Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. The ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the downstream BRN2 signaling cascade driving anoikis resistance remains unknown.

We utilized genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibitors targeting the BRN2 protein were employed as probes for quantitative mass spectrometry analysis, and the functional implication of BRN2 inhibition on cell survival, apoptosis and mitochondrial structure and function assessed in vitro experiments.

Inhibition of BRN2 with small molecules in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through increased oxidative phosphorylation and AIFM2 expression. BRN2 inhibition additionally sensitized cells to BRAF-targeted therapy.

Our study validated the role of BRN2 in anoikis resistance and identified that BRN2 drives anoikis resistance by promoting mitochondrial dysfunction. For the first time in melanoma models, small molecule inhibitors against BRN2 were demonstrated to reverse anoikis resistance and sensitize cells to BRAF-targeted therapy. Inhibition of BRN2 may prevent the seeding of metastatic disease by reversing anoikis resistance.

Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.