Ibrutinib Blocks YAP1 Activation and Reverses BRAF Inhibitor Resistance in Melanoma Cells

Abstract

Most B-Raf proto-oncogene (BRAF)-mutant melanoma tumors respond initially to BRAF inhibitor (BRAFi)/mitogen-activated protein kinase kinase 1 inhibitor (MEKi) therapy, although few patients have durable long-term responses to these agents. The goal of this study was to use an unbiased computational approach to identify inhibitors that reverse an experimentally derived BRAFi resistance gene expression signature. Using this approach, we found that ibrutinib effectively reverses this signature, and we demonstrate experimentally that ibrutinib resensitizes a subset of BRAFi-resistant melanoma cells to vemurafenib. Ibrutinib is used clinically as an inhibitor of the Src family kinase Bruton tyrosine kinase (BTK); however, neither BTK deletion nor treatment with acalabrutinib, another BTK inhibitor with reduced off-target activity, resensitized cells to vemurafenib. These data suggest that ibrutinib acts through a BTK-independent mechanism in vemurafenib resensitization. To better understand this mechanism, we analyzed the transcriptional profile of ibrutinib-treated BRAFi-resistant melanoma cells and found that the transcriptional profile of ibrutinib was highly similar to that of multiple Src proto-oncogene kinase inhibitors. Since ibrutinib, but not acalabrutinib, has appreciable off-target activity against multiple Src family kinases, it suggests that ibrutinib may be acting through this mechanism. Furthermore, genes that are differentially expressed in ibrutinib-treated cells are enriched in Yes1-associated transcriptional regulator (YAP1) target genes, and we showed that ibrutinib, but not acalabrutinib, reduces YAP1 activity in BRAFi-resistant melanoma cells. Taken together, these data suggest that ibrutinib, or other Src family kinase inhibitors, may be useful for treating some BRAFi/MEKi-refractory melanoma tumors. SIGNIFICANCE STATEMENT: MAPK-targeted therapies provide dramatic initial responses, but resistance develops rapidly; a subset of these tumors may be rendered sensitive again by treatment with an approved Src family kinase inhibitor-ibrutinub-potentially providing improved clinical outcomes.

Fig. 1.

Ibrutinib resensitizes BRAFi-resistant cells to vemurafenib. (A) The unfiltered in silico compound library contains 12,441 compounds that span a diversity of chemotypes and molecular targets; many of these compounds are FDA approved or at one point entered clinical trials. Compounds were first filtered to only include compounds that significantly reverse a BRAFi resistance signature (sRGES <−0.3), resulting in 214 compounds that passed this cutoff. These compounds were further filtered to remove compounds that are broadly cytotoxic and to remove compounds lacking a well annotated mechanism of action (MoA). From the resulting ranked list of 71 compounds, we selected nine compounds whose primary target had not been previously implicated in BRAFi resistance for further experimental validation. These compounds were selected primarily based upon the mechanism of action, with the goal of identifying new biology underlying BRAFi resistance. (B) The BRAFi resistance signature was computed by comparing BRAFi-resistant cell lines and normal tissue samples. Red boxes indicate upregulated genes, and blue boxes indicate downregulated genes. Loxoprofen was included as a control since this compound was not predicted to reverse the BRAFi resistance signature. For compounds with multiple gene expression profiles, the profile with the median RGES was chosen for visualization. The sRGES values for the BRAFi resistance signature and the compound-treated signatures are listed above the heatmap. A negative sRGES indicates reversal of the BRAFi resistance signature by the indicated compound. (C) M229P/R, UACC62P/R, and M238P/R cells were treated in a dose-response matrix of ibrutinib (top concentration 10 µM, 1/2 dilution series) and vemurafenib (top concentration 10 µM, 1/2 dilution series). After 72 hours, viability was measured with CellTiter-Glo (n = 3 biologic replicates). (D) M229P/R cells were treated with ±2 µM vemurafenib and ±1 or 5 µM ibrutinib for 72 hours. The cells were stained and analyzed by flow cytometry as described in Materials and Methods (n = 3 biologic replicates). Significant differences of G0/G1 for compound-treated samples versus the relevant DMSO control are indicated (one-way ANOVA, *P < 0.01 versus M229P-DMSO; #P < 0.01 versus M229R-DMSO).

Fig. 2.

BTK deletion or inhibition does not alter vemurafenib sensitivity. (A) M229P/R BTK knockout cells were generated as described in Materials and Methods. Sanger sequencing was performed to measure the extent of BTK deletion in M229P/R cell pools. The fraction of cells with functional BTK deletion was quantified with TIDE (n = 3 biologic replicates). (B) M229P/R sgControl and sgBTK cells were treated with 14 concentrations of vemurafenib (10 µM top concentration, 1/2 dilution series), and, after 72 hours, viability was measured with CellTiter-Glo as described in Materials and Methods (n = 3 biologic replicates). (C) M229P/R cells were treated with seven different concentrations of acalabrutinib (10 µM top concentration, 1/2 dilution series) and 14 different concentrations of vemurafenib (10 µM top concentration, 1/2 dilution series). After 72 hours, viability was measured with CellTiter-Glo (n = 3 biologic replicates).

Fig. 3.

Transcriptional response to ibrutinib and vemurafenib treatment in BRAFi-resistant cells. (A) M229R cells were treated with DMSO, vemurafenib (2 µM), ibrutinib (5 µM), acalabrutinib (5 µM), or the combinations as indicated. After 24 hours RNA was extracted and RNA-Seq was performed as described in Materials and Methods. The number of differentially expressed (DE) genes compared with DMSO control–treated cells is shown for each treatment condition. (B) A heatmap of the BRAFi resistance signature is shown in leftmost column, and the impact of compound treatments on reversal of BRAFi signature gene expression is shown in all other columns in the heatmap. For each treatment condition, the fold change in gene expression was compared with the DMSO control. The median expression value for each gene from three biologic replicates was used. For each treatment group the fold change in gene expression was compared with the DMSO control. Red boxes indicate that the gene is upregulated, and blue boxes indicate that the gene is downregulated. Of all treatments, vemurafenib + ibrutinib significantly reversed the BRAFi resistance signature (Spearman correlation = −0.25, P = 0.0007). (C) LISA analysis of differentially expressed genes in the ibrutinib and vemurafenib + ibrutinib treatment groups for prediction of transcriptional regulators. Data analysis was performed as described in Materials and Methods. x- and y-axis values are enrichment P values. Highly predicted transcription regulators are indicated with YAP1 and its transcriptional partners, TEAD1 and TEAD4, are indicated as red dots. (D) Similarity scores for CMap class analysis was performed as described in Materials and Methods. Transcriptional signatures of ibrutinib, vemurafenib, or vemurafenib + ibrutinib were compared with transcriptional signatures in the CMap data set.

Fig. 4.

Ibrutinib blocks YAP1 nuclear localization. All cells were treated with ibrutinib or acalabrutinib at 5 µM or vehicle control for 24 hours as indicated prior to being fixed and stained. (A) M229P/R cells were stained with an anti-YAP1 antibody as described in Materials and Methods. The percentage of cells with nuclear, cytosolic, or pan-cellular YAP1 localization was quantified as described in Materials and Methods. (B) Representative images from the experiment in Fig. 4A. M238P/R (C) or UACC62P/R (D) cells were stained with an anti-YAP1 antibody as described in Materials and Methods. The percentage of cells with nuclear, cytosolic, or pan-cellular YAP1 localization was quantified as described in Materials and Methods. Statistical analysis (one-way ANOVA) was performed on percentage of cells with nuclear YAP1 localization where P < 0.01 was considered statistically significant. Bars marked with # indicate a statistically significant difference when compared with DMSO-treated parental cells, and bars marked with * indicate a statistically significant difference when compared with DMSO-treated resistant cells (n = 3 biologic replicates for all imaging experiments).