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Melanoma

E322: Transcription Factor E3 (TFE3) Regulates the Invasive Phenotype in MITF-low Melanoma

Introduction: Metastatic melanoma 5- year survival rates remain poor at only 20-30%. Microphthalmia-associated transcription factor (MITF) is associated with phenotypic plasticity of melanoma cells. Melanoma cell lines with low expression of MITF have been shown to express an invasive epithelial mesenchymal transition (EMT) phenotype while high MITF expression is associated with a proliferative melanocytic phenotype. Little is known however about the mechanisms that drive metastasis in low MITF states. We hypothesize that there are transcriptional regulators present specific to the MITF-low states.

Methods: To investigate this, we developed MITF knockouts of an SKMEL28 melanoma cell line utilizing a CRISPR-Cas9 system. RNA-Seq, ATAC-seq and CUT&RUN were performed using an antibody that recognizes both MITF and its paralog Transcription Factor E3 (TFE3) to evaluate transcriptional regulation in MITF low states. TFE3 knockouts (TFE3KO) were created in A375 and SKMEL28 lines. Invasion assay and MTT were performed to evaluate cell metastatic and proliferative potential in vitro. RNA-seq and gene set enrichment analysis (GSEA) was performed in TFE3KO to identify downstream pathways.

Results: Using Anti-MITF CUT&RUN sequencing, we identified 46,158 anti-MITF peaks and of these, 6,943 (15%) were unique to MITF-KO cells, presumably gained TFE3 peaks in the absence of MITF. Enhancers bound by these gained paralog peaks were most significantly enriched for the DNA binding motif of TFE3.  TFE3 knockout cell lines were created in MITF-low melanoma call lines (A375) utilizing CRISPR-Cas9 systems and these gained peaks were confirmed to be TFE3 bound through Anti-TFE3 CUT&RUN. In vitro, while these cells demonstrated no difference in cellular proliferation compared to wild type, there was significant decrease in invasive capacity. GSEA of TFE3KO downregulated genes demonstrated significant enrichment for EMT and neural crest migration gene lists.

Conclusions: These findings support TFE3 as a transcriptional driver of the invasive phenotype in MITF-low conditions. Further studies need to be performed to elucidate the downstream targets in this regulatory pathway. This has the potential to be a novel therapeutic target for metastatic melanoma.

Learning Objectives:

  • Upon completion, participant will be able to understand the the role of MITF in melanoma phenotype switching.
  • Upon completion, participant will be able to describe improvements of CUT&RUN sequencing over ChIP-seq.
  • Upon completion, participant will be able to understand TFE3 role in transcriptional regulation of MITF-low melanoma.