Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.

De novo assembly and annotation of the CHOZN® GS-/- genome supports high-throughput genome-scale screening.

Chinese hamster ovary (CHO) cells have been used as the industry standard for the production of therapeutic monoclonal antibodies for several decades. Despite significant improvements in commercial-scale production processes and media, the CHO cell has remained largely unchanged. Due to the cost and complexity of whole-genome sequencing and gene-editing it has been difficult to obtain the tools necessary to improve the CHO cell line. With the advent of next-generation sequencing and the discovery of the CRISPR/Cas9 system it has become more cost effective to sequence and manipulate the CHO genome. Here, we provide a comprehensive de novo assembly and annotation of the CHO-K1 based CHOZN® GS-/- genome. Using this platform, we designed, built, and confirmed the functionality of a whole genome CRISPR guide RNA library that will allow the bioprocessing community to design a more robust CHO cell line leading to the production of life saving medications in a more cost-effective manner.

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4.

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.

The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

Analysis of peripheral nerve expression profiles identifies a novel myelin glycoprotein, MP11.

The myelin sheath insulates axons and allows for rapid salutatory conduction in the nervous system of all vertebrates. The formation of peripheral myelin requires expression of the transcription factor Egr2, which is responsible for inducing such essential myelin-associated genes as Mpz, Mbp, Pmp22, and Mag. Using microarray analysis to compare gene expression patterns in peripheral nerve during development, during remyelination after nerve injury, and in a congenital hypomyelinating mouse model, we identified an evolutionarily conserved novel component of myelin called Mp11 (myelin protein of 11 kDa). The Mp11 genomic locus contains multiple conserved Egr binding sites, and Mp11 induction is regulated by the expression of Egr2. Similar to other Egr2-dependent genes, it is induced during developmental myelination and remyelination after nerve injury. Mp11 is a glycoprotein expressed preferentially in the myelin of the peripheral nervous system versus CNS and is specifically localized to the Schmidt-Lanterman incisures and paranodes of peripheral nerve. The Mp11 protein contains no identifiable similarity to other known protein domains or motifs. However, like other myelin genes, strict Mp11 expression levels are a requirement for the in vitro myelination of DRG neurons, indicating that this previously uncharacterized gene product is a critical component of peripheral nervous system myelin.

Misexpression of Pou3f1 results in peripheral nerve hypomyelination and axonal loss.

Pou3f1/SCIP/Oct-6 is a POU-domain transcription factor that is an important regulator of peripheral nerve myelination by Schwann cells. Pou3f1-deficient mice experience a developmental delay in myelination indicating that transient induction of Pou3f1 is required for normal development of peripheral myelin. The mechanism by which Pou3f1 regulates myelination is unclear, because it can both increase expression of Egr2, a transcription factor that promotes the myelination program, and also repress the promoters of specific myelin genes such as myelin protein zero (MPZ) and myelin basic protein (MBP). Therefore, to investigate the effects of persistent Pou3f1 expression on peripheral nerve myelination, we created a conditional transgenic mouse [condPou3f1:MPZ(Cre)] that constitutively expresses Pou3f1 specifically in peripheral glia. Examination of sciatic nerves from condPou3f1:MPZ(Cre) mice revealed persistent hypomyelination and eventual axonal loss but no evidence of demyelination/remyelination processes or impaired Schwann cell proliferation. Nerves from these mice had normal levels of Egr2 mRNA but decreased levels of MPZ, MBP, and Pmp22 mRNA. Thus, unlike the Pou3f1 null mice, the condPou3f1:MPZ(Cre) mice exhibit persistent hypomyelination, indicating that strict control of Pou3f1 expression is critical to proper myelination. Our findings establish the importance of identifying factor(s) responsible for Pou3f1 downregulation during myelination, because they may play important roles in the development of peripheral neuropathies.

Deciphering adaptor specificity in GFL-dependent RET-mediated proliferation and neurite outgrowth.

Glial cell derived neurotrophic factor (GDNF)-dependent receptor tyrosine kinase RET activity is required for proper development of the nervous system and genitourinary tract. Loss-of-function mutations in RET are associated with enteric nervous system abnormalities (Hirschsprung disease) and renal deficits (Potter's syndrome), whereas activating mutations lead to hereditary cancer syndromes (multiple endocrine neoplasia type 2A and type 2B). RET activation is crucial for the proper regulation of a variety of cellular processes including cell migration, proliferation and neurite outgrowth. By analyzing a series of RET mutants we found that Y1062 was critical for stimulating GDNF-mediated proliferation as well as proliferation stimulated by GDNF-independent oncogenic forms of RET. Studies using small interfering RNA driven by lentivirus to knock-down expression of particular adaptor proteins that interact with RET phospho-Y1062, demonstrated that only Src-homology 2 and growth factor receptor binding protein 2 were necessary for RET-mediated proliferation by wild type and oncogenic forms of RET. Interestingly, we discovered that Y1062 was also required for GDNF-stimulated neurite outgrowth. However, small interfering RNAs to either Src-homology 2 or growth factor receptor binding protein 2 or a panel of other adaptor proteins known to interact with RET Y1062 were incapable of blocking GDNF-stimulated neurite formation, indicating that differential use of intracellular adaptors is responsible for regulating alternative RET-stimulated cellular events such as proliferation versus a differentiation response like neurite outgrowth.

Akt regulates basal and induced processing of NF-kappaB2 (p100) to p52.

NF-kappaB is a family of transcription factors important for innate and adaptive immunity. NF-kappaB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-kappaB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IkappaB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKalpha and IKKbeta. The preferred substrate for IKKbeta is IkappaBalpha, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKalpha activates a non-canonical NF-kappaB pathway in which p100 (NF-kappaB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKalpha and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-kappaB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-kappaB activity.

Tumor necrosis factor activates CRE-binding protein through a p38 MAPK/MSK1 signaling pathway in endothelial cells.

Tumor necrosis factor (TNF) promotes immunity and modulates cell viability, in part, by promoting alterations of cellular gene expression. The mechanisms through which TNF communicates with the nucleus and alters gene expression are incompletely understood. Incubation of human umbilical vein endothelial cells (HUVEC) with TNF induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. Dominant negative CREB, an antagonist antibody directed against the type 1 TNF receptor, or pharmacological inhibition of p38 MAPK signaling blocked TNF-induced CREB activation as determined by phosphorylation and gene reporter assays. From among the kinases that can activate CREB, we found that downstream of p38 MAPK, MSK1 is activated by TNF to promote CREB activation. These observations show that CREB is activated by TNF/TNFR1 signaling through a p38MAPK/MSK1 signaling pathway.

Cell type-specific expression of the IkappaB kinases determines the significance of phosphatidylinositol 3-kinase/Akt signaling to NF-kappa B activation.

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-kappa B through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-kappa B activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-kappa B DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-kappa B is retained in the cytoplasm by inhibitory proteins, I kappa Bs, which are phosphorylated and targeted for degradation by I kappa B kinases (IKK alpha and IKK beta). Expression and the ratios of IKK alpha and IKK beta, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKK alpha (the IKK kinase activated by Akt) to IKK beta were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKK beta diminished the capacity of the inhibitors to block NF-kappa B DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-kappa B DNA binding in Ikk beta-/- but not Ikk alpha-/- or wild-type cells in which the ratio of IKK alpha to IKK beta is low. Thus, noncoordinate expression of I kappa B kinases plays a role in determining the cell type-specific role of Akt in NF-kappa B activation.