Preclinical Evaluation of the Association of the Cyclin-Dependent Kinase 4/6 Inhibitor, Ribociclib, and Cetuximab in Squamous Cell Carcinoma of the Head and Neck

Abstract

Epidermal growth factor receptor (EGFR) overexpression is observed in 90% of human papillomavirus (HPV)-negative squamous cell carcinomas of the head and neck (SCCHN). Cell cycle pathway impairments resulting in cyclin-dependent kinase (CDK) 4 and 6 activation, are frequently observed in SCCHN. We investigated the efficacy of ribociclib, a CDK4/6 inhibitor, in combination with cetuximab, a monoclonal antibody targeting the EGFR, in HPV-negative SCCHN patient-derived tumor xenograft (PDTX) models. The combination of cetuximab and ribociclib was not significantly more active than cetuximab monotherapy in all models investigated. In addition, the combination of cetuximab and ribociclib was less active than ribociclib monotherapy in the cetuximab-resistant PDTX models. In these models, a significant downregulation of the retinoblastoma (Rb) protein was observed in cetuximab-treated mice. We also observed Rb downregulation in the SCCHN cell lines chronically exposed and resistant to cetuximab. In addition, Rb downregulation induced interleukin 6 (Il-6) secretion and the Janus kinase family member/signal transducer and activator of transcription (JAK/STAT) pathway activation that might be implicated in the cetuximab resistance of these cell lines. To conclude, cetuximab is not an appropriate partner for ribociclib in cetuximab-resistant SCCHN models. Our work has significant clinical implications since the combination of anti-EGFR therapy with CDK4/6 inhibitors is currently being investigated in clinical trials.

Keywords: CDK4/6; cetuximab; resistance; retinoblastoma; ribociclib; squamous cell carcinoma of the head and neck.

Figure 1

Ribociclib, cetuximab, and ribociclib/cetuximab activity were evaluated in three HPV-negative SCCHN PDTX models; (a) HNC002, (b) UCLHN01, (c) HNCOO7, (d), HNC010, (e) HNC002-ResCTX. Mice received daily 100 mg/kg ribociclib, or weekly 30 mg/kg cetuximab, a combination of ribociclib and cetuximab, or the vehicles. n.s. (no significance): p > 0.05; * p < 0.05; ** p < 0.001; *** p < 0.0001.

Figure 2

Rb protein expression levels in four HPV-negative SCCHN PDTX models (HNC002, HNC007, HNC010, HNC002-ResCTX); (a) Rb protein expression evaluated by immunohistochemistry (we show one representative mouse per model), and (b) Rb protein expression measured by histoscore in three mice/treatment/model, except for HNC002 where 2 mice/treatment were measured. In the UCLHN01 and HNC007 models, due to the important efficacy of cetuximab and/or the combination treatment, some of the tumors were too small to be analyzed for protein expression. (c) Tumor volumes after 42 days of treatment are represented for each treatment group, except for the tumor volume of HNC007 treated with ribociclib, which was reported after 28 days of treatment due to important tumor size. n.a.; not available, n.s. (no significance): p > 0.05; * p < 0.05; ** p < 0.001; *** p < 0.0001.

Figure 3

CAL27 cell line: Log2-fold change in values determined by proteomic analyses for proteins identified as important regulators of the cell cycle, JAK/STAT, PI3K/Akt and MAPK pathways.

Figure 4

FaDu cell line: Log2-fold change in values determined by proteomic analyses for proteins identified as important regulators of the cell cycle, JAK/STAT, PI3K/Akt, and MAPK pathways.

Figure 5

Protein expression levels of the CAL27 and FaDu cell lines. The expression of proteins associated with (a) the cell cycle pathway and (b) the PI3K/Akt, MAPK and JAK/STAT pathways was investigated in triplicate by immunoblotting. Parental and cetuximab-resistant lines were analyzed as pairs.

Figure 6

(a) IL-6 levels in the cell culture supernatants of the CAL27 and FaDu cetuximab-resistant cell lines compared to parental cells. (b) IL-6 levels in the cell culture supernatants of CAL27 and FaDu cells treated with siRNA targeting RB1. ** p < 0.001; *** p < 0.0001.