Abstract 2051: Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in <i>BRAFV600</i>-mutant metastatic melanoma

Abstract To investigate the landscape of the activated kinome in MM, we applied a proteomic approach that interrogates the activation status of more than half of the entire kinome (∼300), termed multiplexed kinase inhibitor bead affinity chromatography coupled with mass spectrometry (MIB/MS) on 45 MM patient (pt) tumors (BRAFV600, n=25; NRASQ61, n=5, NF1 loss-of-function, n=4; Triple wild-type n=11) in conjunction with whole exome sequencing and RNA sequencing (RNAseq). We performed exploratory analyses (Wilcoxon rank sum test for each kinase) on the untreated tumors to identify differentially activated kinases in BRAFV600-mutant MM vs. all others. While no activated kinases were differentially expressed in BRAFV600-mutant MM after adjustment for multiple testing, AKT1, PDGFRA, and MAP3K1 kinases exhibited higher MIB binding (i.e., more activated) in BRAFV600E-mutant pts (unadjusted p-value &amp;lt; 0.05). Spearman’s rank correlation tests between the activated kinase and its gene expression by RNAseq and adjusted for multiple testing did not identify a significant correlation for any kinase, suggesting a complex mechanism of kinase activation other than sole gene expression. K-means clustering analysis to classify melanomas into 4 subtypes using a 6-gene discriminant expression signature showed that BRAFV600-mutant tumors were more frequently classified as melanocytic (9/24, MLANA/MITF/SOX10high, AXLlow). In contrast, triple wild-type tumors were more frequently classified as transitory (5/10, MITF/SOX10/ETV4high, AXLlow). We then performed similar exploratory analyses on the baseline (untreated) tumors from the subset of the 20 BRAFV600E-mutant pts who received D+T as part of a prospective clinical trial (NCT01726738; median follow-up 30.5 months, range 2.0-116.1 months) to identify differentially activated kinases that would predict shorter (&amp;lt;12 months) vs. longer progression-free survival. Again, while no kinases survived multiple comparison testing, the bromodomain protein TRIM28, known to regulate melanoma plasticity, exhibited a more consistent loss of MIB binding in BRAFV600E-mutant pts who progressed shorter than in pts who progressed longer than 12 months on D+T (unadjusted p-value &amp;lt; 0.05). 7 baseline-progression tumor pairs were evaluated for MIB/MS, and 6 were for RNAseq. Although diverse resistance mechanisms were identified, as previously described, we also identified melanoma subtype switch in 4/6 pt tumors and a consistent loss of MIB binding of the non-receptor tyrosine kinase PTK6 in response to D+T, an off-target of D and vemurafenib but not encorafenib. Our in vitro findings revealed that off-target PTK6 inhibition by D could activate beta-catenin signaling and contribute to the development of resistance. Citation Format: Steve P. Angus, Naim U. Rashid, Andrew J. Walther, David L. Corcoran, Steven D. Rhodes, Frances A. Collichio, C. Paige Jones, Mikaela J. Bauer, Joel S. Parker, Noah Sciaky, 1Alisha R. Coffey, Samantha M. Bevill, Brian T. Golitz, v Timothy J. Stulhmiller, Nancy E. Thomas, David W. Ollila, Norman E. Sharpless, Carrie B. Lee, Gary L. Johnson, Stergios J. Moschos. Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in BRAFV600-mutant metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2051.