Narendra Suhas Jagannathan

Adoptive cell therapies such as CAR T cells have revolutionized cancer treatment and shown successes even with refractory cases of haematological malignancies. There is burgeoning interest in the optimization and improved manufacturing of cell therapy products. For CAR T cell therapy, enrichment of certain phenotypes of T cells in the infusion product have been correlated with improved long-term treatment outcomes. While metabolic control of T cell phenotypic fates has been demonstrated in some contexts, gaps still exist in our knowledge of how T cell metabolic dynamics early during CAR T manufacturing might affect critical quality attributes (CQAs) of the final product (e.g., differentiation/exhaustion/potency). We present a modelling framework that can perform real-time estimation of per-cell metabolic rates of T cells expanded ex vivo in a reactor. We validate our estimated rates using metabolic assays, show how average rates can be deconvoluted to rates of individual T cell phenotypes, and demonstrate applicability to different reactor types. Applying our tool to the expansion of both healthy and patient-derived cells in a perfusion-based microbioreactor, we offer proof-of-principle to show that correlations exist between early metabolic rates of T cells in culture and cellular attributes related to growth, differentiation and exhaustion of the final product. Given the biological variation that exists in the growth and dynamics of patient-derived cells in culture, such modelling contributes to the overarching goal of improving the consistency of cell therapy through Adaptive Process Control (APC).

Bats have unique characteristics compared to other mammals, including increased longevity and higher resistance to cancer and infectious disease. While previous studies have analyzed the metabolic requirements for flight, it is still unclear how bat metabolism supports these unique features, and no study has integrated metabolomics, transcriptomics, and proteomics to characterize bat metabolism. In this work, we performed a multi-omics data analysis using a computational model of metabolic fluxes to identify fundamental differences in central metabolism between primary lung fibroblast cell lines from the black flying fox fruit bat (Pteropus alecto) and human. Bat cells showed higher expression levels of Complex I components of electron transport chain (ETC), but, remarkably, a lower rate of oxygen consumption. Computational modeling interpreted these results as indicating that Complex II activity may be low or reversed, similar to an ischemic state. An ischemic-like state of bats was also supported by decreased levels of central metabolites and increased ratios of succinate to fumarate in bat cells. Ischemic states tend to produce reactive oxygen species (ROS), which would be incompatible with the longevity of bats. However, bat cells had higher antioxidant reservoirs (higher total glutathione and higher ratio of NADPH to NADP) despite higher mitochondrial ROS levels. In addition, bat cells were more resistant to glucose deprivation and had increased resistance to ferroptosis, one of the characteristics of which is oxidative stress. Thus, our studies revealed distinct differences in the ETC regulation and metabolic stress responses between human and bat cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Bats have unique characteristics compared to other mammals, including increased longevity and higher resistance to cancer and infectious disease. While previous studies have analyzed the metabolic requirements for flight, it is still unclear how bat metabolism supports these unique features, and no study has integrated metabolomics, transcriptomics, and proteomics to characterize bat metabolism. In this work, we performed a multi-omics data analysis using a computational model of metabolic fluxes to identify fundamental differences in central metabolism between primary lung fibroblast cell lines from the black flying fox fruit bat ( Pteropus alecto ) and human. Bat cells showed higher expression levels of Complex I components of electron transport chain (ETC), but, remarkably, a lower rate of oxygen consumption (OCR). Computational modeling interpreted these results as indicating that Complex II activity may be low or reversed, similar to an ischemic state. An ischemic-like state of bats was also supported by decreased levels of central metabolites and increased ratios of succinate to fumarate in bat cells. Ischemic states tend to produce reactive oxygen species (ROS), which would be incompatible with the longevity of bats. However, bat cells had higher antioxidant reservoirs (higher total glutathione and higher ratio of NADPH to NADP) despite higher mitochondrial ROS levels. In addition, bat cells were more resistant to glucose deprivation and had increased resistance to ferroptosis, one of the characteristics of which is oxidative stress. Thus, our studies revealed distinct differences in the ETC regulation and metabolic stress responses between human and bat cells.

Uncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (R_G) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Motivation: Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. Results: We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. Availability and implementation: Freely available for download at http://github.com/nsuhasj/Transcompp. Supplementary information: Supplementary data are available at Bioinformatics online.

Proteins with intrinsically disordered regions (IDRs) have large hydrodynamic radii, compared with globular proteins of equivalent weight. Recent experiments showed that IDRs with large radii can create steric pressure to drive membrane curvature during Clathrin-mediated endocytosis (CME). Epsin and Eps15 are two CME proteins with IDRs that contain multiple motifs for binding the adaptor protein AP2, but the impact of AP2-binding on these IDRs is unknown. Some IDRs acquire binding-induced function by forming a folded quaternary structure, but we hypothesize that the IDRs of Epsin and/or Eps15 acquire binding-induced function by increasing their steric volume. We explore this hypothesis in silico by generating conformational ensembles of the IDRs of Epsin (4 million structures) or Eps15 (3 million structures), then estimating the impact of AP2-binding on Radius of Gyration (R_G). Results show that the ensemble of Epsin IDR conformations that accommodate AP2 binding has a right-shifted distribution of R_G (larger radii) than the unbound Epsin ensemble. In contrast, the ensemble of Eps15 IDR conformations has comparable R_G distribution between AP2-bound and unbound. We speculate that AP2 triggers the Epsin IDR to function through binding-induced-expansion, which could increase steric pressure and membrane bending during CME.