OVERVIEW OF HEAD AND NECK SQUAMOUS CELL CARCINOMAS
Head and neck squamous cell carcinomas (HNSCC) are the most common head and neck cancer type and the sixth most common cancer worldwide.1 In 2018, an estimated 890,000 new cases and 450,000 HNSCC-related deaths were reported in the United States. HNSCC manifests in several regions of the head and neck, including the oral cavity, nasopharynx, oropharynx, hypopharynx, base of the tongue and tonsils, larynx, paranasal sinuses, nasal cavity, and salivary glands.2
HNSCC is broadly classified as human papillomavirus (HPV)-positive (HPV+) or HPV-negative (HPV-), which is defined by the presence or absence of HPV in HNSCC tumor cells.2 Most HPV- HNSCC tumors are detected within the larynx and oral cavity, whereas HPV+ HNSCC tumors are found in the oropharynx. The majority of HNSCC cases are HPV- and are associated with risk factors including tobacco use and heavy alcohol consumption.
Although HPV+ and HPV- HNSCC are clinically, histologically, and molecularly distinct, the same therapeutic strategies are used for both, resulting in limited efficacy and clinical benefit.3 First-line treatments include surgery, radiation, and platinum-based chemotherapy. Notably, there are therapy-specific challenges, including long-term physical, psychological, and occupational effects that negatively affect quality of life for patients and their families.1 Up to 50% of patients treated for locally advanced HNSCC will experience recurrence.4 The prognosis remains poor for patients with recurrent and metastatic HNSCC; the estimated median overall survival (OS) for these patients is just 13 to 15 months.
The available United States FDA-approved therapies have limited efficacy, and survival rates for patients with recurrent and metastatic HNSCC have remained stagnant in recent years.3 In the second line, response rates for the 3 FDA-approved therapies for the treatment of HNSCC range from 13% to 16%, with progression-free survival (PFS) of approximately 2 months. Cetuximab, an EGFR inhibitor, was approved in 2006 as the first molecularly targeted agent indicated for the treatment of HNSCC.5,6 For more than a decade, cetuximab remained the only targeted agent available until the PD-1 and PDL-1 immune checkpoint inhibitors nivolumab and pembrolizumab received expanded indications for HNSCC in 2016 and 2019, respectively.5-11
The majority of known genetic alterations in HNSCC cannot be therapeutically targeted, resulting in limitations to the current FDA-approved treatment options.12 HRAS is a driver oncogenic mutation that was identified as a potential therapeutic target. HRAS is mutated in 4% to 8% of patientswith HNSCC.13,14 In February 2021, the FDA granted breakthrough therapy designation to the investigational drug tipifarnib, a farnesyltransferase inhibitor (FTI), for the treatment of patients with recurrent or metastatic HRAS-mutant HNSCC with variant allele frequency of at least 20% after disease progression on platinum-based chemotherapy, based on the results of the RUN-HN phase 2 clinical trial.15
Additional therapeutic options with better response and outcomes than those observed with currently available agents are needed for patients with recurrent and metastatic HNSCC. There is an opportunity to create novel research tools to aid the discovery of actionable molecular profiles from diverse types of HNSCC tumors.2 For example, investigators noted a need for multiplatform genetic analysis that would allow them to screen sufficient numbers of both HPV+ and HPV- tumors to support the identification of actionable biomarkers. Additionally, such a genetic screening tool could support the classification of HNSCC tumors based on their molecular profiles and account for any differences in tumor profiles collected from distinct sites in the body with HNSCC presentation.
This article discusses the dysregulated molecular pathways within HNSCC and potential therapeutic targets within these pathways that may support the development of novel targeted agents for patients with distinct oncogenic driver-mutated HNSCC. Successful targeting of novel pathways, such as the RAS-RAF family pathway, may provide an opportunity to expand the treatment landscape in HNSCC.
HNSCC GENOMIC ALTERATIONS, SIGNALING PATHWAYS, AND ACTIONABLE TARGETS
As the knowledge of the molecular landscape of HNSCC has expanded, so has knowledge of the frequency of specific oncogenic mutations in patients with HNSCC and the mutation burden of distinct HNSCC tumor types (Figure 1).6 HNSCC tumors demonstrate a high rate of genetic heterogeneity results in loss-of-function mutations in tumor suppressor genes (eg, p53 and p16 INK4a) and activation of oncogenes, (eg, EGFR and PIK3CA).3
To identify actionable biomarkers and develop novel therapies for HNSCC, there is an opportunity to better understand the pathogenesis of HNSCC at the molecular level and the influence of signaling within the tumor microenvironment.3,6 Select dysregulated and targetable pathways in HNSCC are shown in Figure 2.4,9,11,13,16-20
EGFR is overexpressed in 80% to 90% of patients with HNSCC and is associated with poor prognosis.2 EGFR signaling is complex; it involves several interconnected cellular signaling pathways and activation of effector molecules within downstream response cascades.
EGFR is an HER/ErbB receptor tyrosine kinase. Soluble ligands and dimerization with other HER family receptors can activate EGFR and stimulate an aggressive, prosurvival cellular signaling response by means of the PI3K/AKT/mTOR and JAK/STAT pathways and the MAPK cascade.4 EGFR also interacts with other receptors that may enhance its oncogenic potential (eg, AXL).2 As a mechanism of radiotherapy resistance, ionizing radiation can cause EGFR translocation to the nucleus, where it acts as a transcription factor and controls expression of specific genes that facilitate resistance.2
EGFR can be targeted by blocking the ligand-binding domain with monoclonal antibodies, such as cetuximab, or by inhibiting the activity of the tyrosine kinase domain using small-molecule tyrosine kinase inhibitors (TKIs).2,17 Cetuximab binding to EGFR on the surface of both normal and tumor cells competitively inhibits the binding of ligands and blocks EGF from phosphorylating and activating EGFR.7,17 Cetuximab used as therapy for HNSCC is not companion diagnostic-dependent and does not require elevated EGFR expression or KRAS wild-type status.12
Although HNSCC tumors show high rates of EGFR overexpression, the efficacy of cetuximab and other EGFR inhibitors has been limited, particularly in patients with advanced HNSCC.12 Treatment with cetuximab as a single-agent for advanced HNSCC has demonstrated an objective response rate of approximately 10%. Panitumumab, another EGFR inhibitor, was studied in combination with chemotherapy as potential frontline therapy for HNSCC but did not demonstrate improvements in OS.
HNSCC has also been shown to develop resistance to cetuximab; however, the mechanisms of cetuximab resistance, such as the effects of mutations that constitutively activate EGFR-mediated signaling, are still being explored.17
Cancer immunotherapy involving checkpoint blockade has demonstrated success in treating a variety of cancers and demonstrated particular success against tumors with high rates of mutation.2 Because HNSCC has demonstrated high rates of mutation and appears to be an immunosuppressive disease, checkpoint inhibitor therapies may be beneficial for some patients.
PD-1, a T-cell coinhibitory receptor, serves as an immune checkpoint and is expressed on various immune cells, including activated and regulatory T cells, natural killer (NK) cells, activated B cells, and macrophages.20,21 PD-L1 is a known ligand of PD-1 that inhibits T-cell activation and limits the response to inflammation.
PD-L1 is expressed differently in HPV+ and HPV- HNSCC and may play a role in the pathogenesis of HPV+ HNSCC.20 Regulatory mechanisms inside and outside of the tumor cell cause overexpression of PD-L1. IFN-γ, the best known potent cytokine inducer, has been shown to induce PD-L1 expression by upregulation of a downstream target of PI3K.
The prognostic value of PD-1/PD-L1 expression in HNSCC has yet to be determined, and there is conflicting data in the current literature available. Results from some studies suggest that elevated PD-L1 expression may be associated with HNSCC tumor size, clinical stage, metastases, and worse OS.20 Conversely, other studies suggest that lower PD-1/PD-L1 expression correlates with fewer recurrences and better outcomes, especially in patients with HPV+ tumors. One meta-analysis offers a third possibility, as no detectable differences were shown in survival outcomes among patients with PD-L1-positive and PD-L1-negative HNSCC tumors.
Nivolumab and pembrolizumab are both monoclonal antibodies that bind to PD-1 receptors and block their interaction with PD-L1 and PD-L2, which releases PD-1 pathway-mediated inhibition of the immune response, including the antitumor immune response.9,11 Theblocking of PD-1 activity might result in decreased tumor growth. The overall response rate to nivolumab and pembrolizumab has been much lower in patients with HNSCC than in those with Hodgkin lymphoma (15% vs 87%).20 An estimated 50% of patients with HNSCC treated with PD-1/PDL-1 inhibitors experience treatment-related adverse events that negatively affect treatment outcomes. Additional research is warranted to understand the pattern and mechanisms of PD-1/PD-L1 expression in HNSCC.
In HNSCC, most tumors demonstrate PIK3CA/RTK/RAS pathway activation through mutations or amplifications of EGFR, ERBB2, the FGFR family, PI3K, or RAS.12 Moreover, the PI3K/AKT/mTOR pathway is upregulated in over 90% of both HPV+ and HPV- HNSCCs, which enables resistance to radiotherapy and to cytostatic drugs.21 Mutations and gene amplifications in this pathway are also associated with worsened prognosis and survival outcomes. PIK3CA is the most commonly dysregulated oncogene in HNSCC. The HRAS/MAPK and PI3K pathways are interdependent in SCCs.
PI3K and RAS proteins are key players in the pathways that coordinate the growth, proliferation, and survival of head and neck cancer cells.15 HRAS requires PI3Kα to transform squamous epithelial cells, and helicase domain PIK3CA mutants must bind RAS to cause cancer. Preclinical data showed that overexpression of mutant or wildtype HRAS drives resistance to PI3K inhibition in PIK3CA-mutant HNSCC cells.18,19
It is possible that blocking a single pathway may not be adequate to achieve therapeutically significant reductions in RAS signaling. Therefore, there is an opportunity for combination therapy regimens that target RAS in concert with other oncoproteins to control uncontrolled tumorigenesis in HNSCC.14 Notably, a combination strategy will be investigated in the phase 1/2 KURRENT trial to determine the safety and efficacy of tipifarnib plus the PI3K inhibitor alpelisib in patients with HNSCC whose tumors have HRAS overexpression or PIK3CA mutation or amplification.23 The study was announced on July 6, 2021, and it is expected to begin this year.
RAS-RAF Family Pathway
The frequency of RAS-RAF family pathway activation observed in HNSCC has made this pathway a target of high therapeutic interest.12 Approximately one-third of all human cancers contain mutations in RAS genes.24,25 KRAS mutations are frequently found in adenocarcinomas; NRAS mutations are common in melanoma and certain leukemias, and HRAS is mutated primarily in squamous cell carcinomas (SCC), including HNSCC.25 Of the 3 genes in the RAS-RAF family (HRAS, KRAS, and NRAS) HRAS has been identified as the predominant mutated RAS isoform in SCCs, including HNSCC, as shown in Figure 1.6,25 HRAS is mutated in 4% to 8% of HNSCC cases.13,14
HRAS is a proto-oncogene that becomes oncogenic due to mutations or increased expression.6 As with all proto-oncogenes, HRAS codes for its protein product that helps regulate cell growth and differentiation. HRAS-mutated HNSCC tumors generally have coincident loss of function mutations in CASP8, enrichment for wildtype TP53, and low copy-number alterations.
HRAS is mutated and overexpressed in certain HNSCC tumors. According to data collected from genomic sequencing and profiling studies in salivary gland cancers, HRAS mutations were identified in up to 20% high-grade histologic subtypes, such as mucoepidermoid carcinoma, adenocarcinoma, and salivary duct carcinoma.26 HRAS-driven tumors are found predominantly in HPV- HNSCC and are more common in certain demographic groups exposed to specific oral carcinogens (eg, tobacco and alcohol).27 In a Taiwan-based study that analyzed qualified DNA samples from male patients with oral squamous cell carcinoma (N = 120), HRAS mutations were identified in 11.7% of patients. The incidence of HRAS mutations was significantly higher among patients aged 56 years and older compared with younger patients (P < .05) and in patients with substantial alcohol intake.
HRAS-mutated HNSCC has been associated with poor prognosis and limited response to treatment. Patients with HRAS-mutated HNSCC tumors have experienced poor overall treatment outcomes with the current treatment options available, including EGFR inhibitors and PD-1/PD-L1 inhibitors. HRAS mutations were associated with less likelihood of achieving complete response or partial response to cetuximab treatment.17 Moreover, HRAS mutations discovered to contribute to treatment resistance with erlotinib, another EGFR inhibitor.28HRAS-mutant HNSCC tumors exhibit a low overall mutation burden, which is associated with poor response to immuno-oncologic agents like PD-1/PD-L1 inhibitors (eg, nivolumab and pembrolizumab).
RAS proteins serve as molecular switches that control intracellular signaling networks and play a critical role in cell proliferation, survival, and differentiation; cytoskeletal dynamism; and gene expression.24 Mutated or amplified RAS genes can result in constitutively activated RAS leading to overexpression of RAS protein products, which can then signal many other effectors, including RAF and PI3K.16
In normal, healthy cells, RAS receives and obeys signals to switch between the active GTP and inactive GDP states.24 RAS guanine nucleotide exchange factors (GEFs) convert RAS from an inactive guanosine diphosphate (GDP)-bound state to an active guanosine triphosphate (GTP)-bound state. Activation of receptor tyrosine kinases can bring RAS GEFs to the plasma membrane and begin this conversion.25 Once GTP-bound, RAS then connects with effector proteins, including the RAF kinases of the MAPK pathway and the p110 subunit of PI3K, which signal the cell to proliferate and to perform other functions. Binding with RAS GTPase-activating proteins (GAPs) then accelerates RAS GTPase activity, which terminates this signaling and returns RAS to an inactive GDP-bound state.
RAS mutations cause RAS proteins to maintain an active GTP state and ignore signals to switch to the inactive GDP state.25 This causes the perpetual activation of effector proteins throughout the RAS pathway to continue cell proliferation and growth. These oncogenic cells do not undergo apoptosis, and, thus, tumors are formed.
RAS proteins (HRAS, NRAS, KRAS) undergo a series of post-translational modifications. Proper overall RAS protein functions, including trafficking and localization of RAS within cells, are dependent on post-translational modifications. Hence, these modifications and the enzymes that catalyze them have become a target of therapeutic investigation.29 Farnesyltransferase and other enzymes catalyze a series of post-translational modifications, which include farnesylation, proteolysis, carboxymethylation, and palmitoylation, equipping RAS proteins with lipid groups, and initiating RAS protein trafficking to and from the plasma membrane.16
Mutant RAS proteins must be localized to the plasma membrane to activate downstream signaling. This localization and activation is dependent upon attachment of a hydrophobic isoprenyl group to the C-terminal tail of RAS proteins. This is the first post-translational modification step, called prenylation.29 The predominant form of RAS prenylation is farnesylation, which is catalyzed by the enzyme farnesyltransferase. Farnesyltransferase adds a hydrophobic farnesyl group to the CAAX motif of the C-terminal tail of RAS proteins, which increases their affinity for membranes and localization of the RAS protein to the inner cell membrane (Figure 2B).13 RAS protein binding to the inner cell membrane allows for subsequent downstream signaling and function in cell pathways.6,29 Without prenylation, the mutant RAS is inactive—without activity to carry out signaling for uncontrolled cell proliferation.
All RAS isoforms are farnesyltransferase substrates that require farnesylation for cellular membrane insertion and the subsequent activity of certain signaling proteins associated with cancer.6,13 On this basis, farnesyltransferase inhibitors (FTIs) could inhibit the post-translational modifications necessary for translocating RAS to the plasma membrane to prevent newly translated RAS proteins from being activated. By inhibiting downstream signaling, FTIs could result in tumor regressions in RAS-dependent malignancies.24
FTI therapy research began in the 1990s, and it was originally conceived with the intent of targeting oncogenic activity in cells with KRAS mutations.6 The clinical activity of FTI therapies studied in KRAS- and NRAS-driven tumors was underwhelming but led to the discovery of mechanisms associated with this limited response. NRAS and KRAS are susceptible to alternative prenylation events, which allow them to maintain membrane localization and pathway activation despite farnesyltransferase inhibition. Both KRAS and NRAS proteins are substrates of geranylgeranyl transferase (GGTase). When geranylgeranylated, KRAS and NRAS undergo an alternative post-translational modification in the prenylation pathway, which circumvents the mechanism and effect of FTIs. Importantly, HRAS cannot be geranylgeranylated; hence, its membrane localization and cellular function could be suppressed by an FTI. When farnesyltransferase is inhibited, KRAS and NRAS can use the alternative pathway to achieve prenylation but HRAS cannot.
Although all RAS isoforms (KRAS, NRAS, and HRAS) are farnesyltransferase substrates, HRAS oncogene activity is exclusively dependent on post-translational farnesylation for membrane localization that is required for activation of downstream signaling.24 It is believed that farnesyltransferase inhibitors targeted to HRAS can prevent growth of HRAS-driven tumors.6 Therefore, HRAS-mutated tumors are predicted to be uniquely susceptible to tipifarnib-mediated inhibition of farnesyltransferase.
In HNSCC patient–derived xenograft models, FTIs induced dramatic regressions in mutant HRAS and not wild-type models. Tipifarnib prevents farnesyltransferase from prenylating the HRAS protein at the CAAX motif, which, in turn, prevents HRAS membrane binding and renders the protein inactive.12 Preclinical studies of tipifarnib yielded promising results in HRAS-mutated HNSCC tumors, which provided the foundation for further investigation in the RUN-HN clinical trial and subsequent clinical trials for tipifarnib (presented in the next article).12,15
There is an unmet need to address treatment challenges in the population of patients with recurrent or refractory HNSCC, as they have poor survival outcomes and limited treatment options. Current therapeutic options, including cetuximab and the 2 FDA-approved immune therapies, are associated with low rates of response in this population.
Advancements in the genomic profiling of HNSCC have greatly expanded understanding of the molecular profiles and distinct pathways that can be targeted for new therapeutic developments. HRAS-mutant HNSCC was identified as a targetable oncogenic mutation in 4% to 8% of the HNSCC population; these patients may benefit from disruption of HRAS oncogenic signaling.13,14 Tipifarnib targets oncogenic activity specifically in HRAS-driven HNSCC tumors.15
Overall, the FDA’s breakthrough-designated therapy status of tipifarnib in 2021 highlights recent progress in the development of RAS pathway-targeted therapeutic approaches; however, further investigation into the mechanisms behind post-translational modifications that regulate RAS function (eg, trafficking, signaling, activation) is needed.16