The Characterization and Application of Pevonedistat as a Novel Therapeutic in Head and Neck Squamous Cell Carcinoma

Abstract

Head and neck squamous cell carcinomas (HNSCC) are malignancies of the mucosal lining of the upper aerodigestive tract. 900,000 new cases of HNSCC are diagnosed worldwide each year. A major etiologic distinction of significant clinical importance in HNSCC is the human papillomavirus (HPV) status of the tumor. HPV- cancers have a significantly worse prognosis. A lack of novel, targeted therapeutics in HPV- HNSCC has, in part, led to virtually no improvements in patient outcomes in the past three decades. NEDDylation is a post-translational protein modification pathway that is frequently overactivated in HNSCC. Given its importance in regulating apoptosis, cell stress response, and DNA damage repair, NEDDylation is a promising anticancer target. Pevonedistat (PEV) is a first in-class inhibitor of NEDD8 Activating Enzyme (NAE), the proximal enzyme in the NEDDylation cascade. The work described in this dissertation is aimed at investigating the use of pevonedistat in conjunction with cisplatin to target HPV- HNSCC. The in vitro investigation of pevonedistat displayed consistent and potent anticancer activity across a multitude of HNSCC cell lines from a variety of anatomic sites. Furthermore, our in vitro data reveal a highly synergistic relationship between cisplatin and pevonedistat in HNSCC cell lines. Vital targets of NEDDylation include the Cullin-RING Ligases (CRLs), a family of E3 ubiquitin ligases which require the addition of a NEDD8 molecule to function appropriately. Upon further investigation, we discovered that Cullin-4A (CUL4A) is of particular importance to the synergistic relationship of pevonedistat with cisplatin. We determined that targeted inhibition of CUL4A via pevonedistat treatment led to significant impairment of DNA repair pathways which are intimately tied to cisplatin-resistance and cancer cell survival. Specifically, pevonedistat treatment and inhibition of CUL4A resulted in the loss of the nucleotide excision repair (NER) pathway function through decreased expression of DDB2, a vital damage recognition protein. Importantly, co-treatment with pevonedistat and cisplatin led to long-term tumor regression and survival in an in vivo xenograft model. Excised tumors displayed loss of DDB2 when exposed to pevonedistat as a monotherapy or in combination with cisplatin. Interestingly, pevonedistat and cisplatin were not only well-tolerated, but provided a level of protection from the off-target toxicities associated with cisplatin monotherapy. Renal toxicity is a major challenge associated with the clinical use of cisplatin. Through RNA sequencing and additional in vitro assays, we were able to confirm that pevonedistat induces the activation of variety of oxidative stress response genes in normal renal cells, leading to decreased cisplatin-induced damage and cell death in this tissue. We also show that pevonedistat causes the downregulation of Thioredoxin-Interacting Protein (TXNIP), a critical modulator of oxidative stress, in primary kidney cells. This loss of TXNIP was shown to be protective against cisplatin-induced oxidative damage. Furthermore, it was demonstrated that TXNIP expression is increased in cancer cells in response to pevonedistat therapy. Analysis of kidneys and tumors taken from mice treated with either monotherapy or the combination confirmed the differential expression changes of TXNIP in vivo. Together, our work provides clear and convincing evidence that pevonedistat is capable of augmenting cisplatin activity in tumor cells while simultaneously allowing for kidney cell survival. Taken together, the work described herein provides sound rationale for the further investigation of pevonedistat for use against HPV- HNSCC in the clinical setting to improve patient outcomes.