# Exploring mechanisms of therapeutic demethylation effects in HPV-associated head and neck cancer

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2022 · $400,632

## Abstract

Abstract
 Oncogenic human papillomaviruses (HPV) are the causative agents of uterine cervical and an increasing
portion of head and neck squamous cell carcinomas (HNSCC), but HNSCC is almost exclusively associated with
HPV type 16. The oncogenic properties of HPV type 16 are largely attributed to two major HPV oncogenes, E6
and E7, that degrade p53 and retinoblastoma (RB) family members, respectively. Degradation of these tumor
suppressors by E6 and E7 results in uncontrolled proliferation, diminished apoptosis and increased genomic
instability that predisposes to malignant transformation. The crucial roles of E6/E7 in HPV-related carcinogenesis
make them an attractive target for anti-cancer therapy, since methods for decreasing their expression or activities
would restore p53 and RB activity in tumors driven by HPV. Our preliminary results indicate that treatment of
HPV-positive (HPV+) HNSCC with the demethylating agent, 5-azacytidine (5-aza) at clinically relevant
concentrations, resulted in remarkable downregulation of all HPV gene expression, including E6 and E7. 5-aza
treatment restored p53 expression and activity in HPV+ head and neck cancer cells, which was partially
responsible for the sensitivity of these cells to 5-aza. In addition to restoration of p53, 5-aza also was toxic to
HPV+ HNSCC through creation of DNA double strand breaks (DSBs). Mechanistically, 5-aza-induced DNA
DSBs in HPV+ HNSCC were dependent on transcription and replication and on overexpression of the cytidine
deaminase, APOBEC3B (A3B). Experimental depletion of A3B inhibited 5-aza toxicity and diminished DSB
formation, but also indicated that untreated HPV+ HNSCC depend on A3B for clonogenic growth. The
observations that untreated HPV+ HNSCC dependent on A3B, but that A3B contributes to 5-aza toxicity and
DSBs, suggests an A3B-dependent synthetic lethality upon treatment with 5-aza, and that A3B may serve as a
biomarker of response. Treatment of mice bearing HPV+ tumors with 5-aza revealed significant tumor growth
inhibition and prevented detection of circulating tumor cells. A window clinical trial in patients with HNSCC using
standard dosing for 5 days achieved demethylation (LINE-1) similar to that seen in our in vitro experiments and
confirmed that 5-aza treatment: 1) significantly decreased expression of HPV genes; 2) reactivated p53; 3)
activated caspases, 4) and inhibited matrix metalloproteinase expression in HPV+ HNSCCs. This proposal is
designed to determine molecular mechanisms of demethylation-induced downregulation of HPV oncogenes,
elucidate the role of A3B in 5-aza-induced synthetic lethality and DNA DSBs formation, determine effect of
demethylation on immune cell infiltration in HPV+ HNSCC, and explore the potential of 5-aza alone or in
combination with chemotherapeutic agents to suppress HPV-associated HNSCC metastasis and inhibit growth
using patient-derived xenografts. These studies will provide a basis for a new rational targeted therapy for HPV+
H...

## Key facts

- **NIH application ID:** 10438568
- **Project number:** 5R01DE027942-04
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Karen S. Anderson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $400,632
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10438568

## Citation

> US National Institutes of Health, RePORTER application 10438568, Exploring mechanisms of therapeutic demethylation effects in HPV-associated head and neck cancer (5R01DE027942-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10438568. Licensed CC0.

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