Mutant-selective degradation by BRAF-targeting PROTACs

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.


Reporting for specific materials, systems and methods
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. All in vitro or cell based experiments were performed two or more independent times. All attempts at replication of experimental results were successful. For mice studies: Mice bearing xenografts of cancer cells were treated with either vehicle or drug to determine the effect on tumor growth. Tumor sizes are represented by mean ± SD in the graphs (n = 3). The replicates for tumor growth measurements indicate biological replicates.
For in vivo tumor growth experiments: implanted mice were treated in a random manner with vehicle, or drug treatment. Randomization was not relevant to the remaining cell based or biochemical experiments.
Investigators were not blinded to the nature of their samples during data collection and analysis. Experiments were designed based on preliminary results.

April 2020
Validation Primary antibody from Lifespan Biosciences: anti-KRAS (LS-C175665), Dilution 1:1000 Secondary antibodies were from ThermoFisher: • anti-rabbit HRP (31460) Dilution 1:50000 • anti-mouse HRP (31444) Dilution 1:10000 All the antibodies used in this study were purchased from commercial sources and had been validated by their respective manufacturer for immunoblotting and/or immunoprecipitation (the applications used in this study) of the human isoform of their cognate antigens.
Cell Signaling Validation Statement: To ensure our antibodies will work in your experiment, we adhere to the Hallmarks of Antibody Validation™, six complementary strategies that can be used to determine the functionality, specificity, and sensitivity of an antibody in any given assay. CST adapted the work by Uhlen, et. al., ("A Proposal for Validation of Antibodies." Nature Methods (2016)) to build the Hallmarks of Antibody Validation, based on our decades of experience as an antibody manufacturer and our dedication to reproducible science. • Binary Model: Antibody signal is measured in model systems with known presence/absence of target signal. Includes wild-type vs. genetic knockout, targeted induction or silencing. • Ranged Expression: Antibody signal strength is measured in cell lines or tissues representing a known continuum of target expression levels. Includes siRNA and heterozygous knockout assays. • Orthogonal Data: Antibody signal is correlated to target expression in model systems measured using antibody independent assays. Includes mass spectrometry and in situ hybridization. • Multiple Antibodies: Antibody signal is compared to the signal observed using antibodies targeting nonoverlapping epitopes of the target. Includes IP, ChIP, and ChIP-seq. • Heterologous Expression: Antibody signal is evaluated in cell lines following heterologous expression of native (or mutated) target protein.
• Complementary Assays: Antibody specificity may be validated using complementary assays. Includes competitive ELISA, peptide dot blots, peptide blocking, or protein array nature research | reporting summary . Inducible expression NIH3T3 cells were maintained in DMEM;50"g ml#1 hygromycin and 0.2"g ml#1 puromycin). All media was supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin and grown in a humidified incubator at 37°C and 5% CO2.
The cell lines purchased from ATCC were purchased as 'Certified Reference Material' stocks. Cell lines received from other labs were not verified. ATCC authenticates cell lines using morphology, karyotyping and STR profiling.
The cell lines used in this study tested negative for mycoplasma contamination according to the MycoAlert test kit from Lonza.
None of the cell lines used in this study are listed in the current version (v.9) of the ICLAC register.
All our mouse studies are conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Memorial Sloan Kettering Cancer Center (MSKCC). Memorial Sloan-Kettering Cancer Center's animal care and use program is administered by the Research Animal Resource Center (RARC). The program has been fully accredited by the Association of Assessment and Accreditation of Animal Care, International (AAALAC) since 1967, is registered with the USDA, and has an approved assurance on file with the Office of Laboratory Animal Welfare, NIH (OLAW). RARC is staffed by board-certified laboratory animal veterinarians and pathologists, veterinary and animal care technicians, management, and administrative support staff. Veterinary staff is available 24 hours a day, 7 days a week to address emergencies. The program is supported by the Laboratory of Comparative Pathology which provides anatomic and clinical pathologic evaluation of animals, tissues, and fluids in support of animal health and the use of animal models.
The animal resource program is housed in three state-of-the-art facilities occupying a total of 62,500 net ft2 of usable space. All vivaria contain barrier rodent housing facilities. Specialized facilities for the use of animal models exposed to biological and hazardous chemical agents and for conducting surgical procedures in large and small animals are available.
Female athymic nu/nu mice, 6-8 weeks old . temperature: 72 degrees F humidity: 50% relative humidity lighting: 12 hours on starting at 7 am/12 hours off starting at 7 pm the mice have access to food and water ad libitum Study did not include wild animals