Targeted Therapy of HPV Positive and Negative Tonsillar Squamous Cell Carcinoma Cell Lines Reveals Synergy between CDK4/6, PI3K and Sometimes FGFR Inhibitors, but Rarely between PARP and WEE1 Inhibitors

Abstract

Human papillomavirus positive (HPV⁺) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have a favorable outcome, but upon relapse, survival is poor and new therapeutical options are needed. Recently, we found synergistic effects by combining the food and drug administration approved (FDA) phosphoinositide 3-kinase (PI3K) and fibroblast-growth-factor-receptor (FGFR) inhibitors BYL719 and JNJ-42756493 on TSCC cell lines. Here this approach was extended and Cyclin-Dependent-Kinase-4/6 (CDK4/6) and Poly-ADP-ribose-polymerase (PARP) and WEE1 inhibitors PD-0332991, and MK-1775 respectively were also examined. HPV⁺ CU-OP-2, -3, -20, and HPV^- CU-OP-17 TSCC cell lines were treated with either BYL719 and JNJ-42756493, PD-0332991 BMN-673 and MK-1775 alone or in different combinations. Viability, proliferation, and cytotoxicity were followed by WST-1 assays and the IncuCyte S3 Live^® Cell Analysis System. All inhibitors presented dose-dependent inhibitory effects on tested TSCC lines. Synergy was frequently obtained when combining CDK4/6 with PI3K inhibitors, but only sometimes or rarely when combining CDK4/6 with FGFR inhibitors or PARP with WEE1 inhibitors. To conclude, using CDK4/6 with PI3K or FGFR inhibitors, especially PD-0332991 with BYL719 presented synergy and enhanced the decrease of viability considerably, while although dose dependent responses were obtained with PARP and WEE1 inhibitors (BMN-673 and MK-1775 resp.), synergy was rarely disclosed.

Keywords: CDK4/6; FGFR; HPV; PARP; PI3K; WEE1; base of tongue cancer; head neck cancer; oropharyngeal cancer; targeted therapy; tonsillar cancer.

Figure 1

WST-1 viability assay on HPV⁺ cell lines CU-OP-2, CU-OP-3 and CU-OP-20 and HPV⁻ cell line CU-OP-17. Cell viability analysis measured as absorbance after 24, 48 and 72 h of single treatment with PD-0332991 (A–D), BYL719 (E–H), JNJ-42756493 (I–L). PD denotes PD-0332991; BYL denotes BYL719; and JNJ denotes JNJ-42756493.

Figure 2

WST-1 viability assay on HPV⁺ cell lines CU-OP-2, CU-OP-3, and CU-OP-20, and HPV⁻ cell line CU-OP-17. Cell viability analysis measured as absorbance after 24, 48, and 72 h of single treatments with BMN-673 (A–D), or MK-1775 (E–H). BMN denotes BMN-673 and MK denotes MK-1775.

Figure 3

WST-1 viability assay on HPV⁺ cell lines CU-OP-2, CU-OP-3, and CU-OP-20, and HPV⁻ cell line CU-OP-17. Cell viability analysis measured as absorbance after 24, 48, and 72 h of combination treatment with PD-0332991 and BYL719 (A–H) or PD-0332991 and JNJ-42756493 (I–P). PD denotes PD-0332991; BYL denotes BYL719; and JNJ denotes JNJ-42756493.

Figure 4

Combinational effects of CDK4/6 inhibitor PD-0332991 and PI3K inhibitor BYL719 (A) and PD-0332991 and FGFR inhibitor JNJ-42756493 (B) after 48 h. Combination indexes (CIs) were shown with the highest single agent approach after 48 h (A,B) and 72 h, where CI > 1 shows a negative combination effect and CI < 1 shows a positive combination effect. CIs were calculated from the mean of three experiments analyzed by WST-1, at 48 h after treatment. CI denotes combinational index; PD denotes PD-0332991; BYL denotes BYL719; and JNJ denotes JNJ-42756493.

Figure 5

WST-1 viability assay on HPV⁺ cell lines CU-OP-2, CU-OP-3, and CU-OP-20 and HPV⁻ cell line CU-OP-17. Cell viability analysis measured as absorbance after 24, 48, and 72 h of combination treatment with BMN-673 and MK-1775 (A–L). BMN denotes BMN-673 and MK denotes MK-1775.

Figure 6

Combinational effects of PARP inhibitor BMN-673 and WEE1 inhibitor MK-1775 after 48 h. Combination indexes (CIs) were shown with the highest single agent approach after 48 h, where CI > 1 shows a negative combination effect and CI < 1 shows a positive combination effect. CIs were calculated from the mean of three experiments analyzed by WST-1, at 48 h after treatment. CI denotes combinational index; BMN denotes BMN-673 and MK denotes MK-1775.

Figure 7

Proliferation response of HPV⁺ CU-OP-2, CU-OP-3, CU-OP-20, and HPV⁻ CU-OP-17 cell lines upon treatment with PD-0332991, BYL719 or JNJ-42756493, BMN-673, and MK-1775. Proliferation responses of HPV⁺ CU-OP-2, CU-OP-3, CU-OP-20, and HPV⁻ CU-OP-17 after treatment with CDK4/6 inhibitor PD-0332991 (A–D), PI3K inhibitor BYL719 (E–H), FGFR inhibitor JNJ-42756493 (I–L), PARP inhibitor BMN-673 (M–P) and WEE1 inhibitor MK-1775 (Q–T). The graphs represent one experimental run per cell line. Confluence (%) denotes proliferation response; BYL denotes BYL719; JNJ denotes JNJ-4275649 and PD denotes PD-0332991; BMN denotes BMN-673; MK denotes MK-1775.

Figure 8

Proliferation response of HPV⁺ CU-OP-2, CU-OP-3, CU-OP-20, and HPV⁻ CU-OP-17 cell lines upon combined treatment with PD-0332991 and BYL719 or JNJ-42756493. Proliferation responses of HPV⁺ CU-OP-2, CU-OP-3, CU-OP-20, and HPV⁻ CU-OP-17 after treatment with CDK4/6 inhibitor PD-0332991 and PI3K inhibitor BYL719 (A–D) or with FGFR inhibitor JNJ-42756493 (E–H), or with the PARP inhibitor BMN-673 and WEE1 inhibitor MK-1775 (I–L). The graphs represent one experimental run per cell line. Confluence (%) denotes proliferation response; PD denotes PD-033299; BYL denotes BYL719; JNJ denotes JNJ-4275649; BMN denotes BMN-673; and MK denotes MK-1775.