Article Figures & Data
Figures
- 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).
Additional Files
Data Supplement
- 331_Supp_PDF.pdf -
Fig. S1: Principal Component Analysis of resistant cell line samples and tumor tissue samples.
Fig S2. Drug sensitivity correlates with sRGES drug response predictions.
Fig S3. Single-agent activity of compounds identified in the computational screen.
Fig. S4. A BRAFi resistance signature is inversely correlated with melanoma overall survival.
Fig. S5 Identification of compounds that reverse a BRAFi resistance gene expression signature.
Fig S6. Identification of compounds which re-sensitize BRAFi-resistant cells to vemurafenib.
Fig. S7. The combination of ibrutinib and vemurafenib reduces colony formation in M229R cells.
Fig S8. The combination of vemurafenib and ibrutinib increases the number of Annexin V-positive cells but does
not alter caspase3/7 activity or PARP cleavage.Fig S9. BTK mRNA is weakly expressed in M229P/R cells.
Fig S10. Quantification of BTK knockout efficiency.
Fig S11. Differential gene expression networks are associated with developmental gene signatures
Fig S12. Expression of ibrutinib targets in M229P/R cells.Fig S13. Ibrutinib does not alter TAZ localization in BRAFi-resistant cells.
- Data Supplement 1 -
Supplemental Data 1: Primers used for PCR amplification of BTK. The PCR primers listed in Supplemental Data 1 were used to amplify the region of the BTK gene which contained the CRISPR cut site. Sequencing was performed with the indicated nested primers. The data from this experiment are summarized in Fig. 2A.
- Data Supplement 2 -
Supplemental Data 2: Results from sRGES analysis. This data table summarizes the results from the computational sRGES analysis. The analytical steps used to generate these data are described in the Materials and Methods section under “OCTAD Datasets and RNA-Sequence processing”, “Disease signature creation”, and “Drug prediction. The results of this analysis are summarized in Figure 1A.
- 331_Supp_PDF.pdf -
