Pancreatic ductal adenocarcinoma (PDAC) is one of the most life-threatening malignancies. Although the deoxycytidine analog gemcitabine has been used as the first-line treatment for PDAC, the primary clinical challenge arises because of an eventual acquisition of resistance. Therefore, it is crucial to elucidate the mechanisms underlying gemcitabine resistance to improve treatment efficacy. To investigate potential genes whose inactivation confers gemcitabine resistance, we performed CRISPR knockout library screening. We found that DCK deficiency is the primary mechanism of gemcitabine resistance, and the inactivation of CRYBA2, DMBX1, CROT, and CD36 slightly conferred gemcitabine resistance. In particular, gene expression analysis revealed that DCK-knockout (KO) cells displayed a significant enrichment of genes associated with MYC targets, folate/one-carbon metabolism and glutamine metabolism pathways. Evidently, chemically targeting each of these pathways significantly reduced the survival of DCK KO cells. Moreover, the pathways enriched in DCK KO cells represented a trend similar to those in PDAC cell lines and samples of patients with PDAC with low DCK expression. We further observed that short-term treatment of parental CFPAC-1 cells with gemcitabine induces the expression of several genes, which promote synthesis and transport of glutamine in a dose-dependent manner, which suggests glutamine availability as a potential mechanism of escaping drug toxicity in an initial response for survival. Thus, our findings provide insights into novel therapeutic approaches for gemcitabine-resistant PDAC and emphasize the involvement of glutamine metabolism in drug-tolerant persister cells. Implications: Our study revealed the key pathways involved in gemcitabine resistance in PDAC, thus providing potential therapeutic strategies.

<p>S1. Supplementary results corresponding to Figures 1 and 2S2. Supplementary results corresponding to Figure 2Cas9-sgRNA-mediated genome editing in the DCK gene detected using direct sequenceS3. Supplementary results corresponding to Figure 2S4. Supplementary results corresponding to Figure 4</p>

<p>Supplementary Table 1.sgRNA barcode PCR primers. PCR, polymerase chain reactionSupplementary Table 2.Pool A and Pool B log2 fold change of >10 upregulated sgRNA oligo sequencesSupplementary Table 3.Primer sequences used for RT-qPCR and CRISPR/Cas9 degradation-resistant HA-tagged DCK gene. RT-qPCR, real-time quantitative polymerase chain reactionSupplementary Table 4.Primer sequences used for sequencing DCK region</p>

<p>S1. Supplementary results corresponding to Figures 1 and 2S2. Supplementary results corresponding to Figure 2Cas9-sgRNA-mediated genome editing in the DCK gene detected using direct sequenceS3. Supplementary results corresponding to Figure 2S4. Supplementary results corresponding to Figure 4</p>

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