MYC/Glutamine Dependency Is a Therapeutic Vulnerability in Pancreatic Cancer with Deoxycytidine Kinase Inactivation-Induced Gemcitabine Resistance

Abstract

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.

Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer

“…The CFPAC-1 cell line was transduced with oligos and corresponded to the sgRNA sequence (Supplementary Table 1 ) of sgDCK#10 and control sgNT1 described in our previous article and named as DCK#10 and NT1 cell [ 10 ]. CFPAC-1, DCK#10 (DCK knockout CFPAC-1 cell), NT1 (CFPAC-1 cell with the non-target gRNA insert), HA-DCK (DCK#10 with stable expression of HA-DCK [ 10 ]), HA-DCK-KD (DCK#10 with stable expression of HA-DCK bearing the kinase-dead mutation [ 10 ]) and EV (DCK#10 with stable expression of empty vector [ 10 ]) cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; FUJIFILM Wako Pure Chemical Corporation [FUJIFILM-Wako]), HPAF-II cells were maintained in Eagle’s minimum essential medium (FUJIFILM-Wako). All cell culture media were supplemented with 10% heat-inactivated FBS (Gibco #10270–106) and 1% penicillin–streptomycin (10,000 U/mL; Thermo Fisher Scientific, #15140–122).…”

“…Western blotting was performed following the protocol described in our previous publication [ 10 ]. Proteins related to mitochondrial complexes were probed using Total OXPHOS Rodent WB Antibody Cocktail (abcam, ab #110413, 1:1000).…”

“…To achieve ectopic overexpression of DCK, DCK was subcloned into the lentiviral expression vector CSII-CMV-MCS-IRES2-Bsd (RIKEN BRC #RDB04385) at the NheI/XhoI site, following the procedure described previously [ 10 ]. Subsequently, CFPAC-1 and HPAF-II cells transduced with the lentivirus were selected in IMDM and EMEM media containing 10 μg/mL of blasticidin S for 6 days.…”

“…The rate-limiting enzyme responsible for gemcitabine activation, deoxycytidine kinase (DCK), is frequently inactivated in PDAC cells with acquired gemcitabine resistance [ 8 , 9 ]. Furthermore, recent genome-wide screen also confirmed the significance of DCK inactivation in gemcitabine resistance in PDAC cells [ 10 ]. Moreover, PDAC patients with decreased DCK expression exhibited shorter overall survival during gemcitabine-based treatment [ 11 ].…”

“…In our previous study, we elucidated on the upregulation MYC/glutamine dependence and OXPHOS-related genes in DCK-deficient gemcitabine-resistant cells [ 10 ]. In this study, further characterization of the DCK-deficient PDAC cells showed that these cells have increased mitochondrial gene set expression associated with a higher OXPHOS activity for energy production.…”