The dual pathway inhibitor rigosertib is effective in direct patient tumor xenografts of head and neck squamous cell carcinomas

Abstract

The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in squamous cell carcinoma of the head and neck (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from eight patients with HNSCC [four HPV-serotype16 (HPV16)-positive]. HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, Western blotting, and immunohistochemistry (IHC). Rigosertib had potent antiproliferative effects on 11 of 16 HPV(-) HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in three of eight HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA-activating event (amplification or mutation) and a p53-inactivating event (either HPV16- or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV(+) and HPV(-) HNSCCs, focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient, for rigosertib sensitivity.

Conflict of interest statement

Conflict of Interest: The authors declare no conflict of interest.

©2013 AACR.

Figures

Figure 1

A, Treated cultures (N=6) were normalized to the vehicle control data for each cell line which was set at 1.0. Cellular viability of 16 HNSCC cell lines as measured by SRB assay at 0.1uM, 1.0uM, and 10.0uM concentrations are represented graphically. Rigosertib is a potent antiproliferative agent at 1.0 μM and 10.0 μM. B, Viability of 2 sensitive (584 and HN11) and the 2 most resistant (MSK921 and UMSCC19) cell lines verified by clonogenic assay at 0.1 μM and 1.0 μM. Treatment at 10.0 μM prevented colony formation in all cell lines. C, Differential Akt and S6 phosphorylation patterns associated with treatment were identified by western blot analysis in vehicle control (C), rigosertib(R), and the PI3K inhibitor ZSTK474 (Z) treated cell culture. PI3K pathway inhibition by rigosertib is inconsistent. D, Representative cell cycle distribution by flow cytometry with ModFit analysis after 24hrs treatment with 1.0μM rigosertib or 1.0μM ZSTK474. The percentage of gated cells in each stage of the cell cycle is associated with each representative graph. Rigosertib treatment induces a pronounced G2 block in the sensitive lines. ZSTK474 induced a G1 block in all cell lines.

Figure 2

A, Mice bearing orthotopically implanted cell line xenograftsof our two sensitive cell lines received daily rigosertib infusions or vehicle for 28 days. Volumes on the final day of treatment are represented as % normalized to the vehicle control group. Regression was observed in 584, and growth reduction was observed for HN11 treated tumors. B, FISH with a probe specific for PIK3CA showed neither engrafted cell line to have significantly increased gene copy number. C, Western blot analysis suggests rigosertib treatment moderately inhibits PI3K pathway activation in only the HN11 tumors. D, IHC scores plotted for each xenografted cell line from the control and rigosertib treated tissue of PI3K pathway endpoints confirms PI3K/Akt pathway response to rigosertib treatment.

Figure 3

A, Mice bearing patient derived tumors received daily rigosertib infusions or vehicle for 28 days. An asterisk denotes HPV16 positivity. Volumes on the final day of treatment are represented as % normalized to the vehicle control group. Growth reduction was identified in CUHN014, CUHN047 and CUHN026. B, Gene amplification was characterized by a FISH score above 4 as exemplified by CUHN013. C, Western blot analysis of tissue taken at 28-days treatmentshow inconsistent inhibition of the PI3K pathway in the vehicle control (C) and rigosertib (R) treated tissue. Inhibition of S6 phosphorylation is observed in CUHN047, CUHN026 and CUHN013. D, IHC scores plotted for each tumor line from the control and rigosertib treated tissue. The graphical representations show a correlation all sensitive lines have a decrease in S6 phosphorylation in the treated tissue. This is also true for CUHN013.