PLX-4720

Cancer Res,2011 Apr 1;71(7):2750-60.

PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression.[Pubmed: 21317224]

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720.
METHODS AND RESULTS:
Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a.
CONCLUSIONS:
In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.

Proc Natl Acad Sci U S A,2010 Jun 8;107(23):10649-54.

B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression.[Pubmed: 20498063]

Although B-Raf(V600E) is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood.
METHODS AND RESULTS:
Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-Raf(V600E) resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-Raf(V600E) cells in which either B-Raf(V600E) or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity.
CONCLUSIONS:
In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.

Proc Natl Acad Sci U S A,2008 Feb 26;105(8):3041-6.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.[Pubmed: 18287029]

Cell lines:COLO205, A375, WM2664, COLO829, HT716, SW620, H460, Calu-6, HCT116, SK-MEL2, SK-MEL3, Lovo, H1299, 1205Lu, and C8161 cells
Concentrations: Dissolved in DMSO, final concentrations ~1 mM
Incubation Time:  24, 48, and 72 hoursBR/> Method:
Cells are treated with various concentrations PLX-4720 for 24, 48, and 72 hours. Cell proliferation is measured by using the CellTiter-Glo Luminescent Cell Viability Assay or MTT assay. For cell cycle analysis, supernatant and cells are collected, pelleted, and fixed with 70% ethanol. Before staining with propidium iodide (10 μg/mL), cells are incubated for 1 hour at 37 °C in 0.5 mg/mL RNase I to rid samples of residual RNA contamination. Samples are then analyzed by using the EPICS XL apparatus. For the assessment of apoptosis, media and cells are harvested and pelleted before staining with annexin-FITC and propidium iodide. Samples are subsequently analyzed by using the EPICS XL apparatus.

Proc Natl Acad Sci U S A,2008 Feb 26;105(8):3041-6.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.[Pubmed: 18287029]

Animal Models: Female athymic mice (NCr nu/nu) implanted s.c. with COLO205 cells, and SCID mice with 1205Lu or C8161 cells
Dosages:5, 20, or 100 mg/kg
Administration: Oral gavage once or twice daily