Fig 2: NADK sustains cell growth and drives stress resistance to promote PDAC(A) Heatmap of metabolites extracted from MIA PaCa-2-expressing shNT (non-targeting) and shNADK #1 and #2.(B) Pathway analysis of MIA PaCa-2 metabolites upon NADK knockdown.(C) Principal-component analysis of MIA PaCa-2 metabolites upon NADK knockdown.(D) Relative oxidized and reduced glutathione levels in MIA PaCa-2 upon NADK knockdown. n ≥5.(E) Relative nucleotide levels as in (D). n ≥5.(F) Relative oxidized and reduced glutathione levels in PANC-1 upon KRAS knockdown ± NADK overexpression. n ≥ 5.(G) Relative nucleotide levels as in (F). n ≥5.(H) Relative levels of global and distinct species of fatty acids upon knockdown of NADK in MIA PaCa-2. n = 5.(I) Colony-forming capacity in MIA PaCa-2 expressing shNADK and treated with 2 mM N-acetyl-L-cysteine (NAC), 5 mg/L nucleosides, or 12 μg/ml LDL and 50 μM oleate, each alone or in combination for the duration of the assay. n = 4.

Fig 3: NADK is critical for NADP(H) maintenance in PDAC(A) Relative NADP(H) levels in pancreatic ductal epithelial cells (PDEs) expressing an ectopic KRASG12V construct for 24 h and relative NADP(H) levels in mouse embryonic fibroblasts (MEFs) expressing KRASG12D allele for 4 days. n ≥ 3. For this and further experiments *p < 0.05, **p < 0.01, ***p < 0.001 and error bars represent ± SEM.(B) Relative NADP(H) levels in PANC-1 cells upon KRAS knockdown by short hairpin RNAs (shRNAs). n ≥ 5.(C) Schematic of the tracing experiment shown in (D).(D) Labeling of NADP+ and NADPH from [2,4,5,6-2H]nicotinamide, [2H]NAM, in PDEs expressing KRASG12V for 24h. n = 3.(E) Relative NADP+ levels in PANC-1 and MIA PaCa-2 cells upon NADK knockdown by shRNAs. n = 6.(F) Colony-forming capacity in MIA PaCa-2 and PANC-1 cells upon NADK knockdown by shRNAs. n ≥ 3.(G) Tumor size of MIA PaCa-2 xenografts (N ≥ 9) upon NADK knockdown and pancreatic ductal organoid number (p53 KO, KrasG12D) upon Nadk knockdown. n = 3.(H) Relative NADP(H) levels in PANC-1 cells following KRAS knockdown and NADK overexpression. n ≥5.

Fig 4: NADK is regulated by KRAS through PKC(A) Relative peak area of indicated NADK phosphorylation sites detected by LC-MS/MS after overnight serum starvation and inducible KRASG12V or GFP expression for 2 h in PDEs. n ≥ 3.(B) Schematic of NADK domains and putative phosphorylation sites.(C) Endogenous NADK band shift as observed by Phos-tag immunoblot, following overnight serum starvation and subsequent stimulation with 500 nM insulin, 100 ng/ml PMA, or 10% serum for 20 min in PDEs. n = 3.(D) Immunoblot of NADK immunoprecipitation (IP) and detection with PKC substrate antibody. PDEs were serum starved overnight followed by 1 h 100 ng/ml PMA treatment or 15-h inducible KRASG12V expression, with 1-h co-treatment with 500 nM PKC inhibitor Sotrastaurin (SOT) where indicated. n = 3.(E) Immunoblot of NADK IP and detection with PKC substrate antibody following 1-h treatment with 500 nM SOT in PANC-1 cells. n = 3.(F) Labeling of NADP+ and NADPH from [2H]NAM. PANC-1 and MIA PaCa-2 cells were serum starved overnight and then treated with 1 h 100 ng/ml PMA and ±500 nM SOT. n = 4.(G) Labeling of NADP+ and NADPH from [2H]NAM. PANC-1 cells, expressing ±shKRAS, were serum starved overnight followed by 1-h treatment with 100 ng/ml PMA or vehicle control. n = 3.(H) Immunoblot of MIA PaCa-2 for the indicated PKCs after treatment with isoform-specific PKC siRNAs. n = 3.(I) Immunoblot of NADK IP and detection with PKC substrate antibody following treatment with the indicated isoform-specific PKC siRNAs in MIA PaCa-2. n= 3.