Attenuation of SRC Kinase Activity Augments PARP Inhibitor–mediated Synthetic Lethality in BRCA2-altered Prostate Tumors

BRCA2 deletion induces SRC kinase activation and increases sensitivity of prostate cancer cells to dasatinib. A and B, LNCaP cells were transduced with three different gRNAs targeting BRCA2. Cells infected with scrambled (SCR) gRNA were used as control. CAS9, total SRC, GAPDH, and RhoGDI served as loading controls. C, Levels of phosphorylated SRC at tyrosine 416 (pSRC Y416), total SRC, androgen receptor (AR), and E-cadherin were assessed by Western blotting in prostate cancer cell lines. GAPDH was used as loading control. D, Cells were treated with 0.3 and 3 μmol/L dasatinib for 7 days. The equivalent volume of DMSO was used as placebo treatment. After 7 days, cells were treated with 0.5 mg/mL MTT and micrographed in 40 × magnification (top). The bar graph shows the changes in cell growth percentage compared with scrambled gRNA- and DMSO-treated samples (bottom). P values were calculated by Student t test. E, LNCaP scramble control and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with indicated concentration of bosutinib (top) or saracatinib (bottom) for 7 days. The equivalent volume of DMSO was used as a placebo treatment. Cells were treated with 0.5 mg/mL MTT and the graphs represent cell viability (MTT count, % control). P values were calculated by Student t test. F, Indicated cells (LNCaP scramble control and BRCA2-gRNA 2) were counted and plated in 96-well plates (2,500 cells/well in 100-μL media) and treated with 0.1 and 1 μmol/L concentration of ipatasertib for 7 days. The equivalent volume of DMSO was used as a placebo treatment. Cells were treated with 0.5 mg/mL MTT, measured in a plate reader at 570 nm, and represented in the form of a bar graph. P values were calculated by Student t test. G, Samples from patients in TCGA Firehose Legacy cohort were divided into four quartiles based on levels of phospho-SRC at Y416, reverse-phase protein arrays (RPPA). Kaplan–Meier curves were used to compare disease-free survival. Log-rank test was performed to examine significance. H, Volcano plot shows genes that were altered in phospho-SRC Y416 high (top quartile, RPPA value) cases compared with samples with phospho-SRC Y416 low (bottom quartile, RPPA value). The bar graph to the right of the volcano plot shows the oncogenic pathways that were altered in phospho-SRC Y416–high versus -low localized prostate cancer tumors of TCGA Firehose Legacy cohort. Pathway analyses were performed using GSEA (c6 oncogenic signature). The altered (based on NES) pathways are shown.

Synergistic effect of dasatinib and PARPis on BRCA2-null prostate cancer cell viability. A and B, PC3M and LNCaP-abl cells were treated with indicated drugs alone or in combination for 4 days. Drug synergy was calculated using Chou-Talalay Method/CompuSyn. Graphs show the combination index plot for dasatinib (dasa) with olaparib (ola)/talazoparib (tala) combination obtained from CompuSyn (left). Tables show dose-dependent combination index and average effect (Fa) values (right). C and D, LNCaP scramble controls and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with dasatinib (0.3 μmol/L) and olaparib (3 μmol/L) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After indicated days, cells were treated with 0.5 mg/mL MTT. The graphs represent the inhibition of cell viability by combination treatment. P values were calculated by Student t test. E, MTT-treated LNCaP-BRCA2-gRNA 2 cells from D were photographed at 40× magnification. Representative images are shown. F, LNCaP-abl cells were counted and plated in 96-well plates (2,500 cells/well in 100 μL media). Cells were treated with bosutinib (0.3 μmol/L) and talazoparib (0.03 μmol/L) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After 7 days, cells were treated with 0.5 mg/mL MTT, measured in a plate reader at 570 nm, and represented as a bar graph. G, LNCaP scramble (SCR) controls and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with dasatinib (0.3 μmol/L) and cisplatin (0.1 μmol/L; dissolved in DMSF) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After the indicated number of days, cells were treated with 0.5 mg/mL MTT. The graph represents the inhibition of cell viability by single agent and combination treatment. P values were calculated by Student t test. H, MTT-treated cells from G (DMSO and combination treatment) were photographed at 40× magnification. Representative images are shown.

Dasatinib induces DNA damage in BRCA2-null prostate cancer cells. A, PC3M cells treated with dasatinib (0.3 μmol/L) for 24 hours. The equivalent volume of DMSO was used as a placebo treatment. Dot plots show the percentage of cells with phospho-ATM, phospho-H2A.X, and both phospho-ATM and phospho-H2A.X (left). Bar graphs compare the percentages of cells between DMSO- and dasatinib-treated conditions (± SD; right). DSB represents the percentage of cells with dual activation of ATM and H2A.X. Total DNA damage represents the percentage of cells with either activated ATM, activated H2A.X, or dual activation. P values were calculated by Student t test. B and C, LNCaP SCR-gRNA and BRCA2-gRNA2 cl7 cells were treated with dasatinib (0.3 μmol/L) for 24 hours. The equivalent volume of DMSO was used as a placebo treatment. Dot plots show the percentage of cells with phospho-ATM, phospho-H2A.X, and both phospho-ATM and phospho-H2A.X (left). Total DNA damage represents the percentage of cells with either activated ATM, activated H2A.X, or dual activation (right). P values were calculated by Student t test. D, Representative IHC images of paraffin-embedded sections of normal prostate from wild-type C57BL/6J mice and localized prostate tumors from PB-Tag mice. Sections were stained IHC with indicated antibodies (brown). Nuclei were costained with hematoxylin (blue). The micrographs were captured in 200 × magnification. Note that prostate tumors of PB-Tag mice exhibit higher DNA damage (increased phosphorylation of γH2AX) and Src activation (Src y416 phosphorylation) compared with normal mice prostate. E and F, TRAMP-C2 cells (2,500 cells/well in 100-μL media) were treated with dasatinib/bosutinib (0.3 μmol/L) and talazoparib (0.03 μmol/L) alone or in combination for 7 days. DMSO was used as a placebo treatment. Cell viability (± SD) was measured by MTT assay. G, TRAMP-C2 cells were treated with dasatinib (0.3 μmol/L) and cisplatin (0.1 μmol/L; dissolved in DMSF) alone or in combination for 5 and 8 days. The equivalent volume of DMSO was used as a placebo treatment. After indicated number of days, cells were treated with 0.5 mg/mL MTT. The graphs represent the inhibition of cell viability by single agent and combination treatment. P values were calculated by Student t test.

SRC activation leads to PARPi resistance. A, Viability (± SD) of 22RV1 cells treated with dasatinib (0.3 μmol/L) ± olaparib (3.0 μmol/L), as assessed by MTT assay. DMSO was used as a placebo control. B, Viability (± SD) of PC3M cells subjected to periodic treatment with talazoparib, followed by dasatinib, or vice versa, as assessed by MTT assay. DMSO was used as a placebo control. See Supplementary Fig. S5A for experimental schematic. P values were calculated by Student t test. C and D, PC3M cells were incubated in either olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) supplemented media for 15 days to develop partial PARPi resistance. PARPi-resistant cells were counted and treated with dasatinib (see Supplementary Fig. S5B for experimental schematic). Cell viability was assessed by MTT assay. Viability (± SD) of PC3M cells after 7 days of dasatinib treatment is shown in C. Representative images of MTT-treated cells are shown in D. P values were calculated by Student t test. E, SRC or scrambled SMARTpool siRNA-transfected PC3M cells were cultured in olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) supplemented medium for 4 days posttransfection. The equivalent volume of DMSO was used as a placebo treatment. Cell viability (± SD) was measured by MTT assay. P values were determined by Student t test. F, PC3M cells that transiently overexpressed Y527F SRC (constitutively active SRC) were treated with olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) for 7 days. Control cells were transfected with GFP-expressing empty vector. Viability (± SD) of cells at 7 days, as determined by MTT assay is shown. P values determined by Student t test.