1. 1 Applied Biosciences Professional Science Masters Program, Track in Medical and Diagnostic Laboratory Sciences, University of Arizona, Tucson, AZ; 2 Department of Pathology, University of Arizona, Tucson, AZ BACKGROUND Targeting BCL2 and MYC DNA Secondary Structures by Novel Compounds Lowers Gene Expression in DLBCL Eric V. Knight 1, Samantha L. Kendrick, PhD 2, Lisa M. Rimsza, MD 2 Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. The most aggressive DLBCL exhibit over-expression of BCL2 and MYC, two oncogenes that are important for resistance to cell death and high proliferation. This combination of characteristics causes resistance to standard chemotherapy, and currently, there is no effective treatment for double- positive BCL2/MYC DLBCL. Special DNA secondary structures exist, such as DNA G-quadruplexes, in promoter regions. Selectively targeting these structures can stabilize them and decrease transcription levels. The flexible hairpin species can be targeted in the BCL2 promoter region to repress transcription (1). Likewise, the MYC G-quadruplex (G4) has been shown to be a target for repressed gene expression (2). RESULTS METHODS REFERENCES (1)Kendrick S, Kang HS, Alam MP, et al. The Dynamic Character of the BCL2 Promoter i‐Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure. J. Am. Chem. Soc. 2014. 136 (11), 4161-4171. (2) Brown RV, Danford FL, Gokhale V, et al. Demonstration that Drug-targeted Down- regulation of MYC in Non-Hodgkins Lymphoma Is Directly Mediated through the Promoter G-quadruplex. J. Biol. Chem. 2011. 286: 41018-41027. Four DLBCL cell lines (Table 1) were used as a model for aggressive DLBCL to test efficacy of small molecule inhibitors. Tissue cultures were individually treated with compound GQC-05 to down regulate MYC expression and compound IMC-76 to down regulate BCL2 expression. Cells were collected at 6, 24, 48, and 72 hour time points. RNA extraction (Roche RNA isolation kit) was performed on the collected cells. cDNA synthesis (Bio-Rad iScript Reverse Transcription) was performed using the isolated RNA. qPCR (Bio-Rad SsoAdvanced Supermix) was then performed in order to determine the relative levels of BCL2 and MYC expression compared to the housekeeping gene TBP. (TaqMan probes FAM for BCL2 and MYC, and VIC for TBP gene expression assay) CELL LINECELL OF ORIGIN PROTEIN*TRANSLOCATION BCL2MYCBCL2MYC HTGCB---- SUDHL4GCB++ +- VALABC+++ ++ U2932ABC+++ -- *Relative levels compared to HT Table 1. Characteristics of the DLBCL cell lines used. Perform further replicates with IMC-76 Confirm that protein levels decrease after treatment Confirm that decreases in BCL2 and MYC expression leads to an increase in chemotherapy sensitivity GQC-05 Targeting MYC IMC-76 Targeting BCL2 Figure 1. The four cell lines treated with GQC-05 and the relative MYC mRNA expression levels compared to TBP levels. (A) HT cells (B) SUDHL4 cells (C) VAL cells (D) U2932 cells shown at the four time points with the respective MYC levels determined by qPCR. Figure 2. (A) VAL cells treated with IMC-76 and the BCL2 expressions levels only at the 24 hour time point; only run in singlicate so far. (B) BCL2 expression levels for U2932 cells at the four time points relative to TBP, determined by qPCR. CONCLUSIONS Treatment with GQC-05 showed a significant knockdown of MYC mRNA levels at 24h in SUDHL4, at 6h in VAL, and at 6h and 24h in U2932 cells, with the HT control cells expectedly showing no effect. We were not able to consistently show a significant knockdown of BCL2 expression in the IMC-76 treated U2932 cells, but the VAL cells show a decrease at 24h. ONGOING/ FUTURE STUDIES A B C D A B