Investigating and Improving the Therapeutic Activity of Propranolol in Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma

Human papillomavirus-associated head and neck squamous cell carcinoma (HPV[+] HNSCC) is rapidly increasing in incidence and, in this regard, the effects of preventative HPV vaccines have yet to be realized. While HPV(+) HNSCC carries a more favorable prognosis than many other tumor types, a significant number of patients still experience treatment failure. Disease recurrence and metastasis are often fatal. Thus, factors that promote disease progression and underlie aggressive tumor phenotypes must be identified, and, most importantly, novel therapeutic approaches must be developed. Interestingly, β-adrenergic signaling plays a tumorigenic role in several cancer types and β-adrenergic antagonists (β-blockers) have recently garnered interest as anti-cancer agents. Herein, we identify increased β2-adrenergic receptor (β2AR) expression in an aggressive, recurrent/metastatic model of HPV(+) HNSCC as compared to the primary tumor model from which it was derived. This led us to hypothesize that β2AR contributes to an aggressive disease phenotype in HPV(+) HNSCC and that β-blockers would serve as an effective therapeutic tool in this setting. Here, we show that propranolol, a non-selective β-blocker, significantly delays primary tumor growth and decreases metastatic burden in mice implanted with this aggressive tumor model. Additionally, genetic deletion studies indicate that the model’s aggressive phenotype is critically dependent on expression of β2AR. Importantly, we identify that these effects are associated with inhibition of tumor cell mitochondrial metabolism and show that propranolol-mediated perturbations in mitochondrial respiration render tumor cells more sensitive to glucose starvation. Thus, we further hypothesized that concurrent glycolytic inhibition would enhance the anti-cancer activity of propranolol. To this end, we have developed a novel, metabolism-targeting treatment combination in propranolol and dichloroacetate (DCA), a glycolytic inhibitor. This combination dramatically attenuates tumor cell metabolism, displays significant anti-cancer effects in vitro, and delays tumor growth in vivo. Together, these data provide support for further investigation into the therapeutic potential of propranolol in the setting of HPV(+) HNSCC, and suggest that its effectiveness may be improved through combination with other metabolism-targeting drugs. Importantly, as a clinically-available agent, propranolol has the potential for swift translation from the bench to the bedside

Propranolol Promotes Glucose Dependence and Synergizes with Dichloroacetate for Anti-Cancer Activity in HNSCC

Tumor cell metabolism differs from that of normal cells, conferring tumors with metabolic advantages but affording opportunities for therapeutic intervention. Accordingly, metabolism-targeting therapies have shown promise. However, drugs targeting singular metabolic pathways display limited efficacy, in part due to the tumor’s ability to compensate by using other metabolic pathways to meet energy and growth demands. Thus, it is critical to identify novel combinations of metabolism-targeting drugs to improve therapeutic efficacy in the face of compensatory cellular response mechanisms. Our lab has previously identified that the anti-cancer activity of propranolol, a non-selective beta-blocker, is associated with inhibition of mitochondrial metabolism in head and neck squamous cell carcinoma (HNSCC). In response to propranolol, however, HNSCC exhibits heightened glycolytic activity, which may limit the effectiveness of propranolol as a single agent. Thus, we hypothesized that propranolol’s metabolic effects promote a state of enhanced glucose dependence, and that propranolol together with glycolytic inhibition would provide a highly effective therapeutic combination in HNSCC. Here, we show that glucose deprivation synergizes with propranolol for anti-cancer activity, and that the rational combination of propranolol and dichloroacetate (DCA), a clinically available glycolytic inhibitor, dramatically attenuates tumor cell metabolism and mTOR signaling, inhibits proliferation and colony formation, and induces apoptosis. This therapeutic combination displays efficacy in both human papillomavirus-positive (HPV(+)) and HPV(−) HNSCC cell lines, as well as a recurrent/metastatic model, while leaving normal tonsil epithelial cells relatively unaffected. Importantly, the combination significantly delays tumor growth in vivo with no evidence of toxicity. Additionally, the combination of propranolol and DCA enhances the effects of chemoradiation and sensitizes resistant cells to cisplatin and radiation. This novel therapeutic combination represents a promising treatment strategy which may overcome some of the limitations of targeting individual metabolic pathways in cancer

CD137 Enhancement of HPV Positive Head and Neck Squamous Cell Carcinoma Tumor Clearance

Standard-of-care cisplatin and radiation therapy (CRT) provides significant tumor control of human papillomavirus (HPV)-mediated head and neck squamous cell carcinomas (HNSCCs); this effectiveness depends on CRT-mediated activation of the patient’s own immune system. However, despite good survival, patients suffer significant morbidity necessitating on-going studies to define novel therapies that alleviate this burden. Given the role of the immune system in tumor clearance, immune modulation may further potentiate the CRT-activated response while potentially decreasing morbidity. CD137, an inducible cell surface receptor found on activated T cells, is involved in differentiation and survival signaling in T cells upon binding of its natural partner (CD137L). A number of studies have shown the effectiveness of targeting this immune-stimulatory pathway in regards to tumor clearance. Here, we test its role in HPV+ HNSCC tumor clearance using a previously characterized mouse model. We show that amplification of this stimulatory pathway synergizes with CRT for enhanced tumor clearance. Interestingly, tumor clearance is further potentiated by local tumor cell expression of CD137L

Characterization of the Immune Response to PD-1 Blockade during Chemoradiotherapy for Head and Neck Squamous Cell Carcinoma

Background: Chemoradiotherapy is a standard treatment for HNSCC. Blockade of the PD-1/L1-2 interaction may represent a target to overcome immune escape during this treatment. Methods: Utilizing a HNSCC mEERL C57BL/6 mouse model, we evaluated a PD-1 blockade alone or in combination with cisplatin-based chemoradiotherapy. Next, we evaluated peripheral blood mononuclear cells (PBMCs) with relative PD-1, TIM-3, and LAG-3 expression, and myeloid-derived suppressor-like (MDSC-like) populations from a clinical trial evaluating PD-1 blockade with chemoradiotherapy in HNSCC. Finally, we analyzed the effect of therapy on human T-cell clonality through T-cell Receptor (TCR) sequencing. Results: Anti-PD-1 monotherapy induced no response in the mEERL model; however, combination with chemoradiotherapy improved tumor clearance and survival. PBMCs from patients treated with this combination therapy demonstrate a decline in circulating T-cell populations with knockdown of PD-1 expressing CD3+CD4+ and CD3+CD8+ T cells during treatment. However, TIM-3, LAG-3 expressing T-cell and MDSC-like populations concordantly rose. During treatment, the TCR repertoire demonstrates overall clonal expansion, with both unique and previously reported T-cell clones. Conclusions: Our murine HNSCC model demonstrates efficacy of PD-1 blockade during chemoradiotherapy. However, while PD-1-expressing T cells decreased with this therapy, human PBMC findings also identified an increase in populations contributing to immune exhaustion. These findings further characterize PD-1 blockade during chemoradiotherapy for HNSCC and highlight potential competing mechanisms of immune evasion