Identification of acquired Notch3 dependency in metastatic Head and Neck Cancer

ARTICLE https://doi.org/10.1038/s42003-023-04828-9 OPEN Identification of acquired Notch3 dependency in metastatic Head and Neck Cancer 1234567890():,; Maria Kondratyev 1 ✉, Aleksandra Pesic1, Troy Ketela1, Natalie Stickle2, Christine Beswick1, Zvi Shalev1, Stefano Marastoni 1, Soroush Samadian1, Anna Dvorkin-Gheva1, Azin Sayad1, Mikhail Bashkurov3, Pedro Boasquevisque1, Alessandro Datti3, Trevor J. Pugh 1, Carl Virtanen2, Jason Moffat4, Reidar A. Grénman5, Marianne Koritzinsky1 & Bradly G. Wouters 1 ✉ During cancer development, tumor cells acquire changes that enable them to invade surrounding tissues and seed metastasis at distant sites. These changes contribute to the aggressiveness of metastatic cancer and interfere with success of therapy. Our comprehensive analysis of “matched” pairs of HNSCC lines derived from primary tumors and corresponding metastatic sites identified several components of Notch3 signaling that are differentially expressed and/or altered in metastatic lines and confer a dependency on this pathway. These components were also shown to be differentially expressed between early and late stages of tumors in a TMA constructed from over 200 HNSCC patients. Finally, we show that suppression of Notch3 improves survival in mice in both subcutaneous and orthotopic models of metastatic HNSCC. Novel treatments targeting components of this pathway may prove effective in targeting metastatic HNSCC cells alone or in combination with conventional therapies. 1 Princess Margaret Cancer Centre University Health Network, Toronto, ON, Canada. 2 Princess Margaret Cancer Center, Bioinformatics and HPC Core, Toronto, ON, Canada. 3 SMART High-Content Screening facility at Network Biology Collaborative Centre, Toronto, ON, Canada. 4 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 5 Turku University Hospital, Turku, Finland. ✉email: ; COMMUNICATIONS BIOLOGY | (2023)6:538 | https://doi.org/10.1038/s42003-023-04828-9 | www.nature.com/commsbio 1 ARTICLE M COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04828-9 etastatic cells acquire new and unique properties that permit them to invade tissues and seed metastases at distant sites1,2. In HNSCC, gene expression studies lead to identification of 102 genes that predict presence of lymph node metastasis in patients3,4. The first attempt to uncover genetic alterations that characterize metastatic HNSCC was performed by targeted sequencing of a collection of 53 recurrent and metastatic HNSCC tumors comparing to matched normal5. This study revealed several molecular alterations that were not previously reported in the primary tumors. Another study performed whole exome sequencing of 13 synchronous lymph node metastases and 10 metachronous recurrent tumors; notably, they were able to compare the advanced tumor samples to both matched normal controls and matched primary tumors from the same patients6. While 60–80% of mutations were shared between primary tumors and either lymph node metastasis or recurrences, several unique alterations were identified. Another group utilized whole exome sequencing as well as RNA sequencing to characterize a collection of HNSCC cell lines developed from primary tumors and matched metastases7. The authors confirmed that UM-SCC lines recapitulate most of the known genomic alterations reported for HNSCC; moreover, they discovered several novel mutations only present in metastatic/recurrent tumor but not in the matched primary. While genome sequencing and expression profiling provides large amounts of data describing biological properties of cancers, these frequently do not reflect genes and pathways that are functionally important for survival and proliferation of tumor cells. Thus, the development of effective targeted therapies will require a better understanding of both the genetic and functional differences within metastatic disease. In this study, we utilized functional genomic and genomic profiling technologies to perform comprehensive analysis of unique collection of “matched” pairs of HNSCC lines derived from primary tumors and corresponding cervical lymph node metastases. All the cell lines were derived from patients with HPV negative tumors that are known to have worse prognosis compared to the HPV positive subpopulation and will likely require a separate type of treatment for their eradication. Interestingly, both functional and genomic analyses identified a differential and key survival role of the Notch3 signaling pathway in the metastatic lines compared to those derived from primary tumors. The Notch signaling pathway is conserved from Drosophila to human and plays a central role in development, self-renewal, and differentiation8–10. The role of Notch signaling in cancer is being extensively investigated and newly developed drugs that target different arms of the pathway show promising results in preclinical studies11–15. Paradoxically, in HNSCC the Notch pathway has been shown to play roles in both oncogenic and tumor suppressor activities. Several reports demonstrate high frequency of loss-of-function mutations in Notch1 in HNSCC tumors, and consequently a tumor-suppressive role of Notch signaling in at least some subtypes of this disease16–20. In fact, close to 19% of HNSCC tumors harbor inactivating mutations in Notch1, making it the second most commonly mutated gene after TP53. However, other recent reports demonstrate an overexpression of Notch pathway components in HNSCC tumors suggesting oncogenic properties of the pathway21–25. Genes with elevated expression included the receptors (Notch1, Notch2 and Notch3), ligands (Jag1, Jag2) and target genes Hes1 and Hey121–25. Importantly, functional consequences of pathway activation have also been reported. Pharmacological inhibition of the Notch signaling by gamma-secretase inhibitors or knocking down Notch1 significantly reduce cell proliferation and invasion of HNSCC cells14,26. While most mutational and expression profiling data in HNSCC has been performed on primary tumors, recent analysis 2 of metastasis-derived samples suggested increased alterations in Notch signaling and reported mutations in Notch3 gene7. These observations suggest a dual role of Notch in HNSCC, which is context dependent and needs to be further investigated. Our findings provide with important insight into HNSCC pathogenesis, suggesting that metastatic cells acquire dependency on Notch3 signaling that may be amenable to targeting metastatic disease. Results Molecular profiling reveals common changes in matched pairs of HNSCC primary tumor and metastasis-derived cell lines. Identification of both genetic and functional differences between primary and metastatic variants of HNSCC would be enabled by in vitro models derived independently from these sites from the same patient. Unfortunately, HNSCC tumor cells have typically been difficult to isolate and adapt to grow in vitro and as a consequence there are few commercially a (...truncated)