S. Nair1, A. Chan2, H. Feng1, L. Yang1, C. W. D. Chen2, and T. M. Williams1; 1Department of Radiation Oncology, City of Hope National Medical Center, Duarte, CA, 2City of Hope, Duarte, CA
Purpose/Objective(s): Resistance to chemotherapy and/ or radiation remains a major challenge in pancreatic cancer leading to treatment failure and poor prognosis in patients. Resistance arises due to the adaptive plasticity of cancer cells to escape therapeutic pressure or the expansion of an inherently resistant population of cells. Therefore, there is a need to identify novel genes regulating inherent tumor cell autonomous chemoradioresistance in pancreatic cancer cells. CRISPR based screens are powerful tools that can be used for repressing or inducing activation (CRISPRa) of genes en masse. Screens can be genome-wide or a focused library targeting genes involved in specific pathways or biological processes. We employed a focused CRISPRa library targeting 328 DNA damage repair genes to identify novel targets activated in inherently resistant pancreatic cancer cells in vitro.Materials/
Methods: Two human pancreatic cancer cells lines MIA-PaCa2 (MP2) and PANC-1 were modified to stably express dCas9-VP64, a transcriptional activator. We then optimized and validated dCas9-VP64-mediated gene activation system by transfecting cells with a guide RNA (gRNA) expressing RFP and assessing the increase in RFP expression over 2 weeks by flow cytometry. We further confirmed the ability of dCas9-VP64 to increase endogenous gene activation using gRNAs to target a ubiquitin-specific protease (USP) gene called USP36. Gene activation was confirmed by qPCR and western blotting. To mimic inherent resistance, we optimized conditions by treating cells with varying doses of gemcitabine and radiation. We then transduced a library with gRNAs targeting 328 DNA damage repair genes with a targeting density of 7 gRNAs per gene and 1000x coverage into dCas9 expressing cells; treated them with our chemoradiation regimen, followed by extraction and NextSeq550 sequencing to identify genes regulating chemoradioresistance. Results: With our system, we achieved over 50% increase in RFP levels in MP2 and PANC-1 expressing dCas9-VP64 compared to parental cells. The Cas9 expressing cells also significantly upregulated the expression of USP36 consistently over 14 days. By optimization, we found that treating cells for 24 hours with 5nM of gemcitabine followed by 3 consecutive days of 1Gy irradiation was the best treatment regimen to generate 5% viable cells, which we consider an inherently resistant population to chemoradiotherapy. Finally, we are awaiting results from our high-throughput sequencing. Conclusion: We successfully established two in vitro models capable of activating 328 DNA damage repair genes after transduction of the gRNA library and expect to present the full results from our screening and high-throughput sequencing at the meeting. We anticipate that our current work will identify new targets of synthetic lethality that will help sensitize pancreatic cancer cells to radiation and/or chemotherapy.
