Filamin-A (FLNA), also called actin-binding protein 280 (ABP-280), was originally identified as a non-muscle actin binding protein, which organizes filamentous actin into orthogonal networks and stress fibers. Filamin-A also anchors various transmembrane proteins to the actin cytoskeleton and provides a scaffold for a wide range of cytoplasmic and nuclear signaling proteins. Intriguingly, several studies have revealed that filamin-A associates with multiple non-cytoskeletal proteins of diverse function and is involved in several unrelated pathways. Mutations and aberrant expression of filamin-A have been reported in human genetic diseases and several types of cancer. In this review, we discuss the implications of filamin-A in cancer progression, including metastasis and DNA damage response.

Centrosomes together with the mitotic spindle ensure the faithful distribution of chromosomes between daughter cells, and spindle orientation is a major determinant of cell fate during tissue regeneration. Spindle defects are not only an impetus of chromosome instability but are also a cause of developmental disorders involving defective asymmetric cell division. In this work, we demonstrate BCCIP, especially BCCIPα, as a previously unidentified component of the mitotic spindle pole and the centrosome. We demonstrate that BCCIP localizes proximal to the mother centriole and participates in microtubule organization and then redistributes to the spindle pole to ensure faithful spindle architecture. We find that BCCIP depletion leads to morphological defects, disoriented mitotic spindles, chromosome congression defects and delayed mitotic progression. Our study identifies BCCIP as a novel factor critical for microtubule regulation and explicates a mechanism utilized by BCCIP in tumor suppression.

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While CAR-T therapy is a growing and promising area of cancer research, it is limited by high cost and the difficulty of consistently culturing T-cells to therapeutically relevant concentrations ex-vivo. Cytokines IL-2, IL-7 and IL-15 have been found to stimulate the growth of T cells, however, the optimized combination of these three cytokines for T cell proliferation is unknown. In this study, we designed an integrated experimental and modeling approach to optimize cytokine supplementation for rapid expansion in clinical applications. We assessed the growth data for statistical improvements over no cytokine supplementation and used a systems biology approach to identify genes with the highest magnitude of expression change from control at several time points. Further, we developed a predictive mathematical model to project the growth rate for various cytokine combinations, and investigate genes and reactions regulated by cytokines in activated CD4+ T cells. The most favorable conditions from the T cell growth study and from the predictive model align to include the full range of IL-2 and IL-7 studied, and at lower levels of IL-15 (6 ng/mL or 36 ng/mL). The highest growth rates were observed where either IL-2 or IL-7 was at the highest concentration tested (15 ng/mL for IL-2 and 80 ng/mL for IL-7) while the other was at the lowest (1 ng/mL for IL-2 and 6 ng/mL for IL-7), or where both IL-2 and IL-7 concentrations are moderate-corresponding to condition keys 200, 020, and 110 respectively. This suggests a synergistic interaction of IL-2 and IL-7 with regards to promoting optimal proliferation and survival of the activated CD4+ T cells. Transcriptomic data analysis identified the genes and transcriptional regulators up/down-regulated by each of the cytokines IL-2, IL-7, and IL-15. It was found that the genes with persistent expressing changes were associated with major pathways involved in cell growth and proliferation. In addition to influencing T cell metabolism, the three cytokines were found to regulate specific genes involved in TCR, JAK/STAT, MAPK, AKT and PI3K-AKT signaling. The developed Fuzzy model that can predict the growth rate of activated CD4+ T cells for various combinations of cytokines, along with identified optimal cytokine cocktails and important genes found in transcriptomic data, can pave the way for optimizing activated CD4 T cells by regulating cytokines in the clinical setting.

In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene‐body of the transcripts, suggesting epigenetic regulation. Indeed, expression changes of epigenetic regulators were observed including writers (Dnmt1, Setdb1), readers (Mecp2), and erasers (Tet3, Kdm3a, Kdm1b/5c) involved in CpG methylation, histone methylation, and heterochromatin maintenance. In addition, we identified putative transcription factors that may be readers or effectors of the epigenetic regulation in these clones. By combining transcriptomics with DNA methylation data, we identified potential processes and factors that may contribute to the variability in cell physiology between different production hosts.

Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Furthermore, we validate sequence decomposition as a cost effective, rapid, and accurate method for assessing mutants and eliminating non-clonal CHO populations using only capillary sequencing.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high‐producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi‐allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS‐knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS‐knockout hosts with desired growth and recombinant protein expression characteristics.

Background Tyrosine sulfation is a post‐translational modification found on many surface receptors and plays an important role in cell‐cell and cell‐matrix interactions. However, tyrosine sulfation of therapeutic antibodies has only been reported very recently. Because of potential potency and immunogenicity concerns, tyrosine sulfation needs to be controlled during the manufacturing process. Methods and results In this study, we explored methods to modulate antibody tyrosine sulfation during cell line development and upstream production process. We found that tyrosine sulfation levels were significantly different in various Chinese hamster ovary (CHO) cell lines due to differential expression of genes in the sulfation pathway including tyrosylprotein sulfotransferase 2 (TPST2) and the sulfation substrate transporter SLC35B2. We also screened chemical inhibitors to reduce tyrosine sulfation in CHO culture and found that sodium chlorate could significantly inhibit tyrosine sulfation while having minimal impact on cell growth and antibody production. We further confirmed this finding in a standard fed‐batch production assay. Sodium chlorate at 16 mM markedly inhibited tyrosine sulfation by more than 50% and had no significant impact on antibody titer or quality. Conclusion These data suggest that we can control tyrosine sulfation by selecting CHO cell lines based on the expression level of TPST2 and SLC35B2 or adding sodium chlorate in upstream production process.