Anaplastic thyroid cancer (ATC) is one of the most lethal human cancers, with some patients succumbing to the disease within weeks of diagnosis. Although a subset of patients with ATC with BRAFV600E mutation respond to the monomeric type I RAF inhibitor (RAFi) dabrafenib in combination with MEK inhibitor (MEKi) trametinib, most rapidly develop adaptive or acquired resistance. These patients, along with those who do not harbor the BRAFV600E alteration, have limited treatment options. To understand the mechanism of resistance to dabrafenib and trametinib, we utilized multi-region whole genome, high-coverage whole exome and single nuclei RNA-sequencing of ATC patient tumours to unravel genomic, transcriptomic, and microenvironmental evolution during type I RAFi and MEKi therapy. Single-cell nuclei RNA sequencing of matched primary and resistant ATC patient tumours identified reactivation of the MAPK-pathway, along with immunosuppressive macrophage proliferation, underlying the development of acquired resistance. Our translational genomics led us that hypothesize that type II RAFi, which inhibit both RAF monomers and dimers, can be efficacious in overcoming treatment resistance. Screening of a panel of type II RAFi revealed that ATC cell lines are exquisitely sensitive to the type II RAFi, naporafenib, by inhibiting EphA2-mediated MAPK-signaling. We further demonstrated that naporafenib, in combination with the MEKi trametinib, can durably and robustly overcome both innate and acquired treatment resistance to dabrafenib and trametinib using ATC cell lines and patient-derived xenograft models. Finally, we describe a novel mechanism of acquired resistance to type II RAFi and MEKi through compensatory mutations in MAST1. Taken together, our work using translational and functional genomics has unraveled the differential mechanisms of treatment resistance to type I and type II RAFi in combination with trametinib and rationalizes the clinical investigation of type II RAFi in the setting of thyroid cancer.

A.C.N has research funding from Novartis Canada, Merck Canada, LabCorp, and Droplet Biosciences for studies that are unrelated to the submitted work. He has equity from and is a consultant for NEED Inc. M.J.C has research funding from Astra Zeneca, Merck and Pfizer. He has received payment for speaker honorarium and/or served on advisory boards for Eli Lilly Merck, Astra Zeneca, and Amgen. M.J.C has equity from and is a consultant for NEED Inc. D.A.P is a consultant and has equity from NEED Inc. E.W has consulted or advised for Merck, Bayer, Eisai, Roche, Ipsen, and EMD Serono. E.W reports research funding from Merck, Novartis, and Lilly through Western University. P.Y.F.Z, J.W.B, J.S.M, and A.C.N hold patents for transcriptional biomarkers in head and neck cancer, unrelated to this work. All other authors have no conflicts of interest to declare.

This work was supported by Canadian Institutes of Health Research grant MOP 487005 to ACN, LG, LA and PCB, MOP 145586 to PCB and by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-125). This work was also supported through generous donations by the: 1) London Health Sciences Foundation (Robert and Sheila Wilkes, Betty and Harry Ostrander, Mervyn and Joyce Dietz) and 2) the Woodstock Foundation (Laila, Arnold, Mario, Andrea, Tony and Tomassina Spina, Susan and Bill George, Piero and Maria Manzini, Henry and Rina Deroo, Harry and Shani Loewith, Franco and Carol Castellucci, Tony and Bill Van Haeren, Billy Stevanovich, Jennifer Peace-Hall, Tom and Pat Baird, Christine and Jeff Nichols, Cliff and Linda Zaluski, Thomas Vandertuin, Laurie and Paul Green, Kelli and Mike Koopman). ACN was supported by the Wolfe Surgical Research Professorship in the Biology of Head and Neck Cancers Fund. LA is supported by an Ontario Institute for Cancer Research Investigator Award (OICR IA-016). PYFZ is supported by a Vanier Canada Graduate Scholarship and PSI foundation fellowship.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study has been approved by the Western University institutional ethics board (REB#108920) and received written consent from the patients.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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All generated bulk RNA-seq, whole exome sequencing, and single nuclei data sets will be made available upon acceptance at EGA. TCGA data was retrieved from the National Cancer Institute Genome Data Commons (https://portal.gdc.cancer.gov/). The cancer cell line encyclopedia was retrieved using CellMinerDB (https://discover.nci.nih.gov/rsconnect/cellminercdb/).