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, 16 (8), 1596-1609

Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells

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Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells

Kayleigh C Ross et al. Mol Cancer Ther.

Abstract

Metastatic melanoma cells commonly acquire resistance to BRAF V600E inhibitors (BRAFi). In this study, we identified serine biosynthesis as a critical mechanism of resistance. Proteomic assays revealed differential protein expression of serine biosynthetic enzymes PHGDH, PSPH, and PSAT1 following vemurafenib (BRAFi) treatment in sensitive versus acquired resistant melanoma cells. Ablation of PHGDH via siRNA sensitized acquired resistant cells to vemurafenib. Inhibiting the folate cycle, directly downstream of serine synthesis, with methotrexate also displayed similar sensitization. Using the DNA-damaging drug gemcitabine, we show that gemcitabine pretreatment sensitized resistant melanoma cells to BRAFis vemurafenib and dabrafenib. We extended our findings to BRAF WT tumor cell lines that are intrinsically resistant to vemurafenib and dabrafenib. Pretreatment of pancreatic cancer and non-small cell lung cancer cell lines with sublethal doses of 50 and 5 nmol/L of gemcitabine, respectively, enhanced killing by both vemurafenib and dabrafenib. The novel aspects of this study are the direct identification of serine biosynthesis as a critical mechanism of BRAF V600E inhibitor resistance and the first successful example of using gemcitabine + BRAFis in combination to kill previously drug-resistant cancer cells, creating the translational potential of pretreatment with gemcitabine prior to BRAFi treatment of tumor cells to reverse resistance within the mutational profile and the WT. Mol Cancer Ther; 16(8); 1596-609. ©2017 AACR.

Figures

Figure 1
Figure 1. Characterization of SK-MEL-28VR1 cells
A) Growth rate comparisons of SK-MEL-28 and SK-MEL-28VR1 cells (n=3). 100000 cells plated at time-point 0. B) Colony formation assays of SK-MEL-28 and SK-MEL-28VR1 cells following treatments with differential doses of vemurafenib (n=5) (p<0.0001). C) Mass spectrometry: FAM129B protein abundance following differential treatments of SK-MEL-28 and SK-MEL-28VR1 cells (n=3). D) Mass spectrometry: Volcano plot displaying differential protein expression following vemurafenib treatments. SK-MEL-28 protein expression is displayed in red; SK-MEL-28VR1 protein expression is displayed in blue (n=3). Proteins displayed on the right side of 0 in the x axis are decreasing in abundance with vemurafenib treatments, and proteins displayed on the left side of the 0 in the x axis are increasing in abundance following vemurafenib treatments. E) Western blots of FAM129B protein expression in differentially treated SK-MEL-28 and SK-MEL-28VR1 cells. Beta actin used as loading control. 50μg of protein loaded in each lane. F) Immunofluorescence (IF) staining: SK-MEL-28VR1 cells were co-stained with DAPI and FAM129B primary antibody. Cells were treated with either vemurafenib (10uM) or DMSO for 48 hours prior to staining. G) Western blots of p-MEK1/2 and p-ERK1/2 protein expressions in differentially treated SK-MEL-28 and SK-MEL-28VR1 cells. Beta actin used as loading control. 50μg of protein loaded in each lane. H) Western blot of PHGDH protein expression in differentially treated SK-MEL-28 and SK-MEL-28VR1 cells. Beta actin used as loading control. 50μg of protein loaded in each lane.
Figure 2
Figure 2. Importance of serine biosynthesis pathway to vemurafenib resistance in SK-MEL-28VR1 cells
A) Colony formation assays of SK-MEL-28 cells following control or PHGDH siRNAs treatments with differential doses of vemurafenib (n=3) (p=0.3052). B) Colony formation assays of SK-MEL-28VR1 cells following control or PHGDH siRNAs treatments with differential doses of vemurafenib (n=3) (p<0.0001) C) Colony formation assays of SK-MEL-28 cells following treatments with differential doses of vemurafenib +/− methotrexate (75nM) (n=3) (p=0.9203). D) Colony formation assays of SK-MEL-28VR1 cells following treatments with differential doses of vemurafenib +/− methotrexate (75nM) (n=3) (p<0.0001). E) Colony formation assays of SK-MEL-28VR1 cells following treatments with differential doses of vemurafenib +/− serine in media (n=3) (p<0.0001).
Figure 3
Figure 3. Gemcitabine sensitizes SK-MEL-28VR1 cells to vemurafenib
A) Colony formation assays of SK-MEL-28 cells following treatments with differential doses of vemurafenib +/− gemcitabine (50nM) (n=3) (p<0.0001). B) Colony formation assays of SK-MEL-28VR1 cells following treatments with differential doses of vemurafenib +/− gemcitabine (50nM) (n=3) (p<0.0001). C) Colony formation assays of SK-MEL-28 cells following control or PHGDH siRNAs treatments with differential doses of vemurafenib +/− gemcitabine (50nM) (n=3) (p=0.9816). D) Colony formation assays of SK-MEL-28VR1 cells following control or PHGDH siRNAs treatments with differential doses of vemurafenib +/− gemcitabine (50nM) (n=3) (p=0.0189). E) Colony formation assays of SK-MEL-28 cells following treatments with differential doses of vemurafenib + gemcitabine (50nM) +/− methotrexate (75nM) (n=3) (p=0.6585). F) Colony formation assays of SK-MEL-28VR1 cells following treatments with differential doses of vemurafenib + gemcitabine (50nM) +/− methotrexate (75nM) (n=3) (p<0.0001). G) Fa-CI plot representing synergy between gemcitabine and vemurafenib. Data points falling below the line indicate synergy between drugs. Data points represent CI calculations at specific doses. Please refer to Supplementary Table S2 for CI values.
Figure 4
Figure 4. Gemcitabine sensitizes pancreatic cancer and NSCLC cell lines to vemurafenib
A) Colony formation assays of BxPC3M1 cells following treatments with differential doses of gemcitabine +/− vemurafenib (1μM) (n=3) (p<0.0001). B) Fa-CI plot representing synergy between gemcitabine and vemurafenib. Data points falling below the line indicate synergy between drugs. Data points represent CI calculations at specific doses. Please refer to Supplementary Table S3 for CI values. C) Colony formation assays of NCI-H2122 cells following treatments with differential doses of gemcitabine +/− vemurafenib (1μM) (n=3) (p<0.0001).
Figure 5
Figure 5. Vemurafenib induces cell proliferation and serine synthesis in pancreatic cancer cell lines
A) Cell proliferation assay of BxPC3 cells treated with vemurafenib (10μM). 100000 cells plated on day 0. B) Cell proliferation assay of BxPC3M1 cells treated with vemurafenib (10μM). 100000 cells plated on day 0. C) Cell proliferation assay of Panc1 cells treated with vemurafenib (10μM). 100000 cells plated on day 0. D) Cell proliferation assay of MiaPaca2 cells treated with vemurafenib (10μM). 100000 cells plated on day 0. E) Mass spectrometry: PHGDH protein expression in pancreatic cancer cells treated with DMSO or vemurafenib (10μM) (n=3). F) Mass spectrometry: PSAT1 protein expression in pancreatic cancer cells treated with DMSO or vemurafenib (10μM) (n=3). G) Mass spectrometry: PSPH protein expression in pancreatic cancer cells treated with DMSO or vemurafenib (10μM) (n=3). H) Mass spectrometry: SARS protein expression in pancreatic cancer cells treated with DMSO or vemurafenib (10μM) (n=3). I) Colony formation assays of BxPC3M1 cells following treatments with differential doses of gemcitabine +/− vemurafenib (1μM) +/− methotrexate (75nM) (n=3) (p=0.0258). J) Colony formation assays of NCI-H2122 cells following treatments with differential doses of gemcitabine +/− vemurafenib (1μM) +/− methotrexate (75nM) (n=3) (p=0020). K) Colony formation assays of BxPC3M1 cells following treatments with differential doses of vemurafenib +/− serine (n=3) (p<0.0001).
Figure 6
Figure 6. Dabrafenib induced sensitization of BxPC3M1, NCI-H2122, and SK-MEL-28VR1 cells to gemcitabine
A) Colony formation assays of BxPC3M1 cells following treatments with differential doses of gemcitabine +/− dabrafenib (1μM) (n=3) (p<0.0001). B) Colony formation assays of NCI-H2122 cells following treatments with differential doses of gemcitabine +/− dabrafenib (1μM) (n=3) (p<0.0001). C) Colony formation assays of BxPC3M1 cells following treatments with differential doses of dabrafenib +/− gemcitabine (50nM) (n=3) (p<0.0001). D) 3D spheroid assays: 5000 cells were plated on day 0. Drugs were added on day 3. On day 4, gemcitabine (50nM) was washed out and dabrafenib (10uM) was added to the combination treatment wells.
Figure 7
Figure 7. Schematic of cancer cell sensitization via sequential combination treatment with gemcitabine and a BRAF V600E inhibitor
Cascade A represents SK-MEL-28 cellular response to BRAF V600E inhibitors (BRAFi) within the BRAF V600E mutation. Cascade B represents acquired BRAFi resistant SK-MEL-28VR1 cellular response to BRAFi within the mutation profile. Acquired resistance causes a paradoxical induction of the MAPK cascade without gemcitabine pre-treatment. Gemcitabine pre-treatment followed by BRAFi leads to induction of the MAPK cascade and induction of serine synthesis while cells are arrested. Induction of serine synthesis leads to an induction of the folate cycle for nucleotide synthesis. These series of events lead to cell death due to conflicting activation of cellular signaling pathway causing cell cycle arrest signal from gemcitabine-induced DNA damage and activation of MAPK signaling pathway by BRAF inhibitors. Cascade C shows sensitization of BRAF WT pancreatic cancer BxPC3M1 and non-small cell lung cancer NCI-H2122 cells to BRAF inhibitors by gemcitabine pre-treatment. In these BRAF WT cell lines, gemcitabine induces cell cycle arrest. Addition of BRAF inhibitors to the arrested cells induces the MAPK cascade leading to increased serine synthesis and folate synthesis. These series of events lead to cell death due to conflicting activation of cellular signaling pathway causing cell cycle arrest from gemcitabine-induced DNA damage and activation of MAPK signaling pathway by BRAF inhibitors. The actual mechanism of cell death is as yet unknown.

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