The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene–containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. Significance: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.

Transcriptional regulation is a tightly regulated, vital process. The transcription factor cyclic AMP-response element-binding protein 1 (CREB1) controls ∼25% of the mammalian transcriptome by binding the CREB1 binding site consensus sequence (CRE) sequence (TGACGTCA). DNA lesions within CRE modulate CREB1 binding negatively and positively. Because appropriate DNA lesions also interact with base excision repair proteins, we investigated whether CREB1 and repair glycosylases compete with each other. We incubated 39-mer CRE-containing double-stranded oligonucleotides with recombinant CREB1 alone or with UNG2 or OGG1, followed by EMSA. The CpG islet within CRE was modified to contain a G/U or 8-oxoG (°G)/C mispair. OGG1 and CREB1 reversibly competed for CRE containing an °G/C pair. Also, OGG1 blocked CREB1 from dimerizing by 69%, even when total CREB1 binding was reduced only by 20–30%. In contrast, bound CREB1 completely prevented access to G/U-containing CRE by UNG2 and, therefore, to base excision repair, whereas UNG2 exposure prevented CREB1 binding. CREB1 dimerization was unaffected by UNG2 when CREB1 bound to CRE, but was greatly reduced by prior UNG2 exposure. To explore physiological relevance, we microinjected zebrafish embryos with the same oligonucleotides, as a sink for endogenous CREB1. As predicted, microinjection with unmodified or lesion-containing CRE, but not scrambled CRE or scrambled CRE with a G/U mispair, resulted in increased embryo death. However, only the G/U mispair in native CRE resulted in substantial developmental abnormalities, thus confirming the danger of unrepaired G/U mispairs in promoters. In summary, CREB1 and DNA glycosylases compete for damaged CRE in vitro and in vivo, thus blocking DNA repair and resulting in transcriptional misregulation leading to abnormal development.

Background/Objectives: PAX3 is a transcription factor that drives melanoma progression by promoting cell growth, migration, and survival, while inhibiting cellular terminal differentiation. However, known PAX3 target genes are limited and cannot fully explain the wide impact of PAX3 function. The PAX3 protein can regulate DNA through two separate binding domains, the Paired Domain (PD) and Homeodomain (HD), which bind different DNA motifs. It is not clear if these two domains bind and work together to regulate genes and if they promote all or only a subset of downstream cellular events. Methods: PAX3 direct downstream targets were identified using Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assays in SK-MEL-5 melanoma cells. PAX3-binding genomic regions were identified through MACS2 peak calling, and peaks were categorized based on the presence of PD and/or HD binding sites (or neither) through HOMER motif analysis. The peaks were further characterized as Active, Primed, Poised, Repressed, or Closed based on ATAC-seq data and CUT&RUN for histone Post-Translational Modifications H3K4me1, H3K4me3, H3K27me3, and H3K27Ac. Results: This analysis revealed that most of the PAX3 binding sites in the SK-MEL-5 cell line were primarily through the PD and connected to Active genes. Surprisingly, PAX3 does not commonly act as a repressor in SK-MEL-5 cells. Pathway analysis identified genes involved with transcription, RNA modification, and cell growth. Peaks located in distal enhancer elements were connected to genes involved in neuronal growth, function, and signaling. Conclusions: Our results reveal novel PAX3 regulatory regions and putative genes in a melanoma cell line, with a predominance of PAX3 PD binding on active sites.

Vasculogenic mimicry (VM) is the phenomenon whereby non-vascular tumor cells develop vascular-like structures. VM is linked to more aggressive tumor phenotypes including higher rates of metastasis and invasion and is potentially resistant to anti-angiogenic cancer therapies. VM is investigated in vitro using 3D assays with microscopy images capturing the resulting VM structures, including loops, branch points, and tubes. The standard method to quantify endpoint data is to count various structural features manually, which is time-consuming and open to bias. At present, no software solutions have been developed to specifically address the analysis and quantification of VM structures. To address this limitation, we developed an open source, Python-based application, VaMiAnalyzer, allowing straightforward quantification of several VM structural features. The application follows a two-step approach that optionally corrects and enhances the raw input images and then analyzes and quantifies the VM features. VaMiAnalyzer is stand-alone software that allows automated measurement of VM structural features from phase-contrast microscopy images. It produces results that are strongly consistent with manual counts but in a significantly shorter time, allowing quick, non-biased analysis of VM from microscopy images.