Disruption of the HER3-PI3K-mTOR oncogenic signaling axis and PD-1 blockade as a multimodal precision immunotherapy in head and neck cancer

Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but <20% of patients achieve durable responses. Persistent activation of the PI3K/AKT/mTOR signaling circuitry represents a key oncogenic driver in HNSCC; however, the potential immunosuppressive effects of PI3K/AKT/mTOR inhibitors may limit the benefit of their combination with ICB. Here we employ an unbiased kinome-wide siRNA screen to reveal that HER3, is essential for the proliferation of most HNSCC cells that do not harbor PIK3CA mutations. Indeed, we find that persistent tyrosine phosphorylation of HER3 and PI3K recruitment underlies aberrant PI3K/AKT/mTOR signaling in PIK3CA wild type HNSCCs. Remarkably, antibody-mediated HER3 blockade exerts a potent anti-tumor effect by suppressing HER3-PI3K-AKT-mTOR oncogenic signaling and concomitantly reversing the immune suppressive tumor microenvironment. Ultimately, we show that HER3 inhibition and PD-1 blockade may provide a multimodal precision immunotherapeutic approach for PIK3CA wild type HNSCC, aimed at achieving durable cancer remission.


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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Sample sizes were chosen based on the historical data of the variability of tumor growth and treatment response observed and determined to be adequate based on the consistency of measurable differences within and between groups. Additionally, our previous publication was used as a reference: DOI: 10.1038/s41467-019-13471-0 No data were excluded from these analyses.
Every experiment was replicated at least three times with near-identical results.
Based on the tumor volumes on the first day of treatment, tumor bearing mice were randomly assigned to treatment groups such that each treatment group or time point/treatment group had the same average tumor volume.
The data presented did not require the use of blinding. Data reported for mouse experiments were not subjective but rather based on quantitative analyses All flow cytometry antibodies from BioLegend, San Diego, CA: For human experiments, we used CD45 (HI30), CD3 (HIT3A), CD8a (HIT8A), CD4 (RPA-T4), Ep-CAM (9CA), E-cadherin (67A4); all antibodies were used at a 1:100 dilution. For mouse experiments, we used CD45 (30-F11) (1:100), CD90.2 (30-H12) (1:200) , CD8a (53-6.7) (1:100), CD4 (RM4-4) (1:400), Ep-CAM (G8.8) (1:100); The flow cytometry antibody used for FITC-HER3 detection was obtained from Celldex Therapeutics and used at a 1:100 dilution. The antibodies for CyTOF are the following: B220 ( All antibodies were validated by the supplier and were checked in the lab by comparing to the manufacturer's or in-house results. All antibodies were validated by the supplier and were checked in the lab by comparing to the manufacturer's or in-house results. Statement from BioLegend: BioLegend antibodies undergo an extensive series of testing to ensure quality at every step in the nature research | reporting summary Note that full information on the approval of the study protocol must also be provided in the manuscript.

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4MSOC1 syngeneic cell lines were generated in-house and manipulated according to our previous publicatoin: DOI: 10.1038/ s41467-019-13471-0. MOC1 cells were generously provided by Dr. R. Uppaluri lab. Human HNSCC cell lines Cal27, HN12, SCC47 and Detroit 562 were obtained from the NIDCR (National Institute of Dental and Craniofacial Research) cell collection.
DNA authentication of cell lines was confirmed by multiplex STR profiling (Genetica DNA Laboratories, Inc. Burlington, NC) to ensure the consistency of cell identity.
All cell lines are frequently tested for mycoplasma contamination. No presence of mycoplasma was found according to Mycoplasma Detection Kit-QuickTest from Biomake (Houston, TX, USA).
No commonly misidentified cells were used.
Female athymic mice and C57Bl/6 mice (4-6 weeks of age and weighing 16-18g) were purchased from Charles River Laboratories (Worcester, MA, USA). All the animal studies using HNSCC tumor xenografts and oral carcinogenesis studies were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, San Diego, with protocol ASP #S15195. Animal housing conditions are described in the manuscript.
Study did not involve wild animals.
Study did not involve samples from the field. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, San Diego, and mouse procedures were performed following ACP guidelines.
The relevant covariate characteristics for the surgical specimen are as follows: the data presented in Figure 1 E and 1F are from a representative fresh surgical specimen of a Stage II T2N0M0 primary tongue squamous cell carcinoma. This particular experiment examined the expression of HER3 within the tumor and immune compartments in the tumor-microenvironment; and, in this context, additional covariate population characteristics are not relevant. This information also appears in legend