Abstract MicroRNAs are a group of noncoding RNAs that can regulate gene expression by targeting the mRNA of a gene. Many microRNAs respond to different stimuli such as growth signals or cellular stress. The tumor-suppressive miR-29 family (miR-29a, b and c), for example, is induced during aging and senescence as a p53-dependent response to DNA damage. Given the tumor-suppressive potential of miR-29, we sought to examine if its expression is induced by oncogenic stress in a p53-dependent manner. Ectopic overexpression of oncogenic Braf-V600E and Kras-G12D in mouse embryonic fibroblasts (MEFs) using retroviral transduction resulted in increased expression of pri-miR-29a-b1 and, to a lesser extent, pri-miR-29b2~c. While ectopic Kras-G12D overexpression stimulated p53 activity, Braf-V600E failed to elicit a p53 response, suggesting that this oncogene provokes miR-29 expression in a p53-independent manner. Moreover, expression of Braf-V600E at physiologic levels induced miR-29 in the absence of a p53 response. Accordingly, expression of endogenous Braf-V600E stimulated miR-29 in MEFs even when p53 was silenced by RNAi. When Braf-V600E MEFs were treated with a MEK inhibitor, miR-29 levels decreased in both the presence and absence of p53, suggesting that the MAPK pathway downstream of Braf-V600E directly regulates miR-29 transcription. Importantly, TPA-mediated stimulation of the MAPK pathway increased miR-29 levels in human melanocytes. Similarly, expression of Braf-V600E induced miR-29 in human melanocytes and MEK inhibition rescued this effect. These results strongly support the notion that miR-29 transcription is induced by MAPK signaling and suggest that oncogenic BRAF provokes a tumor-suppressive miR-29 checkpoint that needs to be inactivated for further tumor progression. Indeed, human melanoma cells lines express lower levels of miR-29 compared to human melanocytes, and inactivation of miR-29 by a lentiviral sponge in Braf-V600E mutant melanocytes and melanoma cells promotes cell transformation. We are now utilizing our ESC-GEMM platform to generate Braf-V600E; Pten-deficient mice expressing the miR-29 sponge and will assess if counteracting the induction of miR-29 by Braf-V600E promotes melanoma formation in vivo. Our results suggest a biphasic role of miR-29 during melanomagenesis where Braf-V600E-induced miR-29 upregulation prevents the progression of early lesions until miR-29 is downregulated, likely by inactivation of the ARF-p53 axis. This abstract is also being presented as Poster B24. Citation Format: Olga Vera, Ilah Bok, Neel Jasani, Oluwashanu Balogun, Koji Nakamura, Florian Karreth. Regulation of the tumor-suppressive miR-29 family by oncogenic MAPK signaling in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR12.