MYC-DEPENDENT TRANSCRIPTIONAL CONTROL IN HUMAN LYMPHOMA

Deregulated expression of the oncogenic transcription factor MYC is a widespread feature in cancer and is required for tumor maintenance. Despite substantial research efforts, major gaps persist in our understanding of primary MYC-dependent events in tumors. To address this issue, we inserted an in-frame cassette encoding an Auxin-inducible degron (AID) into the oncogenic MYC allele of three human B-cell lymphoma cell lines, thus directing expression of a conditionally degradable MYC-AID fusion protein. Treatment of the cells with Auxin caused an immediate MYC-AID drop, resulting in gradual proliferative arrest and cell death. While residual MYC-AID levels supported 3-4 residual division cycles, no long-term adaptation occurred. Hence, partial MYC inhibition may suffice to elicit potent anti-tumoral effects. RNA-seq profiling of nascent RNA showed that MYC loss elicited rapid changes in transcriptional activity at several hundred loci in each lymphoma cell line, with a conserved core of 187 MYC-dependent genes showing immediate and persistent down-regulation (from 1 to 8 hours after treatment) followed by consistent reductions in the corresponding mature mRNAs. Other groups of genes underwent immediate gains in transcriptional activity, and yet others were activated or repressed at later times; however, none of these groups showed consistent overlaps among the different lymphoma cell lines. Hence MYC was directly required to support transcription at a discrete set of immediate MYC-dependent genes. Since transcription factors also impact co- and post-transcriptional processes, and ultimately translation, we profiled polysome-associated RNAs following Auxin treatment. This revealed that the changes in polysome engagement closely reflected those in total RNA levels, thus providing no evidence for differential effects on translation at MYC-regulated loci. To start addressing the mechanisms underlying MYC-dependent transcription, we established genome-wide chromatin association profiles for MYC, RNA-PolII and several histone marks. Our analyses showed that MYC-AID removal resulted in rapid losses of RNA-PolII from MYC-dependent loci. These effects were proportionate at promoters and gene bodies, suggesting a role of MYC for RNA-PolII loading at those loci. Altogether, the above datasets shall provide us with a dynamic view of MYC-regulated transcription and of the mechanisms underlying oncogene addiction in MYC-driven Lymphoma, paving the way for the identification of new therapeutic targets.