Co-Targeting of DTYMK and PARP1 as a Potential Therapeutic Approach in Uveal Melanoma

Abstract

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with no standardized treatment for advanced disease. Based on preliminary bioinformatical analyses DTYMK and PARP1 were selected as potential therapeutic targets. High levels of both proteins were detected in uveal melanoma cells and correlated with increased tumor growth and poor prognosis. In vitro tests on MP41 (BAP1 positive) and MP46 (BAP1 negative) cancer cell lines using inhibitors pamiparib (PARP1) and Ymu1 (DTYMK) demonstrated significant cytotoxic effects. Combined treatment had synergistic effects in MP41 and additive in MP46 cell lines, reducing cell proliferation and inhibiting the mTOR signaling pathway. Furthermore, the applied inhibitors in combination decreased cell motility and migration speed, especially for BAP1-negative cell lines. Our hypothesis of the double hit into tumoral DNA metabolism as a possible therapeutic option in uveal melanoma was confirmed since combined targeting of DTYMK and PARP1 affected all tested cytophysiological parameters with the highest efficiency. Our in vitro findings provide insights into novel therapeutic avenues for managing uveal melanoma, warranting further exploration in preclinical and clinical settings.

Keywords: BAP1; DTYMK; PARP1; immunohistochemistry; inhibitors; intraocular tumor; uveal melanoma.

Figure 1

In silico analysis of DTYMK and PARP1 RNA expression in UM. (A) Kaplan–Meier estimates of survival probability for UM patients on days from cancer diagnostics grouped by optimal cut point of DTYMK RNA expression level. (B) Kaplan–Meier estimates of survival probability for UM patients on days from cancer diagnostics grouped by optimal cut point of PARP1 RNA expression level. (C) UMAP visualization of MLANA, SOX10, BAP1, DTYMK, and PARP1 expression in UVM tumor tissues (GSE139829). (D) Dot plot summarizing DTYMK expression in 11 UM samples included in GSE139829 dataset. Dot radius represents percent of tumors expressing given gene. Color intensity of dots reflects level of expression of given gene. (E) Dot plot summarizing PARP1 expression in 11 UM samples included in GSE139829 dataset. Dot radius represents percent of tumors expressing given gene. Color intensity of dots reflects level of expression of given gene.

Figure 2

Immunohistochemistry of DTYMK and PARP1 in uveal melanoma patients and correlation between DTYMK and PARP1 H-scores. (A) Low cytoplasmic DTYMK expression in uveal melanoma cells (200×). (B) High DTYMK immunoreactivity in neoplastic cells. Tumoral vessels are negative (yellow arrows) (200×). (C) Lack of PARP1 expression in tumoral cells (200×). (D) Enhanced nuclear PARP1 reactivity in uveal melanoma cells (200×). (E) Plot showing DTYMK H-score values versus PARP1 H-score values (every point is a single patient). There is a very weak correlation (Pearson’s r = 0.2, p-value 0.013). (F) Dependency between DTYMK H-score and BAP1 box plots show the distribution of DTYMK H-score separated by BAP1 immunostaining. Wilcoxon rank-sum test (p-value = 0.23) shows no significant difference between values of DTYMK for patients with loss of nuclear expression and those with retained nuclear expression of BAP1 (F). (G) Kaplan–Meier analysis revealed a negative impact of enhanced expression of DTYMK as a single marker and (H) in combination with PARP1 on shorter overall survival of uveal melanoma patients. Patients with overexpression of both proteins were characterized by the worst long-term prognosis.

Figure 3

Detection of PARP1 and DTYMK and analysis of their localization in MP41 and MP46 cells. (A) Detection of PARP1 and DTYMK. A total of 15 µg of protein was loaded onto a single lane. Membranes were probed with specific antibodies to PARP1 and DTYMK. GAPDH was used as a sample loading control. (B,C) Analysis of PARP1 and DTYMK localization in MP41 (B) and MP46 (C) cells. Phalloidin-conjugated Alexa Fluor™ 568 labeled F-actin (yellow). Hoechst 33242 staining was used to visualize cells’ nuclei (blue), whereas MitoTracker™ labeled mitochondria (red). Cells were also incubated with antibodies directed to PARP1 and DTYMK and next with secondary antibodies conjugated with fluorophores: Alexa Fluor™ 647 (red) and Alexa Fluor™ 488 (green). The scale bar represents 20 µm. Arrows indicate particularly evident colocalization of DTYMK and mitochondria.

Figure 4

Cytotoxic effect of pamiparib and Ymu1 on MP41 and MP46 cell viability and its IC50 values. (A–C) Cytotoxic effect of pamiparib on MP41 (A) and MP46 (B) cells viability and its IC50 value in the range 10–50 µM (C). The same amount of DMSO was used for the vehicle control, which was present when cells were treated with 50 µM pamiparib. (D–G) Cytotoxic effect of Ymu1 on MP41 (D) and MP46 (E) cells viability and its IC50 values. (F,G, respectively) in the range 2–30 µM. The same amount of DMSO was used for the vehicle control, which was present when cells were treated with 30 µM Ymu1. Cells were incubated with inhibitors for 72 h and then subjected to the XTT assay. Results are presented as a mean (% of vehicle control) ± SD of four replicates. Asterisks above the bars express significance vs. vehicle control: p ≤ 0.01 (**) and p ≤ 0.0001 (****).

Figure 5

Cytotoxic effect of pamiparib and Ymu1 combinations on MP41 (A) and MP46 (B) cells viability and evaluation of synergy of examined drugs’ combinations for MP41 (C) and MP46 cells (D) calculated based on the results shown in (A,B). (A,B) For the vehicle control, the same amount of DMSO was used, which was present when cells were treated with 25 µM pamiparib (P) and 15 µM Ymu1 (Y). Cells were incubated with inhibitors for 72 h and then subjected to the XTT assay. The combinations of inhibitors selected for further studies are marked in green. Results are presented as a mean (% of vehicle control) ± SD of four replicates. Asterisks above the bars express significance vs. vehicle control: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). (C,D) Synergy scores’ interpretation: a score less than −10 indicates antagonistic interaction, a score between −10 and 10 implies additive interaction, and scores larger than 10 are typical for synergistic interactions. Analysis was performed using SynergyFinder software [27].

Figure 6

EdU incorporation for MP41 (A) and MP46 cells (B) after administration of selected tested inhibitor concentrations and the effect of pamiparib and Ymu1 on the amount and activity of S6 (C,D). (A,B) The same amount of DMSO was used for the vehicle control, which was present when cells were treated with 25 µM pamiparib (P) and 15 µM Ymu1 (Y). A total of 48 h after the addition of inhibitors to cells, EdU reagent was added to the cells for the next 24 h. After that, the cells were processed according to the manufacturer’s protocol to assess the amount of incorporated EdU. Results are presented as mean ± SD of three replicates. Asterisks above the bars express significance vs. vehicle control; p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****). (C,D) A total of 15 µg of proteins was loaded onto a single lane. Cells were incubated with indicated concentrations of inhibitors for 24 or 48 h. The same amount of DMSO was used for the vehicle control, which was present when cells were treated with 25 µM pamiparib (P) and 15 µM Ymu1 (Y). Membranes were probed with specific antibodies to total and phosphorylated forms of S6. Representative replicates are shown.

Figure 7

Distance, velocity, and directionality were calculated from spontaneous migration assay for MP41 (A–C) and MP46 (D–F) cells after treatment with selected inhibitors. The experiment was run for 72 h using the IncuCyte^® system. Images were collected every two hours, and after the experiment was ready, the trajectories of movement of single cells were prepared (40 cells per condition) (Figure S3). Based on the trajectories, distance, velocity, and directionality of motile cells were assessed (n = 3). The same amount of DMSO was used for the vehicle control, which was present when cells were treated with 25 µM pamiparib (P) and 15 µM Ymu1 (Y). Results are presented as mean ± SD. Asterisks above the bars express significance vs. vehicle control; p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) p ≤ 0.0001 (****).