays with doxycycline-inducible SMYD2-knockdown T47D cell lines using a dose response of alpelisib for a treatment period of 5 days. p values are indicated as in (A) (n = 3 biological replicates, representative shown, mean, SD).

(C) MTT assays with a shSMYD2 MCF7 cell line treated with fulvestrant, alpelisib, and the combination for a treatment period of 10 days. Drug concentrations were as follows: fulvestrant 100 nM, alpelisib 1 μM. Indicated p values were generated with an unpaired, two-tailed Student’s t test (n = 3 biological replicates, representative shown).

(D) MTT proliferation assays with shSMYD2 MCF7 cell lines using a dose response of the AKT inhibitor capivasertib for a treatment period of 5 days. p values are indicated as in (A) (n = 3 biological replicates, representative shown, mean, SD).

(E) MTT assays in MCF7 cells to assess the combination of a single dose of SMYD2 catalytic inhibitors AZ506 (10 μM) and AZ39 (10 μM) with a dose response of alpelisib for a treatment period of 5 days. p values are indicated as in (A) (n = 3 biological replicates, representative shown, mean, SD).

(F) MTT assays in T47D cells to assess the combination of a single dose of SMYD2 catalytic inhibitors AZ506 (5 μM) and AZ39 (10 μM) with a dose response of alpelisib for a treatment period of 5 days. p values are indicated as in (A) (n = 3 biological replicates, representative shown, mean, SD).

(G) CTG assays of PDX-derived organoids treated for 14 days with 5 μM AZ506, alpelisib 1 μM, fulvestrant 1 μM, or the combination of AZ506 and alpelisib. Statistical analysis was conducted using an unpaired, two-tailed Student’s t test (n = 3 biological replicates, mean, SEM).

(H) Images using 4× magnification microscopy of organoids treated as described in (G). Scale bar: 200 μm.

(I) Xenograft study using the doxycycline-inducible shSMYD2 cell line. Mice were treated daily with vehicle or alpelisib (25 mg/kg) for the indicated time course. p values were calculated using a two-sided Mann-Whitney U test (n = 5 per group, mean, SEM).

(J) Western blot of xenograft tumor samples validating doxycycline-induced knockdown of SMYD2 and catalytic inhibition of PI3Kα as measured by AKT pS473.

(K) Kaplan-Meier curves showing survival of TCGA ER+ breast cancer patients stratified by the presence or absence of SMYD2 amplification. p value calculated using log rank test.