Abstract 3340: Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities
Authors: Dammert Marcel A. , Sos Martin L. 
Published: August 2018

Abstract
MYC activation reshapes cellular transcription, promotes cell growth, cell cycle progression and transformation and therefore MYC is considered a strong driver of tumor development. The MYC family includes c-MYC, MYCN, and MYCL that are typically associated with distinct tumor types. Based on the structural properties of these transcription factors MYC family members are considered undruggable targets. However, potential vulnerabilities associated with the cellular MYC status may provide a basis for therapeutic intervention. Currently, there is a lack of comparative studies assessing the specific contribution of each MYC family member to tumor development. Established cell lines in which MYC family members are overexpressed differ greatly in their genetic background, thereby impeding the analysis of MYC family member specific effects. Furthermore, the complexity of MYC regulation and MYC biology is not adequately recapitulated by traditional, heterologous MYC overexpression approaches.
Therefore, we set out to establish an endogenous overexpression system using a CRISPR activation (CRISPRa) approach to activate the individual MYC family members for a systematic comparison of their impact on target gene activation, cell growth and drug sensitivity. Using sgRNAs specific to the promoter regions of the particular MYC family members in combination with a nucleolytically inactive Cas9-VP64 fusion protein, we successfully achieved transcriptional upregulation of all three MYC family members in multiple cell lines including NIH3T3 and MEF cells. The increase in MYC mRNA resulted in markedly elevated MYC protein levels. In addition, MYC activation enhanced transcription of canonical MYC target genes like NPM1, FBL, and 45S pre-rRNA suggesting a functional impact of upregulated MYC. Moreover, activation of c-Myc led to increased anchor-independent growth of cells in a soft-agar colony formation assay indicating that high c-Myc levels induce transformation in contrast to N-Myc or L-Myc. In line with previous studies that implicated upregulated c-Myc as an initiator of genome instability, we observed elevated levels of DNA damage by monitoring phosphorylated histone variant H2AX (γH2AX) in cells with activated c-Myc whereas N-Myc or L-Myc failed to induce γH2AX. Activation of c-Myc further led to increased sensitivity to cisplatin and other chemotherapeutic agents.
In conclusion, we established an endogenous MYC overexpression model system that enables a systematic comparison of the different MYC family members and their specific contribution to tumor initiation, growth and drug sensitivity. The system successfully recapitulated previous observations of MYC activation and can serve as a platform to probe for MYC family member specific vulnerabilities and thereby pave the way for novel, targeted therapy approaches in MYC-driven cancer.
Citation Format: Marcel A. Dammert, Martin L. Sos. Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3340.