Nucleus-Translocated GCLM Promotes Chemoresistance in Colorectal Cancer through a Moonlighting Function

Emerging evidence indicates that metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. In this study, utilizing a genome-scale metabolic CRISPR-Cas9 knockout library screen, we observed that loss of Glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably heightens the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B-repressing factor (NKRF), a known inhibitor of NF-?B signaling, to promote NF-?B activity and subsequently facilitate chemoresistance. In response to platinum drug treatment, P38 phosphorylates GCLM at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM is evident in CRC tissues and correlates with poor prognosis and heightened P38 activity. Overall, our findings shed light on the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-?B activity and subsequent chemoresistance. Overall design: The human metabolic enzyme sgRNA library, targeting 1773 metabolic enzyme-encoding genes with 4 sgRNAs per gene, was designed and synthesized by GENEWIZ (Suzhou, China) and was then packaged into lentiviruses as previously described. For the in vitro CRISPR-Cas9 knockout screenin the presence of oxaliplatin, 4×107 HCT116 cells were plated, transduced with the lentiviral library and selected with puromycin (0.8 µg/mL) as previously reported3. The selected cells were divided into 4 groups and treated with PBS or 10 µM oxaliplatin for 7 days. Thereafter, the remaining surviving cells were harvested, and their genomic DNA was isolated and amplified by 2-step PCR using NEBNext Ultra II Q5 Master Mix (M0544L, New England Biolabs, Ipswich, MA, USA) and the primers listed in Supplementary Table 4. The PCR products containing the sgRNA sequences were extracted from the gel, quantified, sequenced, and analyzed as previously described