Combination of PSMA targeting alpha-emitting radioligand [212Pb]Pb-AB001 with BET bromodomain inhibitors in in vitro prostate cancer models

Abstract

The alpha-emitting radioligand [²¹²Pb]Pb-AB001, targeting prostate-specific membrane antigen (PSMA), is a promising therapy approach for prostate cancer. Bromodomain and extra-terminal (BET) protein inhibitors, such as AZD5153 and JQ1, disrupt oncogenic transcriptional programs by altering chromatin structure. This study evaluated whether BET inhibition enhances the efficacy of radioligand therapy. Cytotoxic effects of [²¹²Pb]Pb-AB001 alone and in combination with BET inhibitors were assessed in 2D monolayers and a 3D spheroid model of PSMA-positive C4-2 prostate cancer cells. Cell viability, cell cycle alterations, and DNA damage were assessed using viability assays and flow cytometry. Spheroid growth and viability were assessed by fluorescence microscopy. AZD5153 was more potent than JQ1 in reducing cell proliferation. [²¹²Pb]Pb-AB001 induced activity- and time-dependent cytotoxicity with a delayed apoptotic response. BET inhibitors induced G1 arrest, while [²¹²Pb]Pb-AB001 caused G2 arrest. Combination treatment reduced cell viability in an additive manner but did not further affect cell cycle distribution or increase apoptosis compared with [²¹²Pb]Pb-AB001 alone. γH2AX staining in 2D models showed an activity- and time-dependent increase in DNA damage at 1, 3 and 6 days post-treatment with [²¹²Pb]Pb-AB001. BET inhibitors alone induced minimal γH2AX, and combination treatments did not enhance DNA damage beyond [²¹²Pb]Pb-AB001 alone. In 3D spheroids, combination treatment led to synergistic growth suppression. In conclusion, these findings indicate that therapeutic inhibition of BET bromodomain in combination with the alpha-emitting radioligand [²¹²Pb]Pb-AB001 could significantly enhance tumour control and should be further evaluated in metastatic prostate cancer models.

Keywords: Bromodomain and extra-terminal proteins; Cell cycle; Combination treatment; Targeted alpha therapy; Targeted radionuclide therapy.

Fig. 1

Effects of [²¹²Pb]Pb-AB001 and BET inhibitors alone and in combination on metabolic activity in 2D models. a C4-2 cells were treated with AZD5153 and JQ1 alone for 3 or 7 days continuously. b For radioligand treatment, [²¹²Pb]Pb-AB001 (RL) was added for 4 h, followed by media replacement. For combination treatment, cells were pre-treated with BET inhibitors for 3–4 h, followed by 4-h RL exposure, and then maintained in BET inhibitor-containing medium for 7 days. The metabolic cell activity was assessed using the CellTiter-Glo® Luminescent Cell Viability assay, and the results are presented as relative luminescence units normalized to the untreated control group (set to 1). Data are presented as a mean of three independent experiments, each performed in triplicate

Fig. 2

Effects of [²¹²Pb]Pb-AB001 and BET inhibitors on growth and viability of C4-2 spheroids. C4-2 spheroids were treated with AZD5153 or JQ1 continuously for 21 days. For radioligand treatment, [²¹²Pb]Pb-AB001 was administered for 4 h, followed by medium replacement. For combination treatment, spheroids were pre-treated with BET inhibitors for 3–4 h, exposed to [²¹²Pb]Pb-AB001 for 4 h, and then maintained in medium containing the respective BET inhibitor for the remainder of the 21-day period. a Cross-sectional area measurements of C4-2 spheroids over time, b Representative images and c the doubling times of treated spheroids. Images were captured using 4 × magnification, with a scale bar of 200 µm. The images shown are representative of a single experiment. Live–dead staining was performed using fluorescein diacetate (FDA) and propidium iodide (PI). FDA is a membrane-permeable dye that is hydrolyzed by intracellular esterases in viable cells to produce green fluorescence. PI is membrane-impermeable and intercalates into DNA of membrane-compromised (dead or dying) cells, emitting red fluorescence. Spheroids were stained on day 21 post-treatment to assess viability distribution

Fig. 3

Effects of [²¹²Pb]Pb-AB001 combination with BET inhibitors on growth and viability of C4-2 spheroids. C4-2 spheroids were treated with AZD5153 or JQ1 continuously for 21 days. For radioligand treatment, [²¹²Pb]Pb-AB001 was administered for 4 h, followed by medium replacement. For combination treatment, spheroids were pre-treated with BET inhibitors for 3 – 4 h, exposed to [²¹²Pb]Pb-AB001 for 4 h, and then maintained in medium containing the respective BET inhibitor for the remainder of the 21-day period. a Cross-sectional area measurements of C4-2 spheroids over time following treatment with [²¹²Pb]Pb-AB001 (RL), AZD5153 or JQ1 alone and in combination. b Fold-change in spheroid cross-sectional area, normalized to control, after combination treatments of 25 nM AZD5153 with 1 kBq/mL [²¹²Pb]Pb-AB001 on day 21 (top) and 100 nM JQ1 with 2 kBq/mL [²¹²Pb]Pb-AB001 on day 14 (bottom), with vehicles representing untreated controls, RL = treatment with [²¹²Pb]Pb-AB001, AZD5153 or JQ1 for BET inhibitors, RL + AZD5153 or JQ1 for combination treatments, and "additive" for calculated theoretical additive effects according to the Bliss model. P-value indicates 2 -tailed t-test against the observed values for combination treatment. p < 0.05 indicates synergism. c Cross-sectional area plotted against activity for combination treatments on days 14 and 21 with [²¹²Pb]Pb-AB001 alongside 25 nM AZD5153 or 100 nM JQ1. d Representative images of spheroid morphology captured on day 21. Live–dead staining was performed using fluorescein diacetate (FDA) and propidium iodide (PI). FDA is a membrane-permeable dye that is hydrolyzed by intracellular esterases in viable cells to produce green fluorescence. PI is membrane-impermeable and intercalates into DNA of membrane-compromised (dead or dying) cells, emitting red fluorescence. Spheroids were stained on day 14 and 21 post-treatment to assess viability distribution

Fig. 4

Effect of [²¹²Pb]Pb-AB001 and BET inhibitors on cell cycle phase responses and DNA damage. C4-2 cells were treated with AZD5153 or JQ1 continuously for 1, 3 or 6 days. For radioligand treatment, [²¹²Pb]Pb-AB001 was administered for 4 h, followed by medium replacement. For combination treatment, cells were pre-treated with BET inhibitors for 3–4 h, exposed to [²¹²Pb]Pb-AB001 for 4 h, and then maintained in medium containing the respective BET inhibitor for the remainder of period. a Cell viability, b DNA damage and c, d cell cycle distribution in C4-2 cells post-treatment were analysed by flow cytometry. a Viability assessed using Annexin V FITC and propidium iodide (PI) staining on days 1, 3 and 6 to distinguish viable, apoptotic and necrotic cells. b DNA damage was assessed by comparing the fold change in the geometrical mean fluorescence intensity of γH2AX-positive cells between untreated controls and treated groups at 1, 3, and 6 days post-treatment. The red line indicates the untreated control (c) Cell cycle distribution following continuous treatment with BET inhibitors (10 nM AZD5153 or 125 nM JQ1) and a 4-h exposure to [²¹²Pb]Pb-AB001, individually and in combination. d Mitotic activity evaluated 1 h after a 4-h incubation with [²¹²Pb]Pb-AB001. Data represent mean ± SD from three independent experiments