Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Developing outcome measures for pediatric mitochondrial disorders: Which complaints and limitations are most burdensome to patients and their parents?

Since some drug intervention effects are only experienced by the patient, organizations such as the Food and Drug Administration prefer clinically meaningful outcome measures. Here, we evaluated which symptoms and limitations in daily life are most burdensome to pediatric patients with mitochondrial disorders and their parents, using two questionnaires. In a study of 78 patients, the most burdensome complaints included fatigue, behavior and speech disturbances, epilepsy and muscle weakness and a high degree of limitations in daily activities was found. Importantly, there was a discrepancy between what symptoms metabolic pediatricians estimated would be most burdensome compared to the patients'/caretakers' opinion. To include feasible and relevant outcome measures in intervention studies, the experience and opinions of patients and caretakers should therefore be heard.     

Female Fertilization: Effects of Sex-Specific Density and Sex Ratio Determined Experimentally for Colorado Potato Beetles and Drosophila Fruit Flies

If males and females affect reproduction differentially, understanding and predicting sexual reproduction requires specification of response surfaces, that is, two-dimensional functions that relate reproduction to the (numeric) densities of both sexes. Aiming at rigorous measurement of female per capita fertilization response surfaces, we conducted a multifactorial experiment and reanalyzed an extensive data set. In our experiment, we varied the density of male and female Leptinotarsa decemlineata (Colorado potato beetles) by placing different numbers of the two sexes on enclosed Solanum tuberosum (potato plants) to determine the proportion of females fertilized after 3 or 22 hours. In the reanalysis, we investigated how the short-term fertilization probability of three Drosophila strains (melanogaster ebony, m. sepia, and simulans) depended on adult sex ratio (proportion of males) and total density. The fertilization probability of female Leptinotarsa decemlineata increased logistically with male density, but not with female density. These effects were robust to trial duration. The fertilization probability of female Drosophila increased logistically with both sex ratio and total density. Treatment effects interacted in m. sepia, and simulans. These findings highlight the importance of well-designed, multifactorial experiments and strengthen previous experimental evidence for the relevance of sex-specific densities to understanding and prediction of female fertilization probability.     

The E3 ubiquitin ligase,HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.     

PABPN1-Dependent mRNA Processing Induces Muscle Wasting

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.     

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HC_ABD/LC_ABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LC_ABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.     

Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the transporter associated with antigen processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

Cortisol administration to pregnant sows affects novelty-induced locomotion, aggressive behaviour, and blunts gender differences in their offspring

Several behavioural effects of prenatal stress are reported in literature, and these seem to depend, among other factors, on the gender studied and the period of gestation in which prenatal stress is applied. In the present study, oral administration of hydrocortisone-acetate (HCA) to 41 pregnant sows was used as a model for prenatal stress, since corticosteroids are considered a key mediator in the effects of prenatal stress. HCA was orally administered to pregnant sows during three periods of gestation: 21-50 (period 1, P1, n = 10), 51-80 (period 2, P2, n = 10) and 81-110 (period 3, P3, n = 10) days after insemination (term 115 days). Control sows (n = 11) received vehicle from 21 to 110 days after insemination. Between days 9 and 48 after birth, treatment effects on male and female piglet behaviour were determined in the home pen and in four different behavioural tests. During the backtest, no gender differences were observed in vocalisations in HCA-piglets, while control males vocalised more than control females. In the home pen at 14 days of age, HCA-piglets spent less time in social interactions than control piglets. During the novel environment test, P1- and P3-piglets walked more than control piglets, but this was not observed during the novel object test, four days later. At weaning, P2- and P3-piglets performed less individual play. Prior to mixing with an unfamiliar piglet (male piglets only), HCA-piglets had lower salivary cortisol concentrations than control piglets, but no difference was observed after mixing. P1-, P2-, and P3- piglets had fewer non-aggressive encounters, and P2-piglets continued fighting longer than control piglets. The present study demonstrates that elevated maternal cortisol concentrations during gestation affect piglet behaviour, and effects do differ between male and female piglets. In addition, effects depend on the period of cortisol administration.