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Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression., Significance: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis., (©2023 American Association for Cancer Research.)

Background: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance., Methods: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8 + T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment., Results: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8 + T cells. Activation of CD8 + T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R + CD11c + MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME., Conclusion: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination., Competing Interests: Competing interests: JH was paid consultant for Bristol-Myers Squibb and MSD Sharpe & Dohme and has received other commercial research support from CureVac A G and PROGEN Biotechnik, Research funding from AstraZeneca, outside the scope of this work (to LGTM). LGTM is an inventor on a patent held by Memorial Sloan Kettering related to tumor mutational burden and immunotherapy. MS is an employee and stockholder of Astra Zeneca. JF received Honoraria from Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme, Merck Serono, Innate pharma, Roche, serve as an advisor in, Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme, Merck Serono, Innate pharma, Roche, has a research fund by Bristol-Myers Squibb, and had travel grants from Astra Zeneca, Bristol-Myers Squibb, Merk Sharp & Dohme., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)