# Project 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response

> **NIH NIH P50** · YALE UNIVERSITY · 2024 · $432,279

## Abstract

SUMMARY
Human papillomavirus (HPV)-associated neck squamous cell carcinoma (HNSCC) represents an increasing
proportion of HNSCC. The incidence of HPV+ HNSCC has dramatically increased over the last 2 decades and
in 2012 surpassed uterine cervical cancer as the most common HPV-related malignancy in the U.S. Despite
the HPV vaccine, it is estimated that the “epidemic” of HNSCC caused by HPV will not diminish until 2060.
HPV+ HNSCCs occur in younger individuals and prognosis for patients with these tumors is better compared
to patients with classical HNSCC; however, ~25% of patients recur with few effective therapeutic options.
Based on observed hypermethylation of HPV+ HNSCC from TCGA, and understanding that HPV uses
hypermethylation to impede the innate immune response, effects of the demethylating agent, 5-azacytidine (5-
azaC), were tested on HPV+ HNSCC. We found that HPV+ HNSCC cells in culture and xenografts are
sensitive to 5-azaC, and that 5-azaC caused double strand breaks (DSB) that were not observed after 5-azaC
therapy in HPV-negative HNSCC, even with much higher doses. We found that following 5-azaC therapy,
APOlipoprotein B mRNA-Editing enzyme Catalytic polypeptide 3B (APOBEC3B) was associated with
chromatin in HPV+ HNSCC, but not HPV-negative cells. CRISPR knockdown of A3B prevented DSB and
protected cells from 5-azaC-induced death. Despite being required for DSBs and cellular toxicity caused by 5-
azaC, A3B was also required for clonogenic survival of untreated HPV+ HNSCC. These data showing that A3B
is required for survival of HPV+ HNSCC cells, but that following demethylation A3B mediates toxicity and DSB.
In addition, 5-azaC therapy increased type I interferon signaling as measured by increased expression of
interferon-stimulated genes. These exciting pre-clinical data led to a window trial of 5days of 5-azaC. Analysis
of tumor specimens confirmed in vitro data showing that 5-azaC resulted in cellular toxicity. Immunofluorescent
staining of an HPV+ patient tumors pre- and post-5-azaC showed a marked increase in tumor-associated
lymphocytes, possibly driven through activation of type I interferon combined with increased expression of
neoantigens. In this YHN-SPORE project, we hypothesize 5-azaC therapy will enhance response to nivolumab
(Nivo) through its ability to cause cell death, increase neoantigen expression, increase A3B-driven mutational
load, and enhance T cell infiltration through increased type I interferon signaling. These hypotheses will be
tested using established and novel in vitro assays, as well as through examination of pre- and post-therapy
tumor specimens from a 3-armed clinical trial. In Aim 1, tumor specimens from the SPORE window trial will be
analyzed to determine effects of 5-azaC, Nivo, or the combination on cell death, cell proliferation, immune
infiltration and immune activation. Aim 2 will employ standard and novel assays to explore the role of A3B in
cellular toxicity exposed by 5-azaC therapy. In Aim ...

## Key facts

- **NIH application ID:** 10889241
- **Project number:** 5P50DE030707-05
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Karen S. Anderson
- **Activity code:** P50 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $432,279
- **Award type:** 5
- **Project period:** 2020-09-22 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10889241

## Citation

> US National Institutes of Health, RePORTER application 10889241, Project 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response (5P50DE030707-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10889241. Licensed CC0.

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