Paola Y. Anguiano Quiroz

Head and neck squamous cell carcinoma (HNSCC) represents a highly prevalent and deadly malignancy worldwide. The prognosis for locoregionally advanced HNSCC has not appreciably improved over the past 30 years despite advances in surgical, radiation, and targeted therapies and less than 20% of HNSCC patients respond to recently approved immune checkpoint inhibitors. The Hippo signaling pathway, originally discovered as a mechanism regulating tissue growth and organ size, transduces intracellular and extracellular signals to regulate the transcriptional co-activators YAP and TAZ. Alterations in the Hippo pathway resulting in persistent YAP and TAZ activation have emerged as major oncogenic drivers. Our analysis of the human HNSCC oncogenome revealed multiple genomic alterations impairing Hippo signaling and activating YAP and TAZ, which in turn contribute to HNSCC development. This includes mutations and deletions of the FAT1 gene (29%) and amplification of the WWTR1 (encoding TAZ, 14%) and YAP1 genes (8%), together representing one of the most genetically altered signaling mechanisms in this malignancy. Here, we discuss key elements of the mammalian Hippo pathway, detail mechanisms by which perturbations in Hippo signaling promote HNSCC initiation and progression and outline emerging strategies to target Hippo signaling vulnerabilities as part of novel multimodal precision therapies for HNSCC.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

Abstract Metastatic melanoma is the most aggressive malignancy of the skin and BRAFV600E mutation is the most common genetic alteration in skin cutaneous melanoma (SKCM). Despite the advent of immune checkpoint inhibition (ICI) immunotherapy, only 40% of patients show long-term responses. As such, combined therapy with BRAF and MEK inhibitors (BRAFi + MEKi) remains the standard of care for BRAFV600E+ SKCM. In the present study, transcriptome analysis of BRAFV600E+ SKCM tumors derived from patients revealed that activation of extracellular matrix signaling, including focal adhesion signaling, is highly enriched in patients who experienced disease progression on BRAFi + MEKi therapy. Consistent with these results, we found increased activation of focal adhesion kinase (FAK) in human BRAFV600E+ SKCM A375 cells treated with BRAFi, MEKi or the RAF/MEK clamp avutometinib. Mechanistically, we discovered that avutometinib-mediated inhibition of the RAF/MEK/ERK pathway decreased RhoE/Rnd3 expression, thereby unleashing RhoA/FAK/AKT signaling. Thus, we hypothesized that FAK activation represents a resistance mechanism to BRAFi + MEKi and that FAK inhibition (FAKi) might overcome resistance to BRAFi/MEKi. Indeed, avutometinib demonstrated synergistic antiproliferative and proapoptotic activity when combined with FAKi in human BRAFV600E+ SKCM A375 cells. Importantly, we found that the combination of FAKi + avutometinib overcame resistance to BRAFi + MEKi in SKCM xenografts and patient-derived cells from SKCM-resistant lesions. Finally, we showed that while BRAFV600E+ SKCM YUMM 1.7 syngeneic tumors failed to respond to ICI therapy, avutometinib ± FAKi inhibited tumor growth. We observed that tumors treated with avutometinib as single agent eventually developed resistance and escaped growth inhibition, but those treated with combined avutometinib and FAKi displayed durable treatment responses, often with complete tumor regression. These findings provide a rationale for the clinical development of avutometinib with defactinib (FAKi) in patients with BRAFV600E+ cutaneous melanoma following progression on BRAFi + MEKi and/or ICI therapy. Citation Format: Simone Lubrano, Farhoud Faraji, Rodolfo Daniel Cervantes-Villagrana, Sydney Ramirez, Nadia Arang, Adam Officer, Damiano C. Rigiracciolo, Paola Y. Anguiano Quiroz, Antonieta Bacchiocchi, Ruth Halaban, Silvia Coma, Sheri Holmen, Claudia Martini, Jonathan Pachter, Andrew Aplin, J. Silvio Gutkind. A novel combination therapy targeting RAF, MEK and FAK to overcome skin cutaneous melanoma treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4745.

Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low-risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. In this study, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified the activation of PKA signaling as the top resistance pathway. We show that metformin mediates PKA activation in HNSCC cells and that PKA inhibition, when combined with metformin treatment, synergistically inhibits HNSCC growth. We found that metformin-induced PKA activation is mediated by a prostaglandin E2 autocrine loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Importantly, COX2 inhibition using nonsteroidal anti-inflammatory drugs (NSAID) combined with metformin treatment synergistically inhibits HNSCC cell growth and prevents the progression of oral premalignant lesions into invasive HNSCC in a model of tobacco-driven oral carcinogenesis. Together, these findings demonstrate that metformin and NSAID combination therapy may represent a promising therapeutic strategy for HNSCC chemoprevention. PREVENTION RELEVANCE: Our findings reveal that using metformin for head and neck cancer chemoprevention leads to compensatory activation of a PKA-driven resistance mechanism that can be blocked by cotreatment with NSAIDs. These findings provide a rationale for combining metformin with NSAIDs as a precision head and neck cancer chemoprevention strategy.