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Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.

SUBMITTER: Wei X

PROVIDER: S-EPMC7668168 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.MethodsIn order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine.ResultsResults from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine.ConclusionThese findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.

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Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.

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