Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor

Abstract

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein response and autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCItreated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

Transcriptomics and cell painting analysis reveals molecular and morphological features associated with fed‐batch production performance in CHO recombinant clones

“…Previous studies have also made the connection between cell size and hyperosmolality, with larger CHO cells also displaying an increased specific productivity (Nasseri et al, 2014; Qin et al, 2019). Deliberate cell cycle inhibition is a strategy previously employed by our group and others (Avello et al, 2022; Chang et al, 2022; Du et al, 2015) to improve CHO cell productivity, further demonstrating the inverse relationship between proliferation and protein production. Interestingly, in this study, the larger cells we observed by cell painting grew at a similar rate under routine cell culture and only showed proliferation defects when subject to fed‐batch culture conditions.…”

“…For this reason, many studies have attempted to shed light on the cellular processes necessary for supporting a high demand for protein production. We and others have used omics‐based approaches to gain insights on the genomic, epigenomic, transcriptional and proteomic landscapes of CHO hosts and high‐performing clones (Chang et al, 2022; Chang et al, 2022; Hausmann et al, 2018; Huhn et al, 2022). An efficient protein production supply chain depends on multiple cellular functions including expression, posttranslational modification, protein folding/assembly, and efficient extracellular delivery.…”

Cyclin-dependent kinase 6 (CDK6) as a potent regulator of the ovarian primordial-to-primary follicle transition

“…In support of this, it was reported that CDK6 inhibition using Quercetin had no negative effect on viability of breast cancer and human adenocarcinoma cells, associating with induction of apoptosis (Yousuf et al, 2020). In contrast, it has been shown that CDK6 inhibition can impair antioxidants and significantly enhance ROS level (Chang et al, 2022). Moreover, it has been reported that CDK6 can regulate stabilization and activation of FOXM1, as a substrate of CDK4/6-Cyclin D complexes, leading to reduction of ROS production (Anders et al, 2011).…”

“…IN summary, these studies support our observations that inhibition of CDK6 could reduce the PI3K/ AKT pathway, which would lead to reduce activation of primordial follicles. If note, and in contrast, it has been determined that CDK6 inhibition induced phosphorylation of AKT1S1 and increased the activation of mTORC1 signaling (Chang et al, 2022), but the details of how this is achieved is not known.…”