Proteomic Dynamics of Breast Cancer Cell Lines Identifies Potential Therapeutic Protein Targets.

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.

PHONEMeS: Efficient Modeling of Signaling Networks Derived from Large-Scale Mass Spectrometry Data.

Post-translational modifications of proteins play an important role in the regulation of cellular processes. The mass spectrometry analysis of proteome modifications offers huge potential for the study of how protein inhibitors affect the phosphosignaling mechanisms inside the cells. We have recently proposed PHONEMeS, a method that uses high-content shotgun phosphoproteomic data to build logical network models of signal perturbation flow. However, in its original implementation, PHONEMeS was computationally demanding and was only used to model signaling in a perturbation context. We have reformulated PHONEMeS as an Integer Linear Program (ILP) that is orders of magnitude more efficient than the original one. We have also expanded the scenarios that can be analyzed. PHONEMeS can model data upon perturbation on not only a known target but also deregulated pathways upstream and downstream of any set of deregulated kinases. Finally, PHONEMeS can now analyze data sets with multiple time points, which helps us to obtain better insight into the dynamics of the propagation of signals. We illustrate the value of the new approach on various data sets of medical relevance, where we shed light on signaling mechanisms and drug modes of action.

SLFN11 informs on standard of care and novel treatments in a wide range of cancer models.

BACKGROUND: Schlafen 11 (SLFN11) has been linked with response to DNA-damaging agents (DDA) and PARP inhibitors. An in-depth understanding of several aspects of its role as a biomarker in cancer is missing, as is a comprehensive analysis of the clinical significance of SLFN11 as a predictive biomarker to DDA and/or DNA damage-response inhibitor (DDRi) therapies. METHODS: We used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting. RESULTS: SLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies. CONCLUSION: SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models.

rMTA: robust metabolic transformation analysis.

MOTIVATION: The development of computational tools exploiting -omics data and high-quality genome-scale metabolic networks for the identification of novel drug targets is a relevant topic in Systems Medicine. Metabolic Transformation Algorithm (MTA) is one of these tools, which aims to identify targets that transform a disease metabolic state back into a healthy state, with potential application in any disease where a clear metabolic alteration is observed. RESULTS: Here, we present a robust extension to MTA (rMTA), which additionally incorporates a worst-case scenario analysis and minimization of metabolic adjustment to evaluate the beneficial effect of gene knockouts. We show that rMTA complements MTA in the different datasets analyzed (gene knockout perturbations in different organisms, Alzheimer's disease and prostate cancer), bringing a more accurate tool for predicting therapeutic targets. AVAILABILITY AND IMPLEMENTATION: rMTA is freely available on The Cobra Toolbox: https://opencobra.github.io/cobratoolbox/latest/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Advances in network-based metabolic pathway analysis and gene expression data integration.

With the emergence of metabolic networks, novel mathematical pathway concepts were introduced in the past decade, aiming to go beyond canonical maps. However, the use of network-based pathways to interpret 'omics' data has been limited owing to the fact that their computation has, until very recently, been infeasible in large (genome-scale) metabolic networks. In this review article, we describe the progress made in the past few years in the field of network-based metabolic pathway analysis. In particular, we review in detail novel optimization techniques to compute elementary flux modes, an important pathway concept in this field. In addition, we summarize approaches for the integration of metabolic pathways with gene expression data, discussing recent advances using network-based pathway concepts.

Direct calculation of minimal cut sets involving a specific reaction knock-out.

MOTIVATION: The concept of Minimal Cut Sets (MCSs) is used in metabolic network modeling to describe minimal groups of reactions or genes whose simultaneous deletion eliminates the capability of the network to perform a specific task. Previous work showed that MCSs where closely related to Elementary Flux Modes (EFMs) in a particular dual problem, opening up the possibility to use the tools developed for computing EFMs to compute MCSs. Until recently, however, there existed no method to compute an EFM with some specific characteristic, meaning that, in the case of MCSs, the only strategy to obtain them was to enumerate them using, for example, the standard K-shortest EFMs algorithm. RESULTS: In this work, we adapt the recently developed theory to compute EFMs satisfying several constraints to the calculation of MCSs involving a specific reaction knock-out. Importantly, we emphasize that not all the EFMs in the dual problem correspond to real MCSs, and propose a new formulation capable of correctly identifying the MCS wanted. Furthermore, this formulation brings interesting insights about the relationship between the primal and the dual problem of the MCS computation. AVAILABILITY AND IMPLEMENTATION: A Matlab-Cplex implementation of the proposed algorithm is available as a supplementary material CONTACT: fplanes@ceit.es SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TreeEFM: calculating elementary flux modes using linear optimization in a tree-based algorithm.

MOTIVATION: Elementary flux modes (EFMs) analysis constitutes a fundamental tool in systems biology. However, the efficient calculation of EFMs in genome-scale metabolic networks (GSMNs) is still a challenge. We present a novel algorithm that uses a linear programming-based tree search and efficiently enumerates a subset of EFMs in GSMNs. RESULTS: Our approach is compared with the EFMEvolver approach, demonstrating a significant improvement in computation time. We also validate the usefulness of our new approach by studying the acetate overflow metabolism in the Escherichia coli bacteria. To do so, we computed 1 million EFMs for each energetic amino acid and then analysed the relevance of each energetic amino acid based on gene/protein expression data and the obtained EFMs. We found good agreement between previous experiments and the conclusions reached using EFMs. Finally, we also analysed the performance of our approach when applied to large GSMNs. AVAILABILITY AND IMPLEMENTATION: The stand-alone software TreeEFM is implemented in C++ and interacts with the open-source linear solver COIN-OR Linear program Solver (CLP).

Context-specific metabolic network reconstruction of a naphthalene-degrading bacterial community guided by metaproteomic data.

MOTIVATION: With the advent of meta-'omics' data, the use of metabolic networks for the functional analysis of microbial communities became possible. However, while network-based methods are widely developed for single organisms, their application to bacterial communities is currently limited. RESULTS: Herein, we provide a novel, context-specific reconstruction procedure based on metaproteomic and taxonomic data. Without previous knowledge of a high-quality, genome-scale metabolic networks for each different member in a bacterial community, we propose a meta-network approach, where the expression levels and taxonomic assignments of proteins are used as the most relevant clues for inferring an active set of reactions. Our approach was applied to draft the context-specific metabolic networks of two different naphthalene-enriched communities derived from an anthropogenically influenced, polyaromatic hydrocarbon contaminated soil, with (CN2) or without (CN1) bio-stimulation. We were able to capture the overall functional differences between the two conditions at the metabolic level and predict an important activity for the fluorobenzoate degradation pathway in CN1 and for geraniol metabolism in CN2. Experimental validation was conducted, and good agreement with our computational predictions was observed. We also hypothesize different pathway organizations at the organismal level, which is relevant to disentangle the role of each member in the communities. The approach presented here can be easily transferred to the analysis of genomic, transcriptomic and metabolomic data.

The PARP1 selective inhibitor saruparib (AZD5305) elicits potent and durable antitumor activity in patient-derived BRCA1/2-associated cancer models.

BACKGROUND: Poly (ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors (PARPi) are targeted therapies approved for homologous recombination repair (HRR)-deficient breast, ovarian, pancreatic, and prostate cancers. Since inhibition of PARP1 is sufficient to cause synthetic lethality in tumors with homologous recombination deficiency (HRD), PARP1 selective inhibitors such as saruparib (AZD5305) are being developed. It is expected that selective PARP1 inhibition leads to a safer profile that facilitates its combination with other DNA damage repair inhibitors. Here, we aimed to characterize the antitumor activity of AZD5305 in patient-derived preclinical models compared to the first-generation PARP1/2 inhibitor olaparib and to identify mechanisms of resistance. METHODS: Thirteen previously characterized patient-derived tumor xenograft (PDX) models from breast, ovarian, and pancreatic cancer patients harboring germline pathogenic alterations in BRCA1, BRCA2, or PALB2 were used to evaluate the efficacy of AZD5305 alone or in combination with carboplatin or an ataxia telangiectasia and Rad3 related (ATR) inhibitor (ceralasertib) and compared it to the first-generation PARPi olaparib. We performed DNA and RNA sequencing as well as protein-based assays to identify mechanisms of acquired resistance to either PARPi. RESULTS: AZD5305 showed superior antitumor activity than the first-generation PARPi in terms of preclinical complete response rate (75% vs. 37%). The median preclinical progression-free survival was significantly longer in the AZD5305-treated group compared to the olaparib-treated group (> 386 days vs. 90 days). Mechanistically, AZD5305 induced more replication stress and genomic instability than the PARP1/2 inhibitor olaparib in PARPi-sensitive tumors. All tumors at progression with either PARPi (39/39) showed increase of HRR functionality by RAD51 foci formation. The most prevalent resistance mechanisms identified were the acquisition of reversion mutations in BRCA1/BRCA2 and the accumulation of hypomorphic BRCA1. AZD5305 did not sensitize PDXs with acquired resistance to olaparib but elicited profound and durable responses when combined with carboplatin or ceralasertib in 3/6 and 5/5 models, respectively. CONCLUSIONS: Collectively, these results show that the novel PARP1 selective inhibitor AZD5305 yields a potent antitumor response in PDX models with HRD and delays PARPi resistance alone or in combination with carboplatin or ceralasertib, which supports its use in the clinic as a new therapeutic option.

Relevance of ATM Status in Driving Sensitivity to DNA Damage Response Inhibitors in Patient-Derived Xenograft Models.

Ataxia-telangiectasia mutated gene (ATM) is a key component of the DNA damage response (DDR) and double-strand break repair pathway. The functional loss of ATM (ATM deficiency) is hypothesised to enhance sensitivity to DDR inhibitors (DDRi). Whole-exome sequencing (WES), immunohistochemistry (IHC), and Western blotting (WB) were used to characterise the baseline ATM status across a panel of ATM mutated patient-derived xenograft (PDX) models from a range of tumour types. Antitumour efficacy was assessed with poly(ADP-ribose)polymerase (PARP, olaparib), ataxia- telangiectasia and rad3-related protein (ATR, AZD6738), and DNA-dependent protein kinase (DNA-PK, AZD7648) inhibitors as a monotherapy or in combination to associate responses with ATM status. Biallelic truncation/frameshift ATM mutations were linked to ATM protein loss while monoallelic or missense mutations, including the clinically relevant recurrent R3008H mutation, did not confer ATM protein loss by IHC. DDRi agents showed a mixed response across the PDX's but with a general trend toward greater activity, particularly in combination in models with biallelic ATM mutation and protein loss. A PDX with an ATM splice-site mutation, 2127T > C, with a high relative baseline ATM expression and KAP1 phosphorylation responded to all DDRi treatments. These data highlight the heterogeneity and complexity in describing targetable ATM-deficiencies and the fact that current patient selection biomarker methods remain imperfect; although, complete ATM loss was best able to enrich for DDRi sensitivity.

Patient-specific Boolean models of signalling networks guide personalised treatments.

Prostate cancer is the second most occurring cancer in men worldwide. To better understand the mechanisms of tumorigenesis and possible treatment responses, we developed a mathematical model of prostate cancer which considers the major signalling pathways known to be deregulated. We personalised this Boolean model to molecular data to reflect the heterogeneity and specific response to perturbations of cancer patients. A total of 488 prostate samples were used to build patient-specific models and compared to available clinical data. Additionally, eight prostate cell line-specific models were built to validate our approach with dose-response data of several drugs. The effects of single and combined drugs were tested in these models under different growth conditions. We identified 15 actionable points of interventions in one cell line-specific model whose inactivation hinders tumorigenesis. To validate these results, we tested nine small molecule inhibitors of five of those putative targets and found a dose-dependent effect on four of them, notably those targeting HSP90 and PI3K. These results highlight the predictive power of our personalised Boolean models and illustrate how they can be used for precision oncology.

How to find the right drug for each patient? Advances and challenges in pharmacogenomics.

Cancer is a highly heterogeneous disease with complex underlying biology. For these reasons, effective cancer treatment is still a challenge. Nowadays, it is clear that a cancer therapy that fits all the cases cannot be found, and as a result the design of therapies tailored to the patient's molecular characteristics is needed. Pharmacogenomics aims to study the relationship between an individual's genotype and drug response. Scientists use different biological models, ranging from cell lines to mouse models, as proxies for patients for preclinical and translational studies. The rapid development of "-omics" technologies is increasing the amount of features that can be measured in these models, expanding the possibilities of finding predictive biomarkers of drug response. Finding these relationships requires diverse computational approaches ranging from machine learning to dynamic modeling. Despite major advances, we are still far from being able to precisely predict drug efficacy in cancer models, let alone directly on patients. We believe that the new experimental techniques and computational approaches covered in this review will bring us closer to this goal.

Logic Modeling in Quantitative Systems Pharmacology.

Here we present logic modeling as an approach to understand deregulation of signal transduction in disease and to characterize a drug's mode of action. We discuss how to build a logic model from the literature and experimental data and how to analyze the resulting model to obtain insights of relevance for systems pharmacology. Our workflow uses the free tools OmniPath (network reconstruction from the literature), CellNOpt (model fit to experimental data), MaBoSS (model analysis), and Cytoscape (visualization).

An in-silico approach to predict and exploit synthetic lethality in cancer metabolism.

Synthetic lethality is a promising concept in cancer research, potentially opening new possibilities for the development of more effective and selective treatments. Here, we present a computational method to predict and exploit synthetic lethality in cancer metabolism. Our approach relies on the concept of genetic minimal cut sets and gene expression data, demonstrating a superior performance to previous approaches predicting metabolic vulnerabilities in cancer. Our genetic minimal cut set computational framework is applied to evaluate the lethality of ribonucleotide reductase catalytic subunit M1 (RRM1) inhibition in multiple myeloma. We present a computational and experimental study of the effect of RRM1 inhibition in four multiple myeloma cell lines. In addition, using publicly available genome-scale loss-of-function screens, a possible mechanism by which the inhibition of RRM1 is effective in cancer is established. Overall, our approach shows promising results and lays the foundation to build a novel family of algorithms to target metabolism in cancer.Exploiting synthetic lethality is a promising approach for cancer therapy. Here, the authors present an approach to identifying such interactions by finding genetic minimal cut sets (gMCSs) that block cancer proliferation, and apply it to study the lethality of RRM1 inhibition in multiple myeloma.

Assessment of FBA Based Gene Essentiality Analysis in Cancer with a Fast Context-Specific Network Reconstruction Method.

MOTIVATION: Gene Essentiality Analysis based on Flux Balance Analysis (FBA-based GEA) is a promising tool for the identification of novel metabolic therapeutic targets in cancer. The reconstruction of cancer-specific metabolic networks, typically based on gene expression data, constitutes a sensible step in this approach. However, to our knowledge, no extensive assessment on the influence of the reconstruction process on the obtained results has been carried out to date. RESULTS: In this article, we aim to study context-specific networks and their FBA-based GEA results for the identification of cancer-specific metabolic essential genes. To that end, we used gene expression datasets from the Cancer Cell Line Encyclopedia (CCLE), evaluating the results obtained in 174 cancer cell lines. In order to more clearly observe the effect of cancer-specific expression data, we did the same analysis using randomly generated expression patterns. Our computational analysis showed some essential genes that are fairly common in the reconstructions derived from both gene expression and randomly generated data. However, though of limited size, we also found a subset of essential genes that are very rare in the randomly generated networks, while recurrent in the sample derived networks, and, thus, would presumably constitute relevant drug targets for further analysis. In addition, we compare the in-silico results to high-throughput gene silencing experiments from Project Achilles with conflicting results, which leads us to raise several questions, particularly the strong influence of the selected biomass reaction on the obtained results. Notwithstanding, using previous literature in cancer research, we evaluated the most relevant of our targets in three different cancer cell lines, two derived from Gliobastoma Multiforme and one from Non-Small Cell Lung Cancer, finding that some of the predictions are in the right track.

A network-based approach for predicting key enzymes explaining metabolite abundance alterations in a disease phenotype.

BACKGROUND: The study of metabolism has attracted much attention during the last years due to its relevance in various diseases. The advance in metabolomics platforms allows us to detect an increasing number of metabolites in abnormal high/low concentration in a disease phenotype. Finding a mechanistic interpretation for these alterations is important to understand pathophysiological processes, however it is not an easy task. The availability of genome scale metabolic networks and Systems Biology techniques open new avenues to address this question. RESULTS: In this article we present a novel mathematical framework to find enzymes whose malfunction explains the accumulation/depletion of a given metabolite in a disease phenotype. Our approach is based on a recently introduced pathway concept termed Carbon Flux Paths (CFPs), which extends classical topological definition by including network stoichiometry. Using CFPs, we determine the Connectivity Curve of an altered metabolite, which allows us to quantify changes in its pathway structure when a certain enzyme is removed. The influence of enzyme removal is then ranked and used to explain the accumulation/depletion of such metabolite. For illustration, we center our study in the accumulation of two metabolites (L-Cystine and Homocysteine) found in high concentration in the brain of patients with mental disorders. Our results were discussed based on literature and found a good agreement with previously reported mechanisms. In addition, we hypothesize a novel role of several enzymes for the accumulation of these metabolites, which opens new strategies to understand the metabolic processes underlying these diseases. CONCLUSIONS: With personalized medicine on the horizon, metabolomic platforms are providing us with a vast amount of experimental data for a number of complex diseases. Our approach provides a novel apparatus to rationally investigate and understand metabolite alterations under disease phenotypes. This work contributes to the development of Systems Medicine, whose objective is to answer clinical questions based on theoretical methods and high-throughput "omics" data.