The Role of Caspase-8 Mutations in HNSCC

The gene encoding caspase-8 is mutated in 12% of patients with head and neck squamous cell carcinoma (HNSCC). We are investigating the impact of these mutations on caspase-8 function and their role in the development and progression of HNSCC. Caspase-8 is a cysteine protease that mediates apoptosis signaling following stimulation of cell surface death receptors, including TNF and TRAIL receptors. Recent evidence indicates that caspase-8 also acts in a noncatalytic manner to stimulate production of cytokines that suppress anti-tumor immunity. We have shown that HNSCC-associated caspase-8 mutants potently inhibit death receptor mediated apoptosis in a dominant-negative fashion. The impact of caspase-8 mutations on apoptosis and necroptosis signaling and sensitivity to chemotherapy drugs and radiation is currently being explored. Preliminary studies indicate that inhibitors of the DNA damage response proteins ATR and ATM may dramatically sensitize cells with caspase-8 mutations to cisplatin and radiation, respectively, offering a possible therapeutic strategy. In further studies we are using cell line and immunocompetent tumor models to determine whether the caspase-8 mutant proteins retain the capacity to stimulate production of immunosuppressive cytokines. The impact of caspase-8 mutations on the tumor microenvironment will be investigated.

Therapeutic Implications of PI3K Pathway Activation in HNSCC

The laboratory has been at the forefront of determining the genomic landscape of head and neck cancer and seeks to leverage this information, along with access to patient specimens and preclinical models, to develop innovative therapeutic approaches. In one such project, we have determined that PIK3CA, the gene encoding the PI3K catalytic subunit, is mutated or amplified in 35% of head and neck squamous cell carinomas (HNSCCs), leading to activation of the PI3K signaling pathway and overexpression of COX2 enzyme. In a retrospective analysis of 266 HNSCC patients we found that regular use of NSAIDs, inhibitors of COX2, was associated with markedly enhanced survival in patients harboring mutated or amplified PIK3CA, but not in individuals with unaltered PIK3CA. Enhanced anti-tumor activity of NSAIDs was seen in preclinical models with altered PIK3CA, including PDXs from our library of over 60 HNSCC PDXs. We are proposing a prospective clinical trial to determine whether aspirin use improves overall survival in HNSCC patients with genetic alterations that lead to activation of the PI3K signaling pathway. Results from this trial may lead to dramatic improvements in survival outcomes for a large subset of patients. Laboratory investigations will seek to determine the impact of PI3K pathway alterations on the tumor microenvironment, and the molecular mechanism responsible for NSAIDs effects in tumors with PI3K pathway alterations. Additional studies are aimed at determining the functional and phenotypic consequences of both canonical and noncanonical PIK3CA mutations, with mass spectrometry used to define the protein interactome of the mutants and CRISPR used to identify synthetic lethal interactions. Collectively, these investigations will facilitate the design of effective precision medicine strategies based on the mutational profile of the patient’s tumor.

Targeting STAT3 in Tumor and Immune Cells with a Novel and Innovative Inhibitor

We have played a leading role in identifying STAT3 transcription factor as a promising therapeutic target in head and neck squamous cell carcinoma (HNSCC). STAT3 is hyperactivated in a majority of HNSCCs, as well as other solid tumor malignancies, where it promotes tumor growth, disease progression, and poor prognosis. Recent studies have shown that STAT3 is also hyperactivated in tumor infiltrating immune cells, where it acts to suppress anti-tumor immunity. Thus, targeting of STAT3 has the potential two-fold benefit of inhibiting HNSCC tumor cell growth and enhancing anti-tumor immunity. As a transcription factor, STAT3 has historically been considered “undruggable”. Drs. Grandis and Johnson are co-inventors of a novel STAT3 inhibitor based on the STAT3 response element in genomic DNA. Their 15-bp STAT3 decoy oligonucleotide competitively inhibits STAT3 binding to genomic DNA, reduces expression of STAT3 target genes, and suppresses the growth of HNSCC cells and tumors. A modified cyclic version of the STAT3 decoy exhibits anti-tumor activity when delivered systemically. Ongoing studies are determining the effects of cyclic STAT3 decoy on tumor and immune cells in the tumor microenvironment using immunocompetent models of HNSCC and cutting-edge methodologies including NanoString and mass cytometry (CyTOF). Additional studies will develop a pharmacokinetic assay for the cyclic STAT3 decoy, enabling clinical evaluation of this unique anti-cancer agent in HNSCC patients.

Molecular factors and pathways regulating adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) of the salivary gland is a rare form of cancer of the head and neck. There are about 1,200 new cases diagnosed yearly in the United States. ACC progresses as a gradual and slow growing tumor that often develops into a highly aggressive cancer. While treatment currently involves surgery and radiation therapy, metastatic recurrence occurs in nearly 50% of cases. These metastases can remain asymptomatic for several years, and the factors that contribute to their dormancy or aggressiveness are not well understood. Furthermore, there are no approved chemotherapeutic or targeted agents available to treat any stage of this disease. Our research program focuses on the identification of molecular factors and pathway/mechanisms, and their role in ACC tumor progression, as it will lead to the development of effective treatment strategies. Projects include the investigation to understand the unique alternative splicing events that occur in ACC and their contribution to ACC tumorigenesis.

We identified alternatively spliced variants of several genes in primary ACC tumors relative to matched normal salivary gland tissue by RNA sequencing analysis. Currently, we are studying the functional impact of novel splice variants of FGFR1 and IGF1R on tumor growth regulation

Exome, methylation, and RNA sequencing of primary ACC tumors coupled with integrated pathway analysis revealed enrichment of genes associated in the wnt/b-catenin pathway. We are investigating the potential targeting of ACC by small molecule inhibitors of the wnt/b-catenin axis using PDX models in collaboration with colleagues at the University of Virginia and the Adenoid Cystic Research Foundation.

Chromosomal rearrangement is a frequent event in ACC; and the expression and fusion of the transcription factors MYB and MYBL1 to NF1B (MYB-NF1B and MYBL1-NF1B) are identified in the majority of ACC. Yet their functional implications remain to be clearly understood. By expressing these fusion constructs in an inducible manner in a validated ACC cell line, experiments are ongoing to profile and identify unique transcriptional and regulatory targets by RNA sequencing. In addition, using the above inducible expression system, we propose to establish MYB/fusion protein interactome by affinity purification based mass spectrometry approach (APMS). Identification of molecular players and pathways will help us better understand the contribution of MYBs and its fusion at the molecular level.

We are also interested in studies with the goal to gain insights into the molecular basis of perineural invasion of ACC. This project, in collaboration with the Knox’s lab (Dr. Sarah Knox, UCSF), utilizes primary patient samples and PDX model along with ex-vivo nerve culture model to understand the nature of ACC neural innervation and invasion.