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Abstract

Pancreatic cancer is one of the deadliest cancers. Alterations in gene expression play an essential role during disease development. Also, variations in gene expression are involved in metastasis. Due to the complexity of gene expression alterations, however, it is still challenging to pinpoint and thus possibly target the functional reason behind the aggressiveness of pancreatic cancer. A high-throughput screening was conducted using the CRISPR-Cas9 whole- genome screening strategy to discover genes that might be essential for metastasis, to define processes that may have the potential to affect the metastatic progression of pancreatic cancer. Whole-genome CRISPR-Cas9 screening was conducted in two cell lines from the same parental cells but with different metastatic potential: S2-007 (highly metastatic) and S2-028 (lowly metastatic). The screening was performed, and samples were analyzed by MAGeCK analysis to select candidate genes. TYMS, MEN1, MYBL2 were selected based on their gene essentiality. In vitro validation was then conducted to confirm the gene essentiality in both cell lines. Cell proliferation, cell viability, soft agar colony formation assay was performed to narrow down the candidate. In vitro validation, MYBL2 showed the most promising potential as an essential gene in the metastatic pancreatic cancer cell. Wound healing assay and invasion assay were done to further validate MYBL2. Next, KEGG pathway analysis and Ingenuity analysis showed that MYBL2 interactions mainly in cell cycle network. Further experiments were performed which focused more on the effect of MYBL2 knockout in the cell cycle. FOXM1 gene knockout was then added to the experiments since FOXM1 plays a role in the G2/M phase. The double transduction was done, followed by cell sorting. Sorted cells were then cultured, and cell viability assay and cell cycle assay were performed. All knockouts were validated using Western blot. MYBL2 knockout showed inhibition in cell proliferation, cell viability, and soft agar colony formation assay in the S2-007 cell line. In addition, MYBL2 loss also showed invasion inhibition in S2-007. The double knockout FOXM1-MYBL2 affects cell viability more than the FOXM1 or MYBL2 single knockout. The cell cycle assay results demonstrated that the FOXM1- MYBL2, FOXM1, and MYBL2 knockout showed cell cycle arrest at G2/M in S2-007. Altogether the results showed that MYBL2 showed potential as an essential gene in the high metastatic pancreatic cancer cell line. However, the mechanism and the reason why MYBL2 showed essentiality only in the S2-007 cell line is still unclear. Therefore, more experiments still need to be conducted to achieve the goal of further elucidating the role of MYBL2 in metastatic pancreatic cancer.