kdm6a inhibitor gskj4  (Tocris)

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Inhibition of KDM6A prevents the H3K27 methyl-to-acetyl switch and gene activation induced by BRAF V600E inhibition. (A) Pharmacological inhibition of KDM6A by GSKJ4 in A375P cells increases H3K27me3 while reducing H3K27ac in a dose-dependent manner. Cells were treated with DMSO or GSKJ4 at the indicated concentration for 24 h. (B-C) GSKJ4 treatment inhibits the BRAF V600E inhibition–induced changes in H3K27me3 and H3K27ac in A375P cells, as shown by Western blot ( B ) and immunofluorescence ( C ). Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. (D) In A375P cells, KDM6A inhibition blunts the PLX4032-mediated alterations in H3K27ac occupancy at the PGC1α, DCT, MET, and WDR19 promoters, as determined by ChIP. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. (E) GSKJ4 treatment abrogates the upregulation of EZH2 target genes (PGC1α, DCT, MET, WDR19) by BRAF V600E inhibition in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. ( F ) KDM6A knockout efficiency in A375P cells was confirmed by qRT-PCR and Western blot. (G-I) CRISPR-mediated KDM6A deletion prevents the changes in H3K27me3 and H3K27ac triggered by BRAF V600E inhibition in A375P cells, as shown by Western blot ( G ) and immunofluorescence ( H and I ). Cells were treated with DMSO or 2 μM PLX4032 for 24 h. (J) KDM6A deletion abolishes the alterations in H3K27ac occupancy at the PGC1α, DCT, MET, and WDR19 promoters in A375P cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (K) CRISPR-mediated KDM6A deletion suppresses the BRAF V600E inhibition–induced upregulation of EZH2 target genes in A375P cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. Data are presented as mean ± SEM and representative of two to three independent experiments with similar results ( n = 3/group/experiment). * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t-test in (F) and by two-way ANOVA in (C), (D), (E), (I), (J), and (K).

Article Snippet: PLX4032 (MedChemExpress, #HY-12057), PLX4720 (AdooQ, #A10002), 3-Deazaneplanocin A (DZnep; MedChemExpress, #HY-14660), JQ1 (AdooQ, #A12729), GSK126 (MedChemExpress, #HY-13470), and GSKJ4 (MedChemExpress, #HY-15648B) were dissolved in DMSO (Sangon Biotech, #A600163) at stock concentrations of 10 or 50 mM.

Techniques: Inhibition, Activation Assay, Concentration Assay, Western Blot, Immunofluorescence, Knock-Out, Quantitative RT-PCR, CRISPR

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Blockade of the KDM6A–H3K27ac–BRD4 axis reprograms melanoma metabolism. (A) Pharmacological inhibition of BRD4 by JQ1 attenuates the PLX4032-induced mitochondrial metabolic program in A375P cells. Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 16 h. (B) CRISPR-mediated BRD4 depletion suppresses the mitochondrial metabolic program triggered by PLX4032 in A375P melanoma cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (C-D) BRD4 inactivation reduces mitochondrial content ( C ) and respiration ( D ) induced by BRAF V600E inhibition in A375P cells, as measured by MitoTracker staining and a Clark-type oxygen electrode, respectively. Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 24 h. (E) Pharmacological inhibition of KDM6A activity by GSKJ4 prevents the PLX4032-mediated increase in the mitochondrial metabolic program in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 16 h. (F) CRISPR-mediated KDM6A depletion suppresses the PLX4032-induced mitochondrial metabolic program in A375P melanoma cells. Cells were treated with DMSO or 2 μM PLX4032 for 16 h. (G-H) KDM6A inhibition decreases mitochondrial content ( G ) and respiration ( H ) following BRAF V600E inhibition in A375P cells. Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. Data are represented as mean ± SEM, and representative of two to three independent experiments with similar results ( n = 3/group/repeat). * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-way ANOVA.

Article Snippet: PLX4032 (MedChemExpress, #HY-12057), PLX4720 (AdooQ, #A10002), 3-Deazaneplanocin A (DZnep; MedChemExpress, #HY-14660), JQ1 (AdooQ, #A12729), GSK126 (MedChemExpress, #HY-13470), and GSKJ4 (MedChemExpress, #HY-15648B) were dissolved in DMSO (Sangon Biotech, #A600163) at stock concentrations of 10 or 50 mM.

Techniques: Inhibition, CRISPR, Staining, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A methyl-to-acetyl switch in H3K27 drives metabolic reprogramming and resistance to BRAF V600E inhibition in melanoma

doi: 10.1016/j.neo.2025.101223

Figure Lengend Snippet: Targeting the H3K27 methyl-to-acetyl switch sensitizes melanoma to BRAF V600E inhibition. (A-B) BRD4 inactivation by JQ1 enhances the efficacy of PLX4032 in suppressing melanoma cell survival, as assessed by cell proliferation ( A ) and apoptosis (indicated by cleaved PARP1) ( B ). Cells were treated with DMSO, 2 μM JQ1, 2 μM PLX4032, or both for 24 h. (C-D) Pharmacological inhibition of KDM6A with GSKJ4 synergizes with PLX4032 to further suppress melanoma cell survival ( C ) and induce apoptotic cell death ( D ). Cells were treated with DMSO, 5 μM GSKJ4, 2 μM PLX4032, or both for 24 h. (E) The BRD4 inhibitor JQ1 significantly sensitizes BRAF V600E -mutant YUMM1.7 melanoma to PLX4032 in vivo . Vehicle ( n = 6), PLX4032 ( n = 5), JQ1 ( n = 5), PLX4032+JQ1 ( n = 10). The diagram illustrates the treatment strategy. (F) Inhibition of KDM6A by GSKJ4 significantly sensitizes BRAF V600E -mutant YUMM1.7 melanoma to PLX4032 in vivo . Vehicle ( n = 4), PLX4032 ( n = 4), GSKJ4 ( n = 4), PLX4032+GSKJ4 ( n = 5). (G-H) Treatment with GSKJ4 in combination with BRAF V600E inhibition reduces tumor progression ( G ) and prolongs survival ( H ) in a genetically engineered melanoma mouse model (Tyr::Cre ERT2 ;Braf CA ;Pten lox/lox ). Mice were induced with 4-hydroxytamoxifen (4-OHT; n = 5 per group for G, n = 6 per group for H). (I ) Diagram summarizing the interplay between BRAF V600E and the H3K27 epigenetic program in melanoma. Data are represented as mean ± SEM, and representative of two to three independent experiments with similar results in (A) and (C) ( n = 3/group/repeat). Statistical significance was determined by two-way ANOVA. Survival curves were estimated using the Kaplan–Meier method and compared using the log-rank (Mantel–Cox) test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: PLX4032 (MedChemExpress, #HY-12057), PLX4720 (AdooQ, #A10002), 3-Deazaneplanocin A (DZnep; MedChemExpress, #HY-14660), JQ1 (AdooQ, #A12729), GSK126 (MedChemExpress, #HY-13470), and GSKJ4 (MedChemExpress, #HY-15648B) were dissolved in DMSO (Sangon Biotech, #A600163) at stock concentrations of 10 or 50 mM.

Techniques: Inhibition, Mutagenesis, In Vivo