CRISPR screens in physiologic medium reveal conditionally essential genes in human cells

Rossiter, Nicholas J.; Huggler, Kimberly S.; Adelmann, Charles H.; Keys, Heather R.; Soens, Ross; Sabatini, David M.; Cantor, Jason R.

doi:10.1101/2020.08.31.275107

Cited by 22 publications

(25 citation statements)

References 86 publications

(114 reference statements)

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“…Many cancer cell types increase glutamine utilization to support the TCA cycle and fatty acid biosynthesis in vitro (Metallo et al, 2012;Mullen et al, 2012); however reproducing this phenotype in vivo has generated mixed results (Davidson et al, 2016;Le et al, 2012;Son et al, 2013;Tardito et al, 2015). One potential explanation for this discrepancy is the difference in nutrient profile of cell culture media compared to in vivo microenvironments (Ackermann and Tardito, 2019;Rossiter et al, 2021;Voorde et al, 2019). Plasma and interstitial fluid contain metabolites that impact cellular metabolism but are routinely excluded from most cell culture medias.…”

Section: Discussionmentioning

confidence: 99%

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

Kumar

Cordes

Thalacker‐Mercer

et al. 2021

Preprint

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Citrate lies at a critical node of metabolism linking tricarboxylic acid metabolism and fatty acid synthesis via acetyl-coenzyme A. Recent studies have linked the sodium citrate transporter (NaCT), encoded by SLC13A5, to dysregulated hepatic metabolism and pediatric epilepsy. To examine how NaCT-mediated citrate metabolism contributes to the pathophysiology of these diseases we applied 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cell lines and primary rat cortical neurons. Exogenous citrate contributed to intermediary metabolism at appreciable levels only under hypoxic conditions. In the absence of glutamine, citrate supplementation increased de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromised citrate uptake and catabolism. Citrate supplementation rescued Huh7 cell viability in response to glutamine deprivation and Zn2+ treatment, and these effects were mitigated by NaCT deficiency. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient limited conditions and may facilitate resistance to metal toxicity.

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Section: Discussionmentioning

confidence: 99%

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

Kumar

Cordes

Thalacker‐Mercer

et al. 2021

Preprint

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“…The genetic dependencies of a cancer cell can be markedly altered by in vitro versus in vivo growth conditions (Rossiter et al 2020), particularly the requirement for cell surface receptors and metabolic enzymes whose function is influenced by the extracellular microenvironment. This issue provided the rationale for developing a robust screening method for interrogating genetic dependencies of human AML cells grown in vivo.…”

Section: A Domain-focused Crispr Screening Methods For Identifying In Vivo-relevant Aml Dependenciesmentioning

confidence: 99%

In vivogenetic screen identifies a SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

Wei

Iyer

Costa

et al. 2020

Preprint

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An enhanced requirement for extracellular nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy for evaluating dependencies in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a unique vulnerability in this disease. In accord with this transport function, we demonstrate that the SLC5A3 dependency reflects a myo-inositol auxotrophy in AML. Importantly, the commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myoinositol biosynthesis, inositol-3-phosphate synthase 1. We used gain- and loss-of-function experiments to demonstrate a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with the presence of IDH1/IDH2 and CEBPA mutations. Collectively, our findings reveal myo-inositol auxotrophy as a novel form of metabolic dysregulation in AML, which is caused by the aberrant silencing of a biosynthetic enzyme.Statement of significanceHere, we show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically-defined subset of AML.

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“…Furthermore, recent studies have shown that cell culture media impacts genetic dependencies observed in CCLs (Cheteh et al , 2017; Li et al , 2019; Rossiter et al , 2021). Nevertheless, despite the lack of immune‐like cells or cancer fibroblasts, it was found that specific metabolites in human plasma‐like medium also influence the set of essential genes in CCLs detected in CRISPR‐based screens (Rossiter et al , 2021). Co‐culturing CCLs with cancer‐associated fibroblasts (CAF) or even CAF‐conditioned medium reduced response to chemotherapeutic treatments and conversely increased cell survival in prostate CCLs (Cheteh et al , 2017).…”

Section: Factors That Might Compromise How Well Cell Lines Represent ...mentioning

confidence: 99%

Computational estimation of quality and clinical relevance of cancer cell lines

Trastulla

Noorbakhsh

Vázquez

et al. 2022

Molecular Systems Biology

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Immortal cancer cell lines (CCLs) are the most widely used system for investigating cancer biology and for the preclinical development of oncology therapies. Pharmacogenomic and genome‐wide editing screenings have facilitated the discovery of clinically relevant gene–drug interactions and novel therapeutic targets via large panels of extensively characterised CCLs. However, tailoring pharmacological strategies in a precision medicine context requires bridging the existing gaps between tumours and in vitro models. Indeed, intrinsic limitations of CCLs such as misidentification, the absence of tumour microenvironment and genetic drift have highlighted the need to identify the most faithful CCLs for each primary tumour while addressing their heterogeneity, with the development of new models where necessary. Here, we discuss the most significant limitations of CCLs in representing patient features, and we review computational methods aiming at systematically evaluating the suitability of CCLs as tumour proxies and identifying the best patient representative in vitro models. Additionally, we provide an overview of the applications of these methods to more complex models and discuss future machine‐learning‐based directions that could resolve some of the arising discrepancies.

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CRISPR screens in physiologic medium reveal conditionally essential genes in human cells

Cited by 22 publications

References 86 publications

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

In vivogenetic screen identifies a SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

Computational estimation of quality and clinical relevance of cancer cell lines

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