Regulation and function of the mammalian tricarboxylic acid cycle.

The tricarboxylic acid (TCA) cycle, otherwise known as the Krebs cycle, is a central metabolic pathway that performs the essential function of oxidizing nutrients to support cellular bioenergetics. More recently, it has become evident that TCA cycle behavior is dynamic, and products of the TCA cycle can be co-opted in cancer and other pathologic states. In this review, we revisit the TCA cycle, including its potential origins and the history of its discovery. We provide a detailed accounting of the requirements for sustained TCA cycle function and the critical regulatory nodes that can stimulate or constrain TCA cycle activity. We also discuss recent advances in our understanding of the flexibility of TCA cycle wiring and the increasingly appreciated heterogeneity in TCA cycle activity exhibited by mammalian cells. Deeper insight into how the TCA cycle can be differentially regulated and, consequently, configured in different contexts will shed light on how this pathway is primed to meet the requirements of distinct mammalian cell states.

A non-canonical tricarboxylic acid cycle underlies cellular identity.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

Glutamine independence is a selectable feature of pluripotent stem cells.

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells (ESCs) in the presence of inhibitors against MEK and GSK3ß to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid (TCA) cycle anaplerosis, enabling ESCs to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the utilization of glutamine-derived carbons in the TCA cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both ESC passage and induced pluripotent cell reprogramming.

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome.

Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis.

Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.