Supplementary Materials1. these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild-type versus mutant forms of MYC protein. encodes an oncogene transcription factor that features prominently in cancer. Across the spectrum of malignancies, activation of MYC is typically driven by overexpression of the wild-type protein, but blood-borne tumors often possess changes to the MYC coding sequence. 1-12 Indeed, ~50% of Burkitts lymphomas (BL) harbor MYC mutations, the majority of which cluster at sites within the amino-terminus of the protein. 5 This region carries an expansive degron that signals MYC proteolysis, and we Vargatef cost have reported that tumor mutations within this segment stabilize MYC, the most-pronounced effects being observed with mutations in a conserved element called Myc box I (MbI). 13 Subsequent studies showed that the core of MbI (residues 58C62) is a phosphorylation-dependent degron for the SCFFbw7 ubiquitin-ligase, 14 and that tumor mutations subvert proteolysis by disabling phosphorylation events within this region. Importantly, functional analyses of tumor-associated MbI mutations reveal that they render MYC profoundly oncogenic, and capable of driving lymphomagenesis without triggering Bim-dependent apoptosis and without selecting for loss of p53. 9 Although it is unclear how such mutations impact MYCs transcriptional activity, it is clear that mutations in MbI induce both quantitative and qualitative changes in MYC that favor tumorigenesis. Despite their widespread prevalence in BL, PTPRR the relevance of tumor-associated MYC mutations to the etiology of the disease remains controversial. On one hand, the canonical t(8:14) translocation in BL is sufficient to drive high levels of MYC expression and places MYC in a hypermutable region of the genome, where random mutations could occur. On the other hand, these mutations do accumulate in specific regions of MYC and clearly enhance its tumorigenic functions, 9 implying that they confer a selective advantage to malignant cells. Much of the difficulty in understanding the significance of these mutations stems from the relatively small number of mutant alleles that have been sequenced, the often complex multi-residue nature of these mutations, and the fact that the best-characterized tumor-associated mutations localize to just a single region of MYC (MbI), leaving open the question of whether the handful of MbI mutations that have been studied to date reflect what occurs in BL patients. Clearly, resolution of this controversy requires analysis of additional tumor MYC alleles, with the most informative being those that lie outside of MbI. Recent BL resequencing efforts 10-12 expanded the number of tumor-associated MYC alleles that have been characterized. Prompted by this work, we collated published reports of missense mutations in BL and other lymphomas. We hypothesized that, Vargatef cost as with MbI, functionally important mutations may cluster in critical regions of the MYC protein, and that identification of clustered mutations in novel regions of MYC would help address the relevance of these mutations to lymphomas. Here, we report the results of this analysis, and identify a novel hotspot for mutations in MYC, spanning residues 243C249 within the central portion of the protein. We show that mutations in this region disrupt a second phosphodegron within MYC, and that they precisely phenocopy effects of mutations within MbI in terms of stability, enhanced tumorigenesis, and immunity to p53-mediated tumor surveillance mechanisms. The remarkable similarity between the effects of tumor-associated mutations in disparate regions of MYC reveals a common Vargatef cost molecular theme in MYC deregulation in lymphoma, and strongly implies that MYC mutations play an active role in the pathophysiology of the disease. RESULTS AND DISCUSSION Identification of a novel hotspot for tumor-derived mutations in MYC To generate a comprehensive view of mutations in lymphoma, we compiled published reports 1-12 of mutations described in patient samples and cultured cell Vargatef cost lines (Supplemental Table S1). Because MYC is subject to multiple mutations in ~50% of cases, we deconvoluted complex mutations and expressed the.