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 < 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 (<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 < 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. 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.

Aging is a consequence of complex molecular changes, but whether a single microRNA (miRNA) can drive aging remains unclear. A miRNA known to be upregulated during both normal and premature aging is miR-29. We find miR-29 to also be among the top miRNAs predicted to drive aging-related gene expression changes. We show that partial loss of miR-29 extends the lifespan of Zmpste24-/- mice, an established model of progeria, indicating that miR-29 is functionally important in this accelerated aging model. To examine whether miR-29 alone is sufficient to promote aging-related phenotypes, we generated mice in which miR-29 can be conditionally overexpressed (miR-29TG). miR-29 overexpression is sufficient to drive many aging-related phenotypes and led to early lethality. Transcriptomic analysis of both young miR-29TG and old WT mice reveals shared downregulation of genes associated with extracellular matrix organization and fatty acid metabolism, and shared upregulation of genes in pathways linked to inflammation. These results highlight the functional importance of miR-29 in controlling a gene expression program that drives aging-related phenotypes. A miRNA screen identifies miR-29 as a driver for aging. Expression of miR-29 is sufficient to accelerate aging while its reduction delays aging in mouse models and transcriptomics reveals miR-29-regulated pathways associated with aging.

Abstract Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal–induced pERK rebound upregulated p38–FRA1–JUNB–CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death–predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction. Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74–93. ©2017 AACR. See related commentary by Stern, p. 20. This article is highlighted in the In This Issue feature, p. 1

Supplementary Figures 1-2 from Expression of p16Ink4a Compensates for p18Ink4c Loss in Cyclin-Dependent Kinase 4/6–Dependent Tumors and Tissues

XLSX file - 32K, Modules/expression signatures analysis of carboplatin/paclitaxel treated C3(1)-T-antigen mouse tumors.

Supplementary Figures S1 - S10, Tables S1 - S2

PDF file - 46K, Univariate and Multivariate Analysis for pCR.

Supplementary Table 1. Clinical and histologic characteristics of cutaneous melanocytic nevi, primary melanomas, and melanoma metastases and their relationship to ITK protein levels

70 genes selected for validation.

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Supplementary Table 6. Significant pathways identified by Ingenuity Pathway Analysis for Reverse Phase Protein Array (RPPA) results of RPMI 8322

PDF file, 53K, MP1U Dosing Regimens.

PDF file - 806K, Supplemental Figure 1. MRI scans at the indicated time points showing tumor burden in EML4-ALK lung cancer mice treated by crizotinib. Multiple scans from a total of 4 mice are shown. Supplemental Figure 2. Representative MRI images showing that EML4-ALK F1174L mutant lung cancers respond to 17-DMAG and TAE684. Note the development of acquired resistance to 17-DMAG and TAE684 after prolonged treatment. Supplemental Figure 3. Acquired resistance to 17-DMAG and TAE684 in mice bearing tumors driven by the EML4-ALK F1174L mutant can be overcome by the combination of the two drugs.

XLSX file - 60K, Gene lists obtained from a study of carboplatin/paclitaxel treated tumors.

XLSX file - 16K, List of mouse C3(1)-T-antigen gene expression microarrays used to derive the murine gene lists.

Kinome and transcriptome responses to combined BRAF and MEK inhibition in murine tumors, patient samples, and human cell lines