Developing effective cancer therapies using compound X, a novel small molecule with cell-killing activtiy biased against non-cancer cells

Our lab manually examined the 1,000 compounds using two breast cancer cell lines (MCF7 and MDA-MB468) and two non-cancer breast cell lines (MCF10A and 184B5), from which 10 compounds were identified as having excellent potential. Subsequently, we examined a variety of different cancers, and found that the top five compounds shared a similar chemical scaffold. The representing compound X effectively killed many different cancerous cells including MCF-7, MDA-MB468, and BT-20; however, along with the two non-cancer cell lines described above, some cancer cell lines such as MDA-MB231 and A549 were resistant to Compound X. An underlying question is why certain cancer cells are exquisitely sensitive while others are resistant to Compound X. Obviously, different genetic background of each cell line is likely resulting in different sensitivity to Compound X. One of my research objectives is to identify the genes that are involved in the sensitivity/resistance to Compound X. I plan to achieve this objective by a synthetic lethal approach. Synthetic lethality refers to the cell is still viable when either of two of genes alone is disabled.  However, ablation/knockdown of both genes results in cell death. We use RNAi approach to attain synthetic lethality. We use all three modules of the Cellecta Decipher shRNA Library targeting approximately 15,377 target mRNAs (signaling pathway, disease associated, cell surface and DNA binding targets) using the shRNA lentivirus system. After transducing lentivirus particles to MDA-MB231 cells using appropriate MOI (Multiplicity of Infection) and selection of stable integration by puromycin as vector has puromycin resistant cassette, cells are treated without drug, vehicle control (DMSO) and  IC20 of Compound X to ensure that drug toxicity does not induce excessive cell death. Amplified product of PCR (Polymerase Chain Reaction) from genomic DNA is sent for next gene sequencing. Barcode is identified as each shRNA (18 nucleotides) has a unique barcode attached to it and converted to a list of genes/hits. Median Absoulte Deviation (MAD) and H score methodology will be implemented to nominate “hits”.  Those “hits” will be validated, and pathway analysis using DAVID software will be carried out.  Selected candidates/hits representative of identified pathways will be validated using multiple individual shRNA constructs with MDA-MB231. Using synthetic lethality approach, we can identify potential novel combination partners with Compound X, which will make the drug more safe and effective to control (metastatic) breast cancer.

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