Multiwell-based G0-PCC assay for radiation biodosimetry.

In cytogenetic biodosimetry, assessing radiation exposure typically requires over 48 hours for cells to reach mitosis, significantly delaying the administration of crucial radiation countermeasures needed within the first 24 hours post-exposure. To improve medical response times, we incorporated the G0-Premature Chromosome Condensation (G0-PCC) technique with the Rapid Automated Biodosimetry Tool-II (RABiT-II), creating a faster alternative for large-scale radiation emergencies. Our findings revealed that using a lower concentration of Calyculin A (Cal A) than recommended effectively increased the yield of highly-condensed G0-PCC cells (hPCC). However, integrating recombinant CDK1/Cyclin B kinase, vital for chromosome condensation, proved challenging due to the properties of these proteins affecting interactions with cellular membranes. Interestingly, Cal A alone was capable of inducing chromosome compaction in some G0 cells even in the absence of mitotic kinases, although these chromosomes displayed atypical morphologies. This suggests that Cal A mechanism for compacting G0 chromatin may differ from condensation driven by mitotic kinases. Additionally, we observed a correlation between radiation dose and extent of hPCC chromosome fragmentation, which allowed us to automate radiation damage quantification using a Convolutional Neural Network (CNN). Our method can address the need for a same-day cytogenetic biodosimetry test in radiation emergency situations.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Elucidating Compound Mechanism of Action and Polypharmacology with a Large-scale Perturbational Profile Compendium.

The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have implemented an efficient integrative experimental and computational framework leveraging the systematic generation and analysis of drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Protein activity-based analyses revealed highly reproducible, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide polypharmacology map, characterization of MoA-related drug clusters and off-target effects, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins. The proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, could help support more systematic and quantitative approaches to precision oncology.

Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.

OncoLoop: A Network-Based Precision Cancer Medicine Framework.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. SIGNIFICANCE: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

The Host Cell ViroCheckpoint: Identification and Pharmacologic Targeting of Novel Mechanistic Determinants of Coronavirus-Mediated Hijacked Cell States.

Most antiviral agents are designed to target virus-specific proteins and mechanisms rather than the host cell proteins that are critically dysregulated following virus-mediated reprogramming of the host cell transcriptional state. To overcome these limitations, we propose that elucidation and pharmacologic targeting of host cell Master Regulator proteins-whose aberrant activities govern the reprogramed state of coronavirus-infected cells-presents unique opportunities to develop novel mechanism-based therapeutic approaches to antiviral therapy, either as monotherapy or as a complement to established treatments. Specifically, we propose that a small module of host cell Master Regulator proteins (ViroCheckpoint) is hijacked by the virus to support its efficient replication and release. Conventional methodologies are not well suited to elucidate these potentially targetable proteins. By using the VIPER network-based algorithm, we successfully interrogated 12h, 24h, and 48h signatures from Calu-3 lung adenocarcinoma cells infected with SARS-CoV, to elucidate the time-dependent reprogramming of host cells and associated Master Regulator proteins. We used the NYS CLIA-certified Darwin OncoTreat algorithm, with an existing database of RNASeq profiles following cell perturbation with 133 FDA-approved and 195 late-stage experimental compounds, to identify drugs capable of virtually abrogating the virus-induced Master Regulator signature. This approach to drug prioritization and repurposing can be trivially extended to other viral pathogens, including SARS-CoV-2, as soon as the relevant infection signature becomes available.

Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay.

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 × 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell: :explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.

Increased erythrophagocytosis induces ferroptosis in red pulp macrophages in a mouse model of transfusion.

Macrophages play important roles in recycling iron derived from the clearance of red blood cells (RBCs). They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (ie, "old") RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs. This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6Chi monocytes, which signaled Ly6Chi monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6Chi monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.

Systematic, network-based characterization of therapeutic target inhibitors.

A large fraction of the proteins that are being identified as key tumor dependencies represent poor pharmacological targets or lack clinically-relevant small-molecule inhibitors. Availability of fully generalizable approaches for the systematic and efficient prioritization of tumor-context specific protein activity inhibitors would thus have significant translational value. Unfortunately, inhibitor effects on protein activity cannot be directly measured in systematic and proteome-wide fashion by conventional biochemical assays. We introduce OncoLead, a novel network based approach for the systematic prioritization of candidate inhibitors for arbitrary targets of therapeutic interest. In vitro and in vivo validation confirmed that OncoLead analysis can recapitulate known inhibitors as well as prioritize novel, context-specific inhibitors of difficult targets, such as MYC and STAT3. We used OncoLead to generate the first unbiased drug/regulator interaction map, representing compounds modulating the activity of cancer-relevant transcription factors, with potential in precision medicine.

Opposing effect of angiopoietin-1 on VEGF-mediated disruption of endothelial cell-cell interactions requires activation of PKC beta.

Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) cooperate in migration and survival of endothelial cells by activation of phosphatidylinositol-3 (PI-3) kinase and mitogen activating protein (MAP) kinase pathways. However, Ang1 opposes the effect of VEGF on vascular permeability. We found that Ang1 also blocks VEGF-mediated diffusion of fluoresin isothiocyanate (FITC)-labeled albumin across an endothelial cell monolayer. VEGF-mediated vascular permeability has been attributed, in part, to activation of phospholipase A(2) and subsequent formation of platelet activating factor. However, Ang1 had no effect on VEGF-induced activation of phospholipase A(2) or the release of arachidonic acid. VEGF-mediated permeability was associated with disruption of endothelial cell junctional complexes, dissociation of beta-catenin from VE-cadherin, and accumulation of beta-catenin in the cytosol. In contrast, Ang1 enhanced the interaction of beta-catenin with VE-cadherin and impaired VEGF-mediated dissociation of this complex. Ang1 also blocked VEGF-induced translocation of protein kinase C (PKC) and beta2 to the membrane, but had no effect on activation of PKC alpha. In addition, staurosporine and a PKC beta inhibitor, LY379196, blocked VEGF-mediated dissociation of beta-catenin from VE-cadherin, diffusion of albumin across the endothelial cell monolayer, and translocation of PKC beta isoforms. These data indicate that VEGF-mediated disruption of endothelial cell-cell interactions requires activation of PKC beta isoforms and that this pathway is blocked by Ang1.