Dimensionality reduction methods for extracting functional networks from large-scale CRISPR screens.

CRISPR-Cas9 screens facilitate the discovery of gene functional relationships and phenotype-specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole-genome CRISPR screens aimed at identifying cancer-specific genetic dependencies across human cell lines. A mitochondria-associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co-essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods-autoencoders, robust, and classical principal component analyses (PCA)-for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low-dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction-based normalization tools.

Dimensionality reduction methods for extracting functional networks from large-scale CRISPR screens.

CRISPR-Cas9 screens facilitate the discovery of gene functional relationships and phenotype-specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole-genome CRISPR screens aimed at identifying cancer-specific genetic dependencies across human cell lines. A mitochondria-associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co-essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods - autoencoders, robust, and classical principal component analyses (PCA) - for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low-dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction-based normalization tools.

Proliferation and Self-Renewal Are Differentially Sensitive to NRASG12V Oncogene Levels in an Acute Myeloid Leukemia Cell Line.

NRAS proteins are central regulators of proliferation, survival, and self-renewal in leukemia. Previous work demonstrated that the effects of oncogenic NRAS in mediating proliferation and self-renewal are mutually exclusive within leukemia subpopulations and that levels of oncogenic NRAS vary between highly proliferative and self-renewing leukemia subpopulations. These findings suggest that NRAS activity levels may be important determinants of leukemic behavior. To define how oncogenic NRAS levels affect these functions, we genetically engineered an acute myeloid leukemia (AML) cell line, THP-1, to express variable levels of NRASG12V. We replaced the endogenous NRASG12D gene with a tetracycline-inducible and dose-responsive NRASG12V transgene. Cells lacking NRASG12V oncoprotein were cell-cycle arrested. Intermediate levels of NRASG12V induced maximal proliferation; higher levels led to attenuated proliferation, increased G1 arrest, senescence markers, and maximal self-renewal capacity. Higher levels of the oncoprotein also induced self-renewal and mitochondrial genes. We used mass cytometry (CyTOF) to define the downstream signaling events that mediate these differential effects. Not surprisingly, we found that the levels of such canonical RAS-effectors as pERK and p4EBP1 correlated with NRASG12V levels. ß-Catenin, a mediator of self-renewal, also correlated with NRASG12V levels. These signaling intermediates may mediate the differential effects of NRASG12V in leukemia biology. Together, these data reveal that oncogenic NRAS levels are important determinants of leukemic behavior explaining heterogeneity in phenotypes within a clone. This system provides a new model to study RAS oncogene addiction and RAS-induced self-renewal in AML. IMPLICATIONS: Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.

Methionine restriction exposes a targetable redox vulnerability of triple-negative breast cancer cells by inducing thioredoxin reductase.

PURPOSE: Tumor cells are dependent on the glutathione and thioredoxin antioxidant pathways to survive oxidative stress. Since the essential amino acid methionine is converted to glutathione, we hypothesized that methionine restriction (MR) would deplete glutathione and render tumors dependent on the thioredoxin pathway and its rate-limiting enzyme thioredoxin reductase (TXNRD). METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control or MR media and the effects on reactive oxygen species (ROS) and antioxidant signaling were examined. To determine the role of TXNRD in MR-induced cell death, TXNRD1 was inhibited by RNAi or the pan-TXNRD inhibitor auranofin, an antirheumatic agent. Metastatic and PDX TNBC mouse models were utilized to evaluate in vivo antitumor activity. RESULTS: MR rapidly and transiently increased ROS, depleted glutathione, and decreased the ratio of reduced glutathione/oxidized glutathione in TNBC cells. TXNRD1 mRNA and protein levels were induced by MR via a ROS-dependent mechanism mediated by the transcriptional regulators NRF2 and ATF4. MR dramatically sensitized TNBC cells to TXNRD1 silencing and the TXNRD inhibitor auranofin, as determined by crystal violet staining and caspase activity; these effects were suppressed by the antioxidant N-acetylcysteine. H-Ras-transformed MCF-10A cells, but not untransformed MCF-10A cells, were highly sensitive to the combination of auranofin and MR. Furthermore, dietary MR induced TXNRD1 expression in mammary tumors and enhanced the antitumor effects of auranofin in metastatic and PDX TNBC murine models. CONCLUSION: MR exposes a vulnerability of TNBC cells to the TXNRD inhibitor auranofin by increasing expression of its molecular target and creating a dependency on the thioredoxin pathway.

Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia.

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.