Chromatin Remodeler SATB2 Reactivates Neural Crest Developmental Program In Melanoma Leading to Invasion and Drug Resistance

Introduction

Epigenetic regulators are emerging as key factors in the pathogenesis of both solid cancers and hematological malignancies. In melanoma, KDM5B has been shown to drive a slow cycling drug-resistant population and ARID2 has been identified among the most recurrently mutated genes in The Cancer Genome Atlas sequencing project. To gain insight into additional epigenetic pathways that might contribute to melanoma progression, we undertook an in vivo transgenesis overexpression screen for chromatin factors that accelerate melanoma development in a genetically engineered zebrafish model.

Materials and methods

We screened 83 epigenetic regulators by transgenesis with an overexpression vector in a primary zebrafish genetic model of melanoma driven by the most frequent melanoma oncogene BRAFV600E and p53 loss of function. We phenotypically characterized a hit from the screen using zebrafish allotransplant assays to assess tumor propagation, metastatic potential, and response to treatment with a standard of care targeted MAPK inhibitor. We validated our results in vitro using genetic perturbation via lentiviral vectors in a panel of human melanoma cell lines combined with proliferation, migration, and invasion assays. To characterize the mechanism of action of our hit we then performed chromatin and transcriptional analysis via ChIP-seq, RNA-seq, and ATAC-seq on primary zebrafish tumors and human melanoma cell lines.

Results

Out of 83 tested factors, we identified Special AT-rich Binding protein 2 (SATB2) as a novel potent accelerator of melanoma. SATB2 is a chromatin remodeler required for vertebrate neural crest development, whose alterations in humans lead to a neurocristopathy known as SATB2-associated syndrome, which is phenocopied by morpholino knockdown in developing zebrafish embryos. Its overexpression in melanoma, which is observed in ~5% of patients, does not affect cell proliferation but leads to the formation of invadopodia and a significant increase in tumor-propagating and metastatic potential in vivo. Chromatin analysis by ChIP-seq and ATAC-seq revealed that SATB2 binding at target loci leads to altered levels of H3K27ac and chromatin accessibility, resulting transcriptionally in the reactivation of an invasive neural crest EMT developmental program. Additionally, we observed a transcriptional switch in SATB2 overexpressing melanoma towards activation of the PDGF-SRC signaling axis and the recently described AXL^highMITF ^low state. This transcriptional phenotype switch functionally results in resistance to targeted MAPK inhibition in allotransplanted primary tumors and A375 human melanoma cells.

Conclusion

In summary, our work illustrates how epigenetic regulation of transcriptional states affects melanoma progression and informs patient treatment with the current standard of care MAPK inhibitors.