Mechanisms of Resistance to EGFR Inhibition in HNSCC

Abstract

Head and neck squamous cell carcinoma (HNSCC) remains a deadly disease with poor prognosis. Developing novel, effective combination therapies have the potential to improve patient survival. However, advancing biomarkers in conjunction with combination therapy will also be essential for efficacy so as to match treatment to the patient. In my thesis, I investigated the hypothesis that co-targeting a specific mechanism of resistance with combination therapy would be more effective than either therapy alone. I focused on identifying resistance mechanisms to cisplatin and EGFR inhibition, which are common HNSCC treatments, in UM-SCC cell lines using CRISPR/Cas9 screening libraries. This approach identified genetic knockouts that sensitized cell lines to either cisplatin or EGFR inhibition. The results of a CRISPR/Cas9 screen nominated NOTCH pathway knockouts and specifically NOTCH1 knockouts as capable of sensitizing cisplatin-resistance cells. Further results suggest that the combination of Notch inhibitors and cisplatin therapy are capable of overcoming cisplatin resistance, and that inactivating mutations in NOTCH1 may be a biomarker of cisplatin sensitivity. I also used genome and kinome CRISPR libraries to identify genetic knockouts that sensitized resistant models to the EGFR inhibitors gefitinib and erlotinib. I observed that PIK3C2A may be an important linchpin in the PI3K pathway for mediating resistance, as well as identified an unexpected set of genes associated with KRAS signaling, nominating KRAS as a potential mediator of resistance to EGFR inhibition in HNSCC. Furthermore, my CRISPR/Cas9 screens also nominated FGF/FGFR knockouts as sensitizing cells to EGFR inhibition. Extending these discoveries, I investigated the potential of dual EGFR and FGFR inhibition by testing multiple UM-SCC cell lines. I observed that FGFR may be a more common compensatory mechanism that previously realized, with 14/22 (63%) of cell lines undergoing cell death when challenged with combination therapy. Surprisingly, neither copy number or expression of FGFRs predicted responsiveness to the combination of EGFR and FGFR inhibition. To explore the mechanism behind this response, I generated an EGFR K/O model and showed that FGFR signaling increases when EGFR protein is lost. Evaluation of the discovery in vivo demonstrated that dual inhibition of EGFR and FGFR was able to significantly decrease tumor volume in a xenograft mouse model, supporting the in vivo relevance of this combination for HNSCC. Consistent with the literature, dual EGFR and FGFR inhibition caused weight loss in animals suggesting a high level of toxicity; thus, this data suggests that new ways to target the pathway are critical to future clinical success. Finally, to evaluate potential clinical relevance, we analyzed transcriptome profiles of tumors from patients who received the EGFR inhibitor cetuximab. We observed changes in the FGFR receptors, KRAS signaling, and PI3K-mTOR signaling, consistent with our profiling data. Overall, my thesis work supports the hypothesis that there are specific and common compensatory pathways to EGFR inhibition and cisplatin, and that co-targeting EGFR or cisplatin with this compensatory pathway is more effective than monotherapy treatments. The CRISPR/Cas9 screens and transcriptome analysis have generated a wealth of data that can continue to be explored to develop novel, effective strategies for combination therapy. Collectively, this body of work represents a step forward in the understanding of how HNSCC tumors compensate in response to two prevalent therapies, and may provide the foundation for opportunities to advance combination therapies and improve survival of HNSCC patients.