Professor of Pharmacology and of Molecular Biophysics and Biochemistry; Co-Leader, Developmental Therapeutics, Yale Cancer Center; Co-Director Therapeutics/Chemotherapy Program
PROJECT 3: Demethylation of HPV-associated head and neck cancer to trigger APOBEC synthetic lethality and enhance immune response
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 3, we will determine effects of 5-azaC on activators of immune recognition and response in the presence or absence of Nivo.
Project 3 Co-Leaders
(University of North Carolina, Chapel Hill)