SUMMARY Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a trans-membrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

Lysine-specific demethylase 1 (LSD1) demethylates nucleosomal histone H3 lysine 4 (H3K4) residues in collaboration with the corepressor CoREST/REST corepressor 1 (Rcor1) and regulates cell fates by epigenetically repressing gene targets. The balanced regulation of this demethylase, if any, is however unknown. We now demonstrate the actions of two other Rcor paralogs, Rcor2 and Rcor3, in regulating LSD1 enzymatic activity and biological function in hematopoietic cells. All three Rcor proteins interact with LSD1 and with the erythro-megakaryocytic transcription factor growth factor independence (Gfi)1b; however, whereas Rcor2, like Rcor1, facilitates LSD1-mediated nucleosomal demethylation, Rcor3 competitively inhibits this process. Appending the SANT2 domain of Rcor1 to Rcor3 confers the ability to facilitate LSD1-mediated demethylation on the chimeric Rcor protein. Consistent with their biochemical activities, endogenous Rcor1, Rcor2, and LSD1 promote differentiation, whereas Rcor3 opposes these processes. Recruitment of Rcor3 to cognate gene targets by Gfi1b and LSD1 leads to inhibition of H3K4 demethylation of chromatin and transcriptional derepression of these loci. Remarkably, profound alterations in Rcor1/3 levels during erythroid versus megakaryocytic differentiation potentiate antagonistic outcomes. In mature erythroid cells, a strong upsurge in Rcor3 and a sharp decline in Rcor1 levels counteract LSD1/Rcor1/2-mediated differentiation. In contrast, the opposite changes in Rcor1/3 levels in megakaryocytes favor differentiation and likely maintain homeostasis between these lineages. Overall, our results identify Rcor3 as a natural inhibitor of LSD1 and highlight a dual mechanism of regulating the enzymatic activity and restraining the epigenetic impact of this robust demethylase during hematopoietic differentiation.

Highlights d ATRX IFFs are redistributed genome wide and are enriched at active promoters d The neuronal silencing transcription factor REST is an ATRX IFF target gene d REST and EZH2 silence neuronal gene programs in ATRX IFF NB d REST loss or EZH2 inhibition induces neuronal gene expression programs and NB cell death