Effect of a novel compound that covalently binds to the HPV16 E6 protein on proliferation of HPV-associated cervical and oropharyngeal cancer cells through p53 effector pathway activation.

Abstract

e15103 Background: The human papillomavirus (HPV) E6 protein is always expressed in HPV-induced malignancies. We identified compounds that specifically bind to the HPV16 E6 protein and irreversibly block its interaction with one of its cellular binding partners, the ubiquitin ligase E6AP. In a trimolecular complex with E6, E6AP ubiquitinates p53, leading to its destruction by the proteasome. KTI-240 specifically induces high levels of wild-type p53 and decreases cell viability in vitro and inhibits tumor growth in vivo only in HPV16 expressing cells. To investigate its mechanism of action, we analyzed the RNA transcriptomes from KTI-240 treated cervical cancer and normal epithelial cells. Methods: HPV16 expressing human cervical cancer cells SiHa, a SiHa clone in which the E6 cysteine targeted by KTI-240 was mutated to serine, and p53 wild-type RPE-1 cells were incubated with KTI-240 or vehicle (n = 3 replicates). Extracted RNA was sequenced with ~50 x 10 6 reads per sample. The data were analyzed in Partek Flow software with DESeq2 (False Discovery Rate ≤ 0.01: Fold Change ≥ 2, and mean transcript count ≥ 10). Results: 192 mRNAs were up- and 218 were down-regulated by ≥ two-fold in KTI-240-treated SiHa cells. KEGG pathway analysis revealed many are p53 pathway and cell cycle regulators such as MDM2, p21WAF1, PAI-1, PUMA, and Fas, which increased by 4.2, 6.9, 3.4, 2.7, and 2.5 fold, respectively. KTI-240 treated RPE-1 cells had 10 up- and 15 down-regulated mRNAs, none of which were p53 related. The E6 cysteine mutation prohibits covalent bonding of KTI-240, and expression of these p53 pathway-related mRNAs in KTI-240 treated mutant SiHa cells did not change. Basal mRNA levels of p53, E6 and E7 transcripts were comparable in SiHa and mutant SiHa cells and were unaltered with KTI-240 treatment, while p53 protein expression was increased by ~2.5-fold in wild type but not in mutant E6 SiHa cells. Only four RNA expression changes were common to the three cell lines, suggesting minor off-target induction. Conclusions: These transcriptomic studies demonstrate that a compound that covalently binds to HPV16 E6 activates p53 transcript programs known to induce apoptosis and senescence. This result was specific to HPV-16 expressing cells and requires the irreversible interaction with a specific cysteine in the E6AP binding pocket of E6. Remarkably, only a small number of RNAs were altered in the normal epithelial cells. The transcriptomic alteration in mutant SiHa cells may be due to effects on the many E6 activities unrelated to the E6AP-p53 pathway. Because HPV16 is identified in ~50% of all cervical and most head and neck cancers, inactivation of the HPV16 E6 protein and induction of cell death and growth arrest represents a novel opportunity for a molecular targeted approach for treatment of HPV16 expressing cervical, anogenital, and oropharyngeal cancers.