A forward genetics screen identifies modifiers of Rocaglate responsiveness

Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as responsive tumor types. In performing a forward genetics screen using a cDNA library, we identified FOXP3 to be a gene of interest. As FOXP3 is a known transcriptional regulator, understandings its impacts on the transcriptional landscape was a logical undertaking in elucidating the resistance mechanis. Here, we report on the RNAseq results in Hap1 cells overexpressing FOXP3 compared to a negative control cell line expressing GFP. We find that certain genes involved in response to drug, such as ABCB1 (P-glycoprotein), are potently upregulated in a FOXP3-overexpression setting. Overall design: We performed RNA-Seq transcriptional profiling of Hap1 cells overexpressing FOXP3 or GFP cDNAs. Briefly, Hap1 CML cells were transduced with vectors encoding our genes of interest. Each cell line (5 x 10^6) was then seeded into 10 cm dishes and allowed to expand for 48 hours. Cells were then washed with PBS, and RNA was harvested using the Qiagen RNeasy Mini Kit with on-column DNase I digestion. Samples were then sent to the Institut de Recherches Cliniques de Montréal (IRCM) for library preparation and deep sequencing. RNA integrity was first assessed on Bioanalyzer RNA Pico chips, then strand-specific, barcoded libraries were prepared via cDNA production, PCR, and ribosomal RNA (rRNA) depletion. Deep sequencing was performed using the Illumina HiSeq 4000 platform with paired-end 50 reads at approximately 50 million reads per sample. Biological triplicates for both GFP- and FOXP3-expressing cells were obtained.