TERT promoter mutation determines apoptotic and therapeutic responses of BRAF-mutant cancers to BRAF and MEK inhibitors: Achilles Heel

Abstract

Combination use of BRAF V600E inhibitor dabrafenib and MEK inhibitor trametinib has become a standard treatment for human cancers harboring BRAF V600E. Its anticancer efficacies vary, however, with dramatic efficacy in some patients and drug resistance/tumor recurrence in others, which is poorly understood. Using thyroid cancer, melanoma, and colon cancer cell models, we showed that dabrafenib and trametinib induced robust apoptosis of cancer cells harboring both BRAF V600E and TERT promoter mutations but had little proapoptotic effect in cells harboring only BRAF V600E. Correspondingly, the inhibitors nearly completely abolished the growth of in vivo tumors harboring both mutations but had little effect on tumors harboring only BRAF V600E. Upon drug withdrawal, tumors harboring both mutations remained hardly measurable but tumors harboring only BRAF V600E regrew rapidly. BRAF V600E/MAP kinase pathway is known to robustly activate mutant promoter of TERT, a strong apoptosis suppressor. Thus, for survival, cancer cells harboring both mutations may have evolved to rely on BRAF V600E-promoted and high-TERT expression-mediated suppression of apoptosis. As such, inhibition of BRAF/MEK can trigger strong apoptosis-induced cell death and hence tumor abolishment. This does not happen in cells harboring only BRAF V600E as they have not developed reliance on TERT-mediated suppression of apoptosis due to the lack of mutant promoter-driven high-TERT expression. TERT promoter mutation governs BRAF-mutant cancer cells' apoptotic and hence therapeutic responses to BRAF/MEK inhibitors. Thus, the genetic duet of BRAF V600E and TERT promoter mutation represents an Achilles Heel for effective therapeutic targeting and response prediction in cancer.

Keywords: BRAF V600E; BRAF inhibitor; MEK inhibitor; TERT promoter mutation; apoptosis.

Fig. 1.

Effects of the combination treatment with dabrafenib and trametinib on apoptosis of cancer cells harboring only BRAF V600E mutation (BRAF-only) or both BRAF V600E and TERT promoter mutations (genetic-duet). (A) Optical microscopic examination (400×) of morphological death changes after drug or control DMSO treatment of BRAF-only cells (SK-Mel-3 and MDA-T41) and genetic-duet cells (M14 and DRO). (B) Flow cytometry analysis of apoptosis, in terms of percentage of Annexin V and PI positivities, in BRAF-only cells (SK-Mel-3, MDA-T41, UKRV-Mel-19c, and HT29) and genetic-duet cells (M14, DRO, K1, and OCUT1) after dabrafenib + trametinib or DMSO treatment. (C) Bar graph summary of the average percentages of Annexin V-positivity in genetic-duet cells and BRAF-only cells after treatments in each cell group corresponding to Fig. 1B. *P < 0.05; n.s.: P > 0.05.

Fig. 2.

Effects of the combination treatment with dabrafenib and trametinib on apoptotic proteins of 19 different cancer cell lines harboring only BRAF V600E mutation (BRAF-only) or both BRAF V600E and TERT promoter mutations (genetic-duet). (A) Western blotting analysis of proteins in the apoptotic signaling, showing changes in total proteins of PARP and Caspase-3 and their corresponding cleaved proteins (C-PARP and C-Caspase-3) in genetic-duet cells and BRAF-only cells after treatments. The protein quantities were matched for correspondingly paired DMSO and dabrafenib + trametinib treatment groups in each cell as shown by the β-actin levels. (B) Bar graph illustration of the relative level of apoptotic proteins in Fig. 2A, representing the average of band intensities of the indicated protein normalized by corresponding β-actin level of the cells in each treatment groups. The color dots represent outliers of the relative expression. *P < 0.05; n.s.: P > 0.05.

Fig. 3.

Effects of the combination treatment with dabrafenib and trametinib on the growth of xenograft tumors harboring only BRAF V600E mutation (BRAF-only) or both BRAF V600E and TERT promoter mutations (genetic-duet). (A) Time courses of the growth of BRAF-only tumors (SK-Mel-3 and HT29) and genetic-duet tumors (M14 and DRO) treated with DMSO (vehicle) or combined dabrafenib and trametinib as described in Materials and Methods. Green arrows indicate day 7 (day 14 for M14 cells; labeled as “Day 7” for unified formality of the x axis of the figure) as the start of the treatment after cell inoculation and red arrows indicate day 28 (actually day 35 for M14 cells) as the termination of the treatment. (B) Bar graph illustration of the average final tumor weights, corresponding to Fig. 2A. *P < 0.05, ***P < 0.001, n.s.: P > 0.05.

Fig. 4.

Photograph of the xenograft tumors surgically removed. Animals in this figure correspond to those in Fig. 3. Specifically, animals with xenograft tumors grown from cancer cells SK-Mel-3 and HT29 harboring only BRAF V600E or cells M14 and DRO harboring both BRAF V600E and TERT promoter mutations were treated with DMSO (vehicle) or combined dabrafenib and trametinib as described in Fig. 3. At the end of the experiment shown in Fig. 3, animals were killed and tumors were surgically removed and photographed. Each group had 5–6 animals.