BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases

Abstract

The development of mutant BRAF inhibitors has improved the outcome for melanoma patients with BRAF V600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes resulting in complete tumor regression, the majority of melanomas become resistant. To study resistance to BRAF inhibition, we developed a novel mouse model of melanoma using a tetracycline/doxycycline-regulated system that permits control of mutant BRAF expression. Treatment with doxycycline leads to loss of mutant BRAF expression and tumor regression, but tumors recur after a prolonged period of response to treatment. Vemurafenib, encorafenib and dabrafenib induce cell cycle arrest and apoptosis in BRAF melanoma cell lines; however, a residual population of tumor cells survive. Comparing gene expression in human cell lines and mouse tumors can assist with the identification of novel mechanisms of resistance. Accordingly, we conducted RNA sequencing analysis and immunoblotting on untreated and doxycycline-treated dormant mouse melanomas and human mutant BRAF melanoma cell lines treated with 2 μM vemurafenib for 20 days. We found conserved expression changes in histone methyltransferase genes ASH2, EZH2 , PRMT5, SUV39H1, SUV39H2, and SYMD2 in P-ERK low, p-38 high melanoma cells following prolonged BRAF inhibition. Quantitative mass spectrometry, determined a corresponding reduction in histone Lys9 and Lys27 methylation and increase in Lys36 methylation in melanoma cell lines treated with 2 μM vemurafenib for 20 days. Thus, these changes as are part of the initiate response to BRAF inhibition and likely contribute to the survival of melanoma cells.

BRAF/EZH2 Signaling Represses miR-129-5p Inhibition of SOX4 Thereby Modulating BRAFi Resistance in Melanoma

“…This provides further evidence that, in melanoma, EZH2 mediates miR-129-5p expression downstream of constitutive active BRAF signaling. Although previous studies showed a reduction of H3K27me3 during treatment with BRAFi or MEKi in melanoma [62,63] we could not confirm these changes in our experiments. These different results may be explained by using low doses of treatment (0.4 µM Vemurafenib or 10 nM Trametinib) or shorter treatment times (48 h).…”

“…We have now demonstrated that, in melanoma, EZH2 represses miR-129-5p, dependent on constitutive active BRAF signaling. After treatment of BRAF mutated melanoma cells with BRAFi or MEKi, we observed a reduction of EZH2 protein as well as mRNA, as shown previously for melanoma [ 62 , 63 ]. Additionally, specific inhibition of EZH2 induces miR-129-5p expression in BRAF mutated melanoma cell lines, independent from their BRAFi response status, while BRAF wildtype cells and normal melanocytes show no changes of expression.…”

“…It controls melanoma growth and metastasis through silencing of distinct tumor suppressors [ 59 , 60 ]. Of note EZH2 was also shown as a mediator of treatment resistance in BRAF mutated melanoma [ 61 , 62 ]. We have now demonstrated that, in melanoma, EZH2 represses miR-129-5p, dependent on constitutive active BRAF signaling.…”

Histone Methylation Related Therapeutic Challenge in Cardiovascular Diseases

“…The methylation of histone H3K4, R8, R17, K26, K36, K79, H4R3, and K12 can activate gene transcription ( 42 , 43 ). However, the methylation of histone H3K9, K27, K56, H4K5, and K20 inhibits gene transcription, confirming the complexity of epigenetic regulation of histone methylation ( 44 ). Interestingly, under different conditions, the methylation of histone H3R2 can activate and inhibit transcription ( 33 ).…”

Interrogating Epigenome toward Personalized Approach in Cutaneous Melanoma

“…These two types of epigenetic changes appear to influence each other in the deposition during mammalian development and carcinogenesis; histone methylation appears to direct DNA methylation patterns, while DNA methylation may serve as a model for establishing certain histone changes [ 32 ]. Moreover, in CM, DNA methylation is gaining increased importance in PD-related immune therapy [ 48 ], while histone alterations are associated with BRAF-targeted therapy [ 49 ].…”