Therapeutic inhibition of ATR in differentiated thyroid cancer

Abstract

Ataxia telangiectasia and Rad3-related protein (ATR) is a critical component of the DNA damage response and a potential target in the treatment of cancers. An ATR inhibitor, BAY 1895344, was evaluated for its use in differentiated thyroid cancer (DTC) therapy. BAY 1895344 inhibited cell viability in four DTC cell lines (TPC1, K1, FTC-133, and FTC-238) in a dose-dependent manner. BAY 1895344 treatment arrested DTC cells in the G2/M phase, increased caspase-3 activity, and caused apoptosis. BAY 1895344 in combination with either sorafenib or lenvatinib showed mainly synergistic effects in four DTC cell lines. The combination of BAY 1895344 with dabrafenib plus trametinib revealed synergistic effects in K1 cells that harbor BRAFV600E. BAY 1895344 monotherapy retarded the growth of K1 and FTC-133 tumors in xenograft models. The combinations of BAY 1895344 plus lenvatinib and BAY 1895344 with dabrafenib plus trametinib were more effective than any single therapy in a K1 xenograft model. No appreciable toxicity appeared in animals treated with either a single therapy or a combination treatment. Our findings provide the rationale for the development of clinical trials of BAY 1895344 in the treatment of DTC.

Keywords: ATR; BAY 1895344; combination therapy; dabrafenib; differentiated thyroid cancer; lenvatinib; sorafenib; trametinib.

Figure 1.. BAY 1895344 reduced cell survival and increased p-H2AX (Ser139) expression in four DTC cell lines.

(A) Cell viability was measured in DTC cells treated with a series of six two-fold dilutions of BAY 1895344 starting from 1000 nmol/L. Dose–response curves were obtained after a 4-day treatment in BHP7-13, K1, FTC-133, and FTC-238 cell lines using LDH assays. (B) The median-effect dose (IC₅₀) of BAY 1895344 on day 4 was determined for four DTC cell lines using CompuSyn software. (C) The levels of p-H2AX (Ser139) were evaluated by immunoblot in BHP7-13, K1, FTC-133, and FTC-238 cells treated with placebo or BAY 1895344 (500 nmol/L) for 24 h and 48 h.

Figure 2.. BAY 1895344 induced G2/M cell cycle arrest and decreased cells in mitosis.

(A) Cell cycle analysis using flow cytometry was done to measure DNA content in BHP7-13 cells treated with placebo or BAY 1895344 (500 nmol/L) for 48 h. (B) Statistical analysis reveal BAY 1895344 treatment significantly arrested cells in the G2/M phase in BHP7-13, K1, FTC-133, and FTC-238 cell lines. (C) BHP7-13 cells were incubated with BAY 1895344 (500 nmol/L) or placebo for 48 h and stained with fluorescent antibodies targeting DAPI (blue), p-Histone H3 (Ser10) (red), and α-tubulin (green). Mitotic chromosomes were visualized using fluorescence microscopy. Mitotic cells at prophase (white arrowhead), prometaphase (yellow arrow), metaphase (white arrow), and anaphase (yellow arrowhead) were identified. (D) The proportion of cells in mitosis was assessed after treatment with BAY 1895344 (500 nmol/L) or placebo for 48 h in BHP7-13, K1, FTC-133, and FTC-238 cell lines. Cells were stained with DAPI, and chromosome features were assessed using immunofluorescence microscopy. The mitotic index was calculated with a minimum of 886 cells counted from 10-15 different fields for each condition. BAY 1895344 significantly decreased the percentage of BHP7-13, K1, FTC-133, and FTC-238 cells in mitosis. Scale bar, 20 μm.

Figure 3.. BAY 1895344 treatment induced caspase-3 activity and stimulated apoptosis in DTC cells.

(A) Caspase-3 activity was determined by a fluorometric assay kit in BHP7-13, K1, FTC-133, and FTC-238 cells treated with BAY 1895344 (500 nmol/L) or placebo for 48 h. BAY 1895344 significantly activated caspase-3 activity in four DTC cell lines. (B) BHP7-13, K1, FTC-133, and FTC-238 cells were treated with BAY 1895344 (500 nmol/L) or placebo for 48 h and early apoptosis was assessed using annexin V FITC kit to detect Annexin V-positive/PI-negative staining by flow cytometry. (C) Statistical analyses revealed BAY 1895344 treatment significantly increased the proportion of early apoptotic cells in four DTC cell lines. (D) Sub-G1 hypoploid apoptotic cells were identified by assessing the DNA content of 10,000 events for each sample using flow cytometry in BHP7-13, K1, FTC-133, and FTC-238 cells treated with BAY 1895344 (500 nmol/L) or vehicle for 48 h. (E) BAY 1895344 significantly increased the proportion of sub-G1 apoptotic cells in four DTC cell lines. (F) DTC cells were treated with BAY 1895344 (500 nmol/L) or vehicle for 24 and 48 h. Western blotting analysis revealed BAY 1895344 treatment increased the levels of cleaved PARP, a marker of apoptosis, by 24 h in four DTC cell lines.

Figure 4.. Combination therapy of BAY 1895344 and targeted therapies in DTC cells.

(A) The cytotoxicity of BAY 1895344 and sorafenib alone and in combination after a 4-day incubation in BHP7-13, K1, FTC-133, and FTC-238 cells was evaluated using LDH assays. (B) The CI of BAY 1895344 and sorafenib was calculated using CompuSyn software. BAY 1895344 plus sorafenib was synergistic in K1 and FTC-238 cells, and ranged synergistic-to-antagonistic in BHP7-13 and FTC-133 cells. (C) The cytotoxicity of BAY 1895344, lenvatinib, and their combination after a 4-day treatment was determined in four DTC cell lines. (D) CompuSyn software was used to calculate the CI of BAY 1895344 and lenvatinib in BHP7-13, K1, FTC-133, and FTC-238 cells. BAY 1895344 plus lenvatinib was synergistic in K1, FTC-133 and FTC-238 cells, and ranged synergistic-to-antagonistic in BHP7-13 cells. (E) The cytotoxicity of BAY 1895344, dabrafenib plus trametinib, and the triple combination of BAY 1895344 with dabrafenib plus trametinib after a 4-day incubation in K1 cells is shown. (F) The triple combination of BAY 1895344 with dabrafenib plus trametinib showed synergistic to antagonistic effects (CI 0.4–2.8) in K1 cells. The horizontal dotted line at CI = 1 illustrates the judgement between synergism (CI < 1) and antagonism (CI > 1).

Figure 5.. BAY 1895344 inhibited K1 tumor growth and sensitized K1 tumors to lenvatinib therapy and dabrafenib plus trametinib therapy.

(A) Nude mice bearing K1 tumors were orally treated for three cycles with: placebo; BAY 1895344 (30 mg/kg, twice a day, 3-days-on and 4-days-off); lenvatinib (30 mg/kg, once a day, 5-days-on and 2-days-off); BAY 1895344 plus lenvatinib; dabrafenib (30 mg/kg) plus trametinib (0.6 mg/kg) (once a day, 5-days-on and 2-days-off); or the triple combination of BAY 1895344 with dabrafenib plus trametinib. BAY 1895344, lenvatinib, BAY 1895344 plus lenvatinib, dabrafenib plus trametinib, and the triple combination substantially repressed K1 tumor growth when compared with placebo treatment (P < 0.001 for all comparisons). BAY 1895344 and lenvatinib combination therapy was superior in reducing K1 tumor growth relative to either alone. Compared to any single regimen treatment, tumor volume was also significantly repressed by the triple combination therapy of BAY 1895344 with dabrafenib plus trametinib. (B) BAY 1895344, lenvatinib, BAY 1895344 plus lenvatinib, dabrafenib plus trametinib, and triple combination therapy did not significantly lead to changes in body weight after a three-week treatment. (C) Representative photographs of K1 xenograft (arrowhead) taken on day 21 after various treatments. (D) Tumor expression of p-H2AX (Ser139), cleaved PARP, and PCNA was evaluated in K1 xenografts after BAY 1895344 treatment using immunoblotting. Black arrow: treatment with placebo, BAY 1895344, lenvatinib, dabrafenib plus trametinib, and combination therapy.

Figure 6.. BAY 1895344 inhibited tumor growth in an FTC-133 xenograft model.

(A) Mice bearing FTC-133 flank tumors were orally treated with placebo, BAY 1895344 (30 mg/kg, twice a day, 3-days-on and 4-days-off), sorafenib (40 mg/kg, once a day, 5-days-on and 2-days-off), lenvatinib (30 mg/kg, once a day, 5-days-on and 2-days-off), BAY 1895344 plus sorafenib, or BAY 1895344 plus lenvatinib for three cycles of treatment. Compared with vehicle treatment, BAY 1895344, sorafenib, lenvatinib, BAY 1895344 plus sorafenib, and BAY 1895344 plus lenvatinib significantly retarded FTC-133 tumor growth. BAY 1895344 and sorafenib in combination did not substantially repress FTC-133 tumor growth over either monotherapy. Tumor volumes were also not significantly more inhibited by the combination therapy of BAY 1895344 plus lenvatinib as compared with either drug alone. (B) BAY 1895344, sorafenib, lenvatinib, BAY 1895344 plus sorafenib, and BAY 1895344 plus lenvatinib did not significantly decrease body weight compared with vehicle treatment. (C) Representative photographs of FTC-133 xenograft (arrowhead) taken on day 21 after treatment. (D) Tumor levels of p-H2AX (Ser139), cleaved PARP, and PCNA were assessed in FTC-133 tumors after BAY 1895344 treatment using immunoblotting. Black arrow: treatment with placebo, BAY 1895344, sorafenib, lenvatinib, or combination therapy.