Effect of TGFb/MEKi treatment on human melanoma cell lines

The TGFß signaling pathway is known for its pleiotropic functions in a plethora of biological processes. In melanoma, TGFß signaling promotes invasiveness and metastasis formation. However, its involvement in the response to therapy is controversial. While several studies have linked TGFß signaling to elevated resistance to targeted therapy in melanoma, separate findings have indicated a favorable treatment response through TGFß-mediated increase of cell death. We now found that the outcome of TGFß signaling in the context of targeted therapy is dose dependent. Unlike low doses, high levels of TGFß signal activation induce apoptosis upon simultaneous MAPK pathway inhibition, even in targeted therapy resistant melanoma cell lines. Using transcriptomic analyses, combined with genomic target identification of the critical TGFß signaling effector SMAD4, we demonstrate that parallel activation of TGFß signaling and MAPK pathway inhibition causes a complete switch of TGFß target genes from promoting pro-invasive processes to fueling pro-apoptotic pathways. Investigations of underlying mechanisms identified a novel apoptosis-inducing gene signature. Functional validation of signature members confirmed the deubiquitinating enzyme BAP1, the ubiquitin conjugation factor UBE4B and the pro-apoptotic BCL2 family member BCL2L11 (BIM) to mediate apoptosis in this condition. Using a modified, synthetic version of the TGFß1 mRNA for intra-tumoral injections, we additionally showcase a potential therapeutic application of this treatment combination. Overall design: In total 6 human melanoma cell lines were treated with 4 different conditions (control, TGFb only, MEKi only or TGFb + MEKi) for 20h before RNA was isolated and transcriptome sequencing was performed. For two cell lines M130830 and M170117 three replicates were sequenced, for the remaining 4 cell lines only one replicate per condition was sequenced.