Loss of HAT1 expression confers BRAFV600E inhibitor resistance to melanoma cells by activating MAPK signaling via IGF1R

Abstract

BRAF inhibitors (BRAFi) have been approved for the clinical treatment of BRAF-mutant metastatic melanoma. Although initial responses to BRAFi are generally favorable, acquired BRAFi resistance emerges rapidly, resulting in treatment failure. Only some of the underlying mechanisms responsible for BRAFi resistance are currently understood. Here, we showed that the genetic inhibition of histone acetyltransferase 1 (HAT1) in BRAF-mutant melanoma cells resulted in BRAFi resistance. Using quantitative immunofluorescence analysis of patient sample pairs, consisting of pre-treatment along with matched progressed BRAFi + MEKi-treated melanoma samples, HAT1 downregulation was observed in 7/11 progressed samples (~63%) in comparison with pre-treated samples. Employing NanoString-based nCounter PanCancer Pathway Panel-based gene expression analysis, we identified increased MAPK, Ras, transforming growth factor (TGF)-β, and Wnt pathway activation in HAT1 expression inhibited cells. We further found that MAPK pathway activation following the loss of HAT1 expression was partially driven by increased insulin growth factor 1 receptor (IGF1R) signaling. We showed that both MAPK and IGF1R pathway inhibition, using the ERK inhibitor SCH772984 and the IGF1R inhibitor BMS-754807, respectively, restored BRAFi sensitivity in melanoma cells lacking HAT1. Collectively, we show that the loss of HAT1 expression confers acquired BRAFi resistance by activating the MAPK signaling pathway via IGF1R.

Fig. 1. Loss of HAT1 expression confers resistance to BRAF inhibitors.

a Indicated melanoma cell lines, expressing either nonspecific (NS) shRNA or HAT1 shRNAs, were analyzed for HAT1 protein expression levels by immunoblotting. ACTINB was used as a loading control. b Indicated melanoma cell lines, expressing either a nonspecific (NS) shRNA or HAT1 shRNAs, were analyzed by qRT-PCR. Actin was used as an internal control. c Relative survival rates of A375 and SKMEL-28 cells, expressing either nonspecific (NS) shRNA or the indicated HAT1 shRNA, upon treatment with vemurafenib for 3 days, as measured by MTT assay. d Relative survival rates of A375 and SKMEL-28 cells, expressing either nonspecific (NS) shRNA or the indicated HAT1 shRNA, upon treatment with dabrafenib for 3 days, were measured by MTT assay. e IC50 values for the MTT data presented in panels c and d. f A375 or SKMEL-28 cells, expressing either a nonspecific (NS) shRNA or HAT1 shRNAs, were treated with DMSO or vemurafenib (1 μM) and analyzed by the soft-agar assay. Scale bar is 100 µm. g Relative colony sizes for the data presented in panel f. Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.000, calculated using Student’s t test.

Fig. 2. HAT1-knockout-mediated resistance leads to the long-term survival of melanoma cells treated with BRAF inhibitor.

a Immunoblotting for the indicated proteins in A375 and SKMEL-28 HAT1-knockout (HAT1-KO) cells. b Clonogenic assay results for A375 and SKMEL-28 cells, expressing non-targeting (NT) or HAT1 sgRNAs, in the presence of vemurafenib (3 μM) (left). Colony number for the data are shown (right). c Clonogenic assay results for A375 and SKMEL-28 cells, expressing NT or HAT1 sgRNAs, in the presence of dabrafenib (100 nM) (left). Colony number for the data presented (right). Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, calculated using Student’s t test.

Fig. 3. HAT1 protein is downregulated in progressed patient-derived melanoma samples following BRAF inhibitor treatment.

a Average AQUA scores for pre-treatment and progressed melanoma samples from patients treated with BRAFi or BRAFi + MEKi. b Representative AQUA immunofluorescence scores and merged images of the indicated matched pre-treatment and progressed melanoma samples, stained for DAPI, S100/HMB45, and HAT1. Data are presented as the mean ± SEM. ***p < 0.001, ****p < 0.0001, calculated using Student’s t test.

Fig. 4. The loss of HAT1 expression leads to the activation of alternative signaling pathways, which contribute to BRAFi resistance in melanoma cells.

a Heatmap for A375 cells, expressing non-targeting (NT) or HAT1 sgRNAs. b Heatmap for A375 cells, expressing nonspecific (NS) or HAT1 shRNAs. c. Heatmap for SKMEL-28 cells, expressing NS or HAT1 shRNAs. d Venn diagram showing the genes that were significantly up- and downregulated in A375 HAT1 knockout (KO), A375 HAT1 knockdown (KD), and SKMEL-28 HAT1 KD cells in the Ras, MAPK, Wnt, and TGF-β signaling pathways. e Fold changes of the genes associated with the Ras, MAPK, TGF-β, and Wnt signaling pathways that were significantly altered in A375 cells expressing NT or HAT1 sgRNAs, A375 cells expressing NS or HAT1 shRNAs, and SKMEL-28 cells expressing NS or HAT1 shRNAs. Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, calculated using Student’s t test.

Fig. 5. Loss of HAT1 expression leads to the IGF1R-dependent activation of the MAPK signaling pathway, which confers BRAFi resistance in melanoma cells.

a Immunoblotting for phospho- and the total ERK in A375 and SKMEL-28 cells expressing nonspecific NS or HAT1 shRNAs. b Relative IGF1R mRNA levels in A375 and SKMEL-28 cells expressing nonspecific NS or HAT1 shRNAs were analyzed by qRT-PCR. Actin was used as an internal control. c Immunoblotting for phospho- and the total IGF1R in A375 and SKMEL-28 cells expressing nonspecific NS or HAT1 shRNAs. d, f A375 or SKMEL-28 cells expressing a nonspecific (NS) shRNA or HAT1 shRNAs were treated with DMSO, BRAF inhibitor vemurafenib (1 μM), IGF1R inhibitor, BMS-754807 (0.1 μM) and ERK inhibitor, SCH772984 (0.2 μM), which was analyzed by soft-agar assay. e, g Relative colony size for data presented in panels d and f. Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, calculated using Student’s t test.

Fig. 6. Pharmacological inhibition of IGF1R and ERK suppresses the growth of BRAFi-resistant cells following the loss of HAT1 expression.

a Immunoblotting for phospho- and total ERK in A375 cells expressing non-targeting NT or HAT1 sgRNAs. b Relative IGF1R mRNA levels in A375 cells expressing non-targeting NT or HAT1 sgRNAs was analyzed by qRT-PCR. Actin was used as internal control. c, e A375 or SKMEL-28 cells expressing a non-targeting (NT) sgRNA or HAT1 sgRNAs were treated with DMSO, BRAF inhibitor vemurafenib (3 μM), IGF1R inhibitor, BMS-754807 (1 μM) and ERK inhibitor, SCH772984 (1 μM) was analyzed by clonogenic assay. d, f Relative colony number for data presented in panels c and e. Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, calculated using Student’s t test.

Fig. 7. Model depicting the mechanism of HAT1 loss-induced BRAFi resistance.

One of the mechanisms by which loss of HAT1 drives acquired BRAFi resistance is via IGF1R-dependent activation of MAPK signaling pathway, and the pharmacological targeting of IGF1R or ERK1/2 restores the sensitivity of melanoma cells with reduced HAT1 expression levels to BRAFi.