Impact of genomic background and developmental state on signaling pathways and response to therapy in glioblastoma patient-derived cells.

Glioblastoma (GBM) tumors represents diverse genomic epigenomic, and transcriptional landscapes, with significant intratumoral heterogeneity that challenges standard of care treatments involving radiation (RT) and the DNA-alkylating agent temozolomide (TMZ). In this study, we employed targeted proteomics to assess the response of a genomically-diverse panel of GBM patient-derived cancer stem cells (CSCs) to astrocytic differentiation, growth factor withdrawal and traditional high fetal bovine serum culture. Our findings revealed a complex crosstalk and co-activation of key oncogenic signaling in CSCs and diverse patterns of response to these external stimuli. Using RNA sequencing and DNA methylation, we observed common adaptations in response to astrocytic differentiation of CSCs across genomically distinct models, including BMP-Smad pathway activation, reduced cholesterol biosynthesis, and upregulation of extracellular matrix components. Notably, we observed that these differentiated CSC progenies retained a subset of stemness genes and the activation of cell survival pathways. We also examined the impact of differentiation state and genomic background on GBM cell sensitivity and transcriptional response to TMZ and RT. Differentiation of CSCs increased resistance to TMZ but not to RT. While transcriptional responses to these treatments were predominantly regulated by p53 in wild-type p53 GBM cells, its transcriptional activity was modulated by the differentiation status and treatment modality. Both mutant and wild-type p53 models exhibited significant activation of a DNA-damage associated interferon response in CSCs and differentiated cells, suggesting this pathway may play a wider role in GBM response to TMZ and RT. Our integrative analysis of the impact of GBM cell developmental states, in the context of genomic and molecular diversity of patient-derived models, provides valuable insights for pre-clinical studies aimed at optimizing treatment strategies.

Protein biomarkers for subtyping breast cancer and implications for future research: a 2024 update.

INTRODUCTION: Breast cancer subtyping is used clinically for diagnosis, prognosis, and treatment decisions. Subtypes are categorized by cell of origin, histomorphology, gene expression signatures, hormone receptor status, and/or protein levels. Categorizing breast cancer based on gene expression signatures aids in assessing a patient's recurrence risk. Protein biomarkers, on the other hand, provide functional data for selecting therapies for primary and recurrent tumors. We provide an update on protein biomarkers in breast cancer subtypes and their application in prognosis and therapy selection. AREAS COVERED: Protein pathways in breast cancer subtypes are reviewed in the context of current protein-targeted treatment options. PubMed, Science Direct, Scopus, and Cochrane Library were searched for relevant studies between 2017 and 17 August 2024. EXPERT OPINION: Post-translationally modified proteins and their unmodified counterparts have become clinically useful biomarkers for defining breast cancer subtypes from a therapy perspective. Tissue heterogeneity influences treatment outcomes and disease recurrence. Spatial profiling has revealed complex cellular subpopulations within the breast tumor microenvironment. Deciphering the functional relationships between and within tumor clonal cell populations will further aid in defining breast cancer subtypes and create new treatment paradigms for recurrent, drug resistant, and metastatic disease.

Functional activation of the AKT-mTOR signalling axis in a real-world metastatic breast cancer cohort.

BACKGROUND: Mutations of the PIK3CA/AKT/mTOR axis are common events in metastatic breast cancers (MBCs). This study was designed to evaluate the extent to which genetic alterations of the PIK3CA/AKT/mTOR can predict protein activation of this signalling axis in MBCs. METHODS: Molecular profiles were generated by CLIA-certified laboratories from a real-world evidence cohort of 171 MBC patients. Genetic alterations of the PIK3CA pathway were measured using next-generation sequencing. Activation levels of AKT and downstream signalling molecules were quantified using two orthogonal proteomic methods. Protein activity was correlated with underlying genomic profiles and response to CDK4/6 inhibition in combination with endocrine treatment (ET). RESULTS: Oncogenic alterations of the PIK3CA/AKT/PTEN pathway were identified in 49.7% of cases. Genomic profiles emerged as poor predictors of protein activity (AUC:0.69), and AKT phosphorylation levels mimicked those of mutant lesions in 76.9% of wild-type tumours. High phosphorylation levels of the PI3K/AKT/mTOR downstream target p70S6 Kinase (T389) were associated with shorter PFS in patients treated with CDK4/6 inhibitors in combination with ET (HR:4.18 95%CI:1.19-14.63); this association was not seen when patients were classified by mutational status. CONCLUSIONS: Phosphoprotein-based measurements of drug targets and downstream substrates should be captured along with genomic information to identify MBCs driven by the PI3K/AKT/mTOR signalling.

Proteomics based selection achieves complete response to HER2 therapy in HER2 IHC 0 breast cancer.

Recent trials have shown the efficacy of trastuzumab deruxtecan (T-DXd) in HER2-negative patients, but there is not yet a way to identify which patients will best respond, especially with the inability of current HER2 IHC and FISH assays to accurately determine HER2 expression in the unamplified setting. Here, we present a heavily pre-treated patient with triple-negative breast cancer (HER2 IHC 0 who had a complete response to T-DXd. In this case, we used a CLIA-certified reverse-phase protein array-based proteomic assay (RPPA) to determine that the patient had moderate HER2 protein expression (HER2Total 2+, 42%) and activation (HER2Y1248 1+, 23%). Using these results, we determined that the patient may benefit from T-Dxd despite being traditionally qualified as HER2 IHC 0. These findings highlight the potential for proteomics-based assays that may more accurately quantitate HER2 and (its activation) in the HER2 unamplified/IHC 0 setting to better select patients whose tumors are classically molecularly defined as HER2 IHC 0, but still could respond to HER2-directed therapy, and give patients access to therapies which for which they otherwise would not be eligible.

Pilot clinical trial and phenotypic analysis in chemotherapy-pretreated, metastatic triple-negative breast cancer patients treated with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency followed by cisplatin and nab paclitaxel.

BACKGROUND: A subset of triple-negative breast cancers (TNBCs) have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. PIKTOR, a combination of TAK-228 (TORC1/2 inhibitor) and TAK-117 (PI3Kα inhibitor), is hypothesized to increase genomic instability and increase DNA damage repair (DDR) deficiency, leading to increased sensitivity to DNA-damaging chemotherapy and to immune checkpoint blockade inhibitors. METHODS: 10 metastatic TNBC patients received 4 mg TAK-228 and 200 mg TAK-117 (PIKTOR) orally each day for 3 days followed by 4 days off, weekly, until disease progression (PD), followed by intravenous cisplatin 75 mg/m2 plus nab paclitaxel 220 mg/m2 every 3 weeks for up to 6 cycles. Patients received subsequent treatment with pembrolizumab and/or chemotherapy. Primary endpoints were objective response rate with cisplatin/nab paclitaxel and safety. Biopsies of a metastatic lesion were collected prior to and at PD on PIKTOR. Whole exome and RNA-sequencing and reverse phase protein arrays (RPPA) were used to phenotype tumors pre- and post-PIKTOR for alterations in DDR, proliferation, and immune response. RESULTS: With cisplatin/nab paclitaxel (cis/nab pac) therapy post PIKTOR, 3 patients had clinical benefit (1 partial response (PR) and 2 stable disease (SD) ≥ 6 months) and continued to have durable benefit in progression-free survival with pembrolizumab post-cis/nab pac for 1.2, 2, and 3.6 years. Their post-PIKTOR metastatic tissue displayed decreased mismatch repair (MMR), increased tumor mutation burden, and significantly lower levels of 53BP1, DAG Lipase ß, GCN2, AKT Ser473, and PKCzeta Thr410/403 compared to pre-PIKTOR tumor tissue. CONCLUSIONS: Priming patients' chemotherapy-pretreated metastatic TNBC with PIKTOR led to very prolonged response/disease control with subsequent cis/nab pac, followed by pembrolizumab, in 3 of 10 treated patients. Our multi-omics approach revealed a higher number of genomic alterations, reductions in MMR, and alterations in immune and stress response pathways post-PIKTOR in patients who had durable responses. TRIAL REGISTRATION: This clinical trial was registered on June 21, 2017, at ClinicalTrials.gov using identifier NCT03193853.

A resample-replace lasso procedure for combining high-dimensional markers with limit of detection.

In disease screening, a biomarker combination developed by combining multiple markers tends to have a higher sensitivity than an individual marker. Parametric methods for marker combination rely on the inverse of covariance matrices, which is often a non-trivial problem for high-dimensional data generated by modern high-throughput technologies. Additionally, another common problem in disease diagnosis is the existence of limit of detection (LOD) for an instrument - that is, when a biomarker's value falls below the limit, it cannot be observed and is assigned an NA value. To handle these two challenges in combining high-dimensional biomarkers with the presence of LOD, we propose a resample-replace lasso procedure. We first impute the values below LOD and then use the graphical lasso method to estimate the means and precision matrices for the high-dimensional biomarkers. The simulation results show that our method outperforms alternative methods such as either substitute NA values with LOD values or remove observations that have NA values. A real case analysis on a protein profiling study of glioblastoma patients on their survival status indicates that the biomarker combination obtained through the proposed method is more accurate in distinguishing between two groups.

Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer.

BACKGROUND: Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined. METHODS: We explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes. FINDINGS: TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer. CONCLUSIONS: Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.

Durability of Viral Neutralization in Asymptomatic Coronavirus Disease 2019 for at Least 60 Days.

A cohort consisting of asymptomatic healthcare workers donated temporal serum samples after infection with severe acute respiratory syndrome coronavirus 2. Analysis shows that all asymptomatic healthcare workers had neutralizing antibodies, that these antibodies persist for ≥60 days, and that anti-spike receptor-binding domain immunoglobulin G levels were correspondingly durable over the same time period.

Proteomic Analysis of Cardioembolic and Large Artery Atherosclerotic Clots Using Reverse Phase Protein Array Technology Reveals Key Cellular Interactions Within Clot Microenvironments.

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Clinical Utility of a Highly Sensitive Lateral Flow Immunoassay as determined by Titer Analysis for the Detection of anti-SARS-CoV-2 Antibodies at the Point-of-Care.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.

Solid Pin Protein Array Printing Platforms.

Reverse phase protein arrays (RPPA) are miniature dot blots constructed using robotic arrayers to deposit protein containing samples onto nitrocellulose-coated glass slides. Reverse phase protein arrays address the challenge of quantifying low-abundance proteins and posttranslationally modified proteins in cellular lysates and body fluids. RPPA technology is ideally suited to biomarker discovery, signal pathway profiling, functional phenotype analysis, and mechanism of action studies for drug discovery. Each array is fabricated with specimens, controls, and calibrators, thus providing a complete assay on each slide. Constructing a reverse phase protein array initially consists of selecting an arrayer, pin type, print head configuration, and nitrocellulose slide that is optimized for the particular specimen type and protein detection method. Herein we present the nuances of RPPA fabrication and study design using a solid pin arrayer and nitrocellulose-coated slides.

Combining the "Sibling Technologies" of Laser Capture Microdissection and Reverse Phase Protein Microarrays.

Reverse phase protein microarrays (RPPA) and laser capture microdissection (LCM) are "sibling" technologies that originated from the same laboratory to overcome the challenge of quantifying low-abundance proteins in heterogeneous tissues. Combining both technologies provides both unique opportunities and unique challenges. Enabling the unprecedented resolution of the activation state of labile biomarkers, such as phosphorylated cell signaling proteins, has had a substantial impact on our understanding of diseases and is playing a significant role in clinical trials. At the same time, quantifying proteins at this sensitivity in very small amounts of material requires cognizance of pre-analytical variability and the limits of downstream detection technologies. Here, we discuss both the potential that the combination of both technologies presents and the potential pitfalls that must be navigated.

A new model isolates glioblastoma clonal interactions and reveals unexpected modes for regulating motility, proliferation, and drug resistance.

Tumor clonal heterogeneity drives treatment resistance. But robust models are lacking that permit eavesdropping on the basic interaction network of tumor clones. We developed an in vitro, functional model of clonal cooperation using U87MG glioblastoma cells, which isolates fundamental clonal interactions. In this model pre-labeled clones are co-cultured to track changes in their individual motility, growth, and drug resistance behavior while mixed. This highly reproducible system allowed us to address a new class of fundamental questions about clonal interactions. We demonstrate that (i) a single clone can switch off the motility of the entire multiclonal U87MG cell line in 3D culture, (ii) maintenance of clonal heterogeneity is an intrinsic and influential cancer cell property, where clones coordinate growth rates to protect slow growing clones, and (iii) two drug sensitive clones can develop resistance de novo when cooperating. Furthermore, clonal communication for these specific types of interaction did not require diffusible factors, but appears to depend on cell-cell contact. This model constitutes a straightforward but highly reliable tool for isolating the complex clonal interactions that make up the fundamental "hive mind" of the tumor. It uniquely exposes clonal interactions for future pharmacological and biochemical studies.

Unlocking bone for proteomic analysis and FISH.

Bone tissue is critically lagging behind soft tissues and biofluids in our effort to advance precision medicine. The main challenges have been accessibility and the requirement for deleterious decalcification processes that impact the fidelity of diagnostic histomorphology and hinder downstream analyses such as fluorescence in-situ hybridization (FISH). We have developed an alternative fixation chemistry that simultaneously fixes and decalcifies bone tissue. We compared tissue morphology, immunohistochemistry (IHC), cell signal phosphoprotein analysis, and FISH in 50 patient matched primary bone cancer cases that were either formalin fixed and decalcified, or theralin fixed with and without decalcification. Use of theralin improved tissue histomorphology, whereas overall IHC was comparable to formalin fixed, decalcified samples. Theralin-fixed samples showed a significant increase in protein and DNA extractability, supporting technologies such as laser-capture microdissection and reverse phase protein microarrays. Formalin-fixed bone samples suffered from a fixation artifact where protein quantification of ß-actin directly correlated with fixation time. Theralin-fixed samples were not affected by this artifact. Moreover, theralin fixation enabled standard FISH staining in bone cancer samples, whereas no FISH staining was observed in formalin-fixed samples. We conclude that the use of theralin fixation unlocks the molecular archive within bone tissue allowing bone to enter the standard tissue analysis pipeline. This will have significant implications for bone cancer patients, in whom personalized medicine has yet to be implemented.

Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

Protein biomarkers for subtyping breast cancer and implications for future research.

INTRODUCTION: Breast cancer subtypes are currently defined by a combination of morphologic, genomic, and proteomic characteristics. These subtypes provide a molecular portrait of the tumor that aids diagnosis, prognosis, and treatment escalation/de-escalation options. Gene expression signatures describing intrinsic breast cancer subtypes for predicting risk of recurrence have been rapidly adopted in the clinic. Despite the use of subtype classifications, many patients develop drug resistance, breast cancer recurrence, or therapy failure. Areas covered: This review provides a summary of immunohistochemistry, reverse phase protein array, mass spectrometry, and integrative studies that are revealing differences in biological functions within and between breast cancer subtypes. We conclude with a discussion of rigor and reproducibility for proteomic-based biomarker discovery. Expert commentary: Innovations in proteomics, including implementation of assay guidelines and standards, are facilitating refinement of breast cancer subtypes. Proteomic and phosphoproteomic information distinguish biologically functional subtypes, are predictive of recurrence, and indicate likelihood of drug resistance. Actionable, activated signal transduction pathways can now be quantified and characterized. Proteomic biomarker validation in large, well-designed studies should become a public health priority to capitalize on the wealth of information gleaned from the proteome.

One-Step Preservation and Decalcification of Bony Tissue for Molecular Profiling.

Bone metastasis from primary cancer sites creates diagnostic and therapeutic challenges. Calcified bone is difficult to biopsy due to tissue hardness and patient discomfort, thus limiting the frequency and availability of bone/bone marrow biopsy material for molecular profiling. In addition, bony tissue must be demineralized (decalcified) prior to histomorphologic analysis. Decalcification processes rely on three main principles: (a) solubility of calcium salts in an acid, such as formic or nitric acid; (b) calcium chelation with ethylenediaminetetraacetic acid (EDTA); or (c) ion-exchange resins in a weak acid. A major roadblock in molecular profiling of bony tissue has been the lack of a suitable demineralization process that preserves histomorphology of calcified and soft tissue elements while also preserving phosphoproteins and nucleic acids. In this chapter, we describe general issues relevant to specimen collection and preservation of osseous tissue for molecular profiling. We provide two protocols: (a) one-step preservation of tissue histomorphology and proteins and posttranslational modifications, with simultaneous decalcification of bony tissue, and (b) ethanol-based tissue processing for TheraLin-fixed bony tissue.

Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain.

Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT™ laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.

Rapamycin modulation of p70 S6 kinase signaling inhibits Rift Valley fever virus pathogenesis.

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.