Malignant peripheral nerve sheath tumors (MPNSTs) are rare, but highly aggressive sarcomas that often occur in patients with neurofibromatosis type I (NF1). Currently, surgical resection is the only treatment, but it is largely ineffective, resulting in a five-year survival rate between 20 and 50 percent. While MPNSTs are genetically and molecularly heterogeneous, mTOR is almost universally overexpressed in this cancer. Targeting the localization and activation of the mTOR complex mTORC1 within this subset of tumors may offer a strong potential treatment. As arginine is implicated in both mTORC1 localization and activation, modulating arginine levels may serve to regulate mTORC1. Importantly, arginine succinate dehydrogenase 1 (ASS1), an enzyme necessary for arginine synthesis, is suppressed in most sarcomas, causing cells to become reliant on extracellular arginine. Arginine deiminase reverts extracellular arginine to citrulline, effectively making arginine inaccessible by cells lacking ASS1. In this study, we set out to determine how arginine deprivation affects mTORC1 activation and localization and the potential impacts of mTORC1 on cellular metabolism. Using ADI-PEG20, a pegylated arginine deiminase drug, we deprived MPNST cells of arginine in vitro. To determine whether ADI-PEG20 could be effective at depriving cells of arginine, we first examined expression of ASS1 in MPNSTs and found heterogeneous expression among established MPNST cell lines. We found that depleting MPNSTs of arginine by ADI-PEG20 affects mTORC1 activation; however, this was independent of ASS1 expression, in contrast to our initial hypothesis. Because mTORC1 potentially interacts with hexokinase II (HK-II), a metabolic regulator, we examined the effect of arginine deprivation on mTORC1 binding to HK-II. mTORC bound HK-II independent of ASS1 expression. However, inactive mTORC1 preferentially bound to HK-II over active mTORC1. Finally, we explored MPNST metabolism, because previous studies have implicated mTORC1 interaction with HK-II in a switch from a glycolytic to OXPHOS phenotype. We found that MPNSTs favor an OXPHOS phenotype, an unusual metabolism in cancers. This association between mTORC1, HK-II, arginine, and metabolism give preliminary data important in understanding this unique cancer’s metabolism and methods of survival and may help to develop a treatment targeting MPNSTs.
Non-genetic cellular heterogeneity is often overlooked in the study of molecular biology. Population averaged measurements of clonal cell populations are made under the assumption that genetic homogeneity implies cellular homogeneity. On the contrary, such assumption discounts the vast variability that exists within a clonal cell population. When gene expression of individual genes is observed across a clonal population of mammalian cells, variation in expression of the same gene differ between cells. This phenomenon is defined as gene expression noise and has been shown to have a functional role in processes like cell fate decisions and viral latency reactivation. While many efforts have been made to measure gene expression noise, less is known about how to control noise. Here, we present our work towards controlling noise using a synthetic genetic circuit we call a noise rheostat. The circuit we built places two small-molecule inducible transcription systems linked in series, driving expression of a green fluorescent protein reporter gene. The inducible transcription system includes an abscisic acid inducible synthetic transcription factor with its cognate promoter and a doxycycline inducible transcription factor with its cognate promoter. By using two inducible transcription systems in series, we create lags in transcription of the terminal output and thus produce different noise levels. We performed transient transfections and characterized the system through dosage experiments using flow cytometry. The datasets analyzed demonstrate that gene expression noise is dialable while maintaining gene expression mean. These results offer a promising prototype for the first mammalian noise rheostat. We propose that this tool will be useful in the study of noise biology as it provides the ability to separate the control of gene expression mean from gene expression noise.
Heterochromatin boundary elements inhibit the spread of repressive histone methylation through gene coding regions to prevent the silencing of nearby genes. Two parallel and redundant pathways are responsible for the function of heterochromatin boundaries in the fission yeast, Schizosacchromyces pombe; A pathway that involves TFIIIC, a transcription factor that associates with specific DNA elements, and a pathway that involves Epe1, a Jmjc domain-containing protein enriched at heterochromatin boundaries. Although TFIIIC and Epe1 are known to regulate heterochromatin boundaries, their mechanisms of action are still relatively unknown. To elucidate the proteins involved in the TFIIIC-dependent pathway of boundary function, chemical mutagenesis was employed using a reporter strain that reads out boundary function and lacks the Epe1-dependent boundary pathway. Mutants that exhibited impaired TFIIIC-dependent boundary function were sequenced to identify individual point mutations in four unique genes, sda1, cog5, dpb2, and byr3. To test if these genes play a role in TFIIIC-dependent boundary function, a CRISPR/Cas9 system was engineered to target wildtype genes in the Epe1-deficient reporter strains and reintroduce the identified mutations. The CRISPR/Cas9 plasmids were successfully amplified with sgRNA inserts capable of targeting Cas9 to sda1+, cog5+, dpb2+, and byr3+. The plasmids and sgRNA sequences were confirmed by CspCI restriction enzyme digest and Sanger sequencing. These plasmids will be transformed into S. pombe to generate reporter strains harboring each mutation, which can be used to verify if these mutations impair TFIIIC-dependent boundary function.
B cell lymphocytes are an immune cell able to generate antigen specific responses. B cell activation occurs through two different receptors, the B cell receptor and CD40, by interaction of antigen and CD40 Ligand, respectively. Following activation through the BCR and CD40, B cells undergo the energetically demanding processes of proliferation and differentiation into either memory cells, which provide a rapid and strong response to a secondary infection, or plasma cells, that produce large amounts of antigen specific antibodies. The immune cell most similar to B cells – T lymphocytes – require a metabolic change to aerobic glycolysis following activation to support effector function and memory differentiation. Due to the similarities in effector and memory classes of the two cell types, we hypothesized that a metabolic change must also occur in B cells to allow for their proliferation and differentiation to memory cells. Specifically, we examined alterations in mitochondrial mass to indicate a metabolic transition after activation. To do this, we stained cells with MitoTracker Green stain and quantified mitochondrial content by flow cytometry. We previously demonstrated that mitochondrial mass increased following stimulation through the BCR and CD40 in the Ramos cells – a germinal center-like Burkitt’s Lymphoma B cell. To generalize these results, we repeated these experiments in BL41, another Burkitt’s lymphoma B cell line. However, we found no increase in mitochondrial mass in BL41 following stimulation through BCR and CD40. We therefore investigated if strength of signaling could effect the cell types differently, and preliminary data suggests that increased BCR stimulation in BL41 may increase mitochondrial mass. We also asked whether a viral CD40 mimic – the Epstein-Barr Virus (EBV) protein LMP1 – can regulate mitochondrial mass in BL41 cells. LMP1 signaling did not increase mitochondrial mass. These results could motivate future studies investigating the mechanism through which mitochondrial mass increases in Ramos cells in response to BCR and CD40 activation, using BL41 as a negative control.