American Association for Cancer Research
21598290cd171263-sup-192218_2_supp_4702098_p9tcjs.pdf (7.63 MB)

Supplementary Material from A Secondary Mutation in BRAF Confers Resistance to RAF Inhibition in a BRAFV600E-Mutant Brain Tumor

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posted on 2023-04-03, 21:40 authored by Jiawan Wang, Zhan Yao, Philip Jonsson, Amy N. Allen, Alice Can Ran Qin, Sharmeen Uddin, Ira J. Dunkel, Mary Petriccione, Katia Manova, Sofia Haque, Marc K. Rosenblum, David J. Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas

Supplementary Figure S1: Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors. Supplementary Figure S2: WES analysis of copy number variation in pre- and postdabrafenib tumors. Supplementary Figure S3: Whole copy number profiles from WES of pre- and posttreatment tumors. Supplementary Figure S4: Homology alignment of BRAF p. L514 with residues in other tyrosine kinases. Supplementary Figure S5: Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing. Supplementary Figure S6: BRAF V600E/L514V reduces dabrafenib sensitivity in NIH- 3T3 cells, related to Figure 2. Supplementary Figure S7: BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G. Supplementary Figure S8: Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H. Supplementary Figure S9: The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B. Supplementary Figure S10: BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib. Supplementary Figure S11: BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation. Supplementary Figure S12: Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. Supplementary Figure S13: Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing BRAF V600E and BRAF V600E/L514V, related to Figure 4C. Supplementary Figure S14: The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D. Supplementary Figure S15: V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A. Supplementary Figure S16: Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S17: Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S18: BGB3245 binds mutant BRAF V600E monomer and second site of V600E/L514V dimer with similar affinity. Supplementary Table S1: Mutations identified by WES of pre-dabrafenib and postdabrafenib tumors. Supplementary Table S2: Secondary mutations associated with acquired resistance and occurring in residues homologous with L514 in BRAF. Supplementary Table S3: BRAF L514V allele frequency determined by ddPCR.



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Marie-Josée and Henry R. Kravis Center for Molecular Oncology of MSKCC



BRAFV600E hyperactivates ERK and signals as a RAF inhibitor–sensitive monomer. Although RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAFV600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAFL514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAFV600E/L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Newer RAF dimer inhibitors and an ERK inhibitor are effective against BRAFL514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that, like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence.Significance: In contrast to receptor tyrosine kinases, in which secondary mutations are often responsible for acquired resistance, second-site mutations in BRAF have not been validated in clinically acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confirm functionally that this mutation is responsible for resistance. Cancer Discov; 8(9); 1130–41. ©2018 AACR.See related commentary by Romano and Kwong, p. 1064.This article is highlighted in the In This Issue feature, p. 1047

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