Fig 2: DCK and DCTPP1 simultaneous knockdown induced no synergistic effect in pancreatic and ovarian cancer cells to NUC-1031. (A) DCK and DCTPP1 protein expression analyzed by Western blot in MiaPaCa2 and A2780 cells transfected with a pX333 plasmid containing sgRNA sequences targeting DCK (sgDCK) and DCTPP1 (sgDCTPP1) or a non-targeting scrambled control sgRNA (sgScr). (B) Dose response curves for MiaPaCa2 and A2780 sgDCK-sgDCTPP1 cells and sgScr-sgScr cells (control), 4d after treatment with NUC-1031 or gemcitabine (n = 6, +/-SEM. MiaPaCa2 sgScr-sgScr vs MiaPaCa2 sgDCKe-sgDCTPP1a: p = 0.0022, A2780 sgScr vs A2780 sgDCK-sgDCTPP1: p = 0.0022; Mann-Whitney test).

Fig 3: DCK expression is associated with outcome in gemcitabine-treated patients. (A) DCTPP1 expression in one tissue core before (left panel) and after (right panel) QuPath analysis based on staining intensity in tumour cells. A histoscore of 115.739 was calculated based on the proportion of positive cells and their staining intensity. DCK expression in a second tissue core before (left panel) and after (right panel) QuPath analysis based on staining intensity in tumour cells. A histoscore of 69.4313 was calculated based on the proportion of positive cells and their staining intensity. (B) KM survival curves for DCTPP1 and DCK expression generated using TMA Navigator. All of the tissue cores were divided in three groups according to their histoscore. Each group was composed of 20 samples presenting histoscores in the indicated ranges. Survival of patients is presented in months.

Fig 4: Genome-Scale CRISPR/Cas9 Screen Implicates DCK and DCTPP1 in NUC-1031 sensitivity. (A) Experimental design of the GECKOv2 screen for gemcitabine and NUC-1031 resistance, performed in MiaPaCa2 cells, in 2 biological replicates. (B) MiaPaCa2 cell number at d0, d14 and d21 after treatment with either dimethyl sulfoxide (DMSO) as control, gemcitabine or NUC-1031. (C) Heat map of sgRNA abundance comparing biological replicates and treatment conditions. (D,E) Distribution of sgRNA reads frequency before treatment (Baseline), in DMSO (Control), NUC-1031 or gemcitabine treated cells after d14 and d21 of exposure. The box extends from the first to the third quartile with the whiskers denoting 1.5 times the interquartile range and show an increased variability of sgRNA frequency in cells treated with NUC-1031 compared DMSO treated cells. (F) Scatterplot of sgRNA read counts in NUC-1031 treated cells compared to control (DMSO) cells showing enrichment of DCK and DCTPP1 sgRNAs after 14d exposure. (G) Identification of candidate genes, targeted by enriched sgRNAs, in NUC-1031 treated cells compared to control cells, using MAGeCK p-value analysis.

Fig 5: DCK mediates NUC-1031 sensitivity in pancreatic and ovarian cancer cells. (A) DCK protein expression analyzed by Western blot in MiaPaCa2 and A2780 cells transduced with independent sgRNAs targeting DCK (sgDCK) or a non-targeting scrambled control sgRNA (sgScr). (B) Dose-response curves for MiaPaCa2 and A2780 cells transduced with individual sgRNAs targeting DCK or a non-targeting scrambled control sgRNA (sgScr) and treated with NUC-1031 or gemcitabine (n = 6, +/-SEM. MiaPaCa2 sgScr vs MiaPaCa2 sgDCK: p = 0.0022, A2780 sgScr vs A2780 sgDCK: p = 0.0022 for NUC-1031 and p = 0.0043 for gemcitabine; Mann-Whitney test).