Darren R. Heintzman’s research while affiliated with Vanderbilt University and other places

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Publications (15)


Impaired oxidative phosphorylation drives primary tumor escape and metastasis
  • Preprint

January 2025

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19 Reads

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Erin Q. Jennings

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Jeffrey C. Rathmell

Metastasis causes most cancer deaths and reflects transitions from primary tumor escape to seeding and growth at metastatic sites. Epithelial-to-mesenchymal transition (EMT) is important early in metastasis to enable cancer cells to detach from neighboring cells, become migratory, and escape the primary tumor. While different phases of metastasis expose cells to variable nutrient environments and demands, the metabolic requirements and plasticity of each step are uncertain. Here we show that EMT and primary tumor escape are stimulated by disrupted oxidative metabolism. Using Renal Cell Carcinoma (RCC) patient samples, we identified the mitochondrial electron transport inhibitor NDUFA4L2 as upregulated in cells undergoing EMT. Deletion of NDUFA4L2 enhanced oxidative metabolism and prevented EMT and metastasis while NDUFA4L2 overexpression enhanced these processes. Mechanistically, NDUFA4L2 suppressed oxidative phosphorylation and caused citric acid cycle intermediates to accumulate, which modified chromatin accessibility of EMT-related loci to drive primary tumor escape. The effect of impaired mitochondrial metabolism to drive EMT appeared general, as renal cell carcinoma patient tumors driven by fumarate hydratase mutations with disrupted oxidative phosphorylation were highly metastatic and also had robust EMT. These findings highlight the importance of dynamic shifts in metabolism for cell migration and metastasis, with mitochondrial impairment driving early phases of this process. Understanding mitochondrial dynamics may have important implications in both basic and translational efforts to prevent cancer deaths.


Figure 1. CyTOF of lung draining lymph nodes of deceased donor patients reveals sex differences in T cells and metabolic protein expression. CyTOF was conducted on human lung draining lymph nodes from deidentified female and male deceased donors. (A) UMAP visualization of cell surface markers in all samples. (B-D) Median expression of metabolic markers on CD4+ T cells, Th1, Th2, Th17, and Treg cells. Gating strategies and population definitions are shown in Figure S1. Data shown are mean ± SEM: n=14 females and n=17 males. *p<0.05, **p<0.01, ns: not significant, two-tailed Mann-Whitney U test. See Supplemental Figures 1-2 and Supplemental Tables 1-2.
Figure 2. AR signaling in CD4+ T cells reduces Th17 driven neutrophilic inflammation during airway inflammation. (A) Model of HDM allergen challenge. Female and male Ar floxed and Cd4 Cre+ Ar floxed mice were challenged intranasally with 40µg of HDM 3 times per week for 3 weeks. BAL fluid and lungs were harvested 24 hours after the last challenge. (B) Quantification of eosinophils and neutrophils in the BAL fluid from mice after HDM challenge. (C) IL-13 and IL-17A protein expression in BAL fluid after HDM challenge. (D) Airway hyperresponsiveness as measured by FlexiVent with increasing doses of methacholine was also conducted on HDM-challenged female and male Ar floxed and Cd4 Cre+ Ar floxed mice 48 hours after last challenge (Data shown are mean ± SEM, n=4-6 mice per group). (E) Representative flow diagrams of IL-13+ Th2 cells and IL-17A+ Th17 cells. (F) Quantification of lung Th2 and Th17 cells after HDM challenge (G-H) Expression of GLUD1, Glut1, HIF1α, and pS6 in lung Th2 cells (top) and Th17 cells (bottom) after HDM challenge. (B-C and E-H) Data are expressed as mean ± SEM: n=7-9 mice per group combined from 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant, ANOVA with post-hoc Tukey testing. See Supplemental Figure 3, 4 and 5.
Figure 4. AR signaling reduces glutamine metabolism in Th17 cells. (A) Pathway Analysis on targeted metabolomics from differentiated Th17 cells from WT male and Ar Tfm male mice (n=5-6 mice per group from 2 independent experiments, statistical analysis by MetaboAnalyst Global test, larger circle indicates larger impact, darker shade indicates increased significance). (B) Intracellular glutamine and glutamate levels from differentiated Th17 cells from WT male and Ar Tfm male mice measured by mass spectrometry (n=5-6 mice per group). (C) Seahorse Substrate Oxidation Assay using CB-839, an inhibitor of glutaminase, on differentiated Th17 cells from wild-type male and Ar Tfm male mice to measure dependance of mitochondrial respiration of Th17 cells on glutamine metabolism. (D) Quantified ΔΔOCR from C. ΔOCR was calculated by determining the difference in measured OCR after DMSO or CB-839 injection. ΔΔOCR was calculated as follows: ΔOCR CB-839 basal -ΔOCR DMSO basal . See Supplemental Figure 5A (E) Quantified from C and calculated as OCR CB-839 Max -OCR DMSO Max . *p<0.05, unpaired T test. See Supplemental Figure 5 and Supplemental Table 3. (F-G) HDM-induced airway inflammation in female and sham or gonadectomized (GNX) male Gls fl/fl and Cd4 Cre+ Gls fl/fl mice were sensitized and challenged with HDM as described in Figure 2A. One day following the last challenge, BAL fluid and lungs were harvested. (F) Eosinophils and neutrophils in BAL fluid and (G) Lung Th2 and Th17 cells quantified by flow cytometry. Data shown are mean ± SEM, n=3-4 mice per group, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant, ANOVA with post-hoc Tukey testing. See Supplemental Figure 8 and 9 and Supplemental Table 3.
Figure 5. AR signaling reduces glutamine uptake in T cells. (A) Model of targeted CRISPR screen using a glutamine library on differentiated Th17 cells from male and female OT-II Cas9 mice in an OVA-induced lung inflammation model. (B) Change in gRNA abundance in lung Th17 cells from male mice (y-axis) and GNX male mice (x-axis) with table showing statistics (Data shown are mean ± SEM, n=4-5 mice, statistical analysis by MAGeCK affected represented by color of dot as shown in legend). (C) Model of F18-Glutamine PET studies in HDM-induced lung inflammation as shown in Figure 2A. (D) Quantification of 18 F-Glutamine concentration in whole lung by PET/CT imaging (n=4-6 mice per group). (E) Quantification of 18 F-Glutamine concentration in lung CD4+ T cells by magnetic separation and gamma counting, normalized to viable cells (Data shown are mean ± SEM, n=4-6 mice per group). ***p<0.001, unpaired T test. See Supplemental Figure 10 and 11.
Figure 7. AR signaling alters H3K27 trimethylation in Th17 cells. CUT & RUN analysis for H3K27me3 was conducted on Th17 cells differentiated from WT male, WT female, and Ar Tfm male mice (n=3 per group). (A) Heatmaps of H3K27me3 to IgG control ratio in ±10-kb regions around promoters of UCSC known genes in Th17 cells measured by CUT&RUN. (B-C) Analysis of differentially methylated areas in Th17 cells from male and Ar Tfm male mice (panel B) or Th17 cells from males and females (panel C). (D) GREAT analysis to find differences in gene ontology pathways in Th17 cells from WT male versus Ar Tfm male mice. (E) GREAT analysis to find differences in H3K27me3 tagged genomic regions in Th17 cells from WT male versus Ar Tfm male mice. (D-E) Pathways and genes shown had a p<0.05. (F) qPCR analysis of Slc1a5 in Th17 cells. Data shown are mean ± SEM, n=3-5 samples, ** p<0.01, ANOVA with Tukey post-hoc analysis. See Supplemental Figure 12.

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Androgen signaling restricts glutaminolysis to drive sex-specific Th17 metabolism in allergic airway inflammation
  • Article
  • Full-text available

October 2024

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20 Reads

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1 Citation

The Journal of clinical investigation

Females have an increased prevalence of many Th17 cell-mediated diseases, including asthma. Androgen signaling decreases Th17 cell-mediated airway inflammation, and Th17 cells rely on glutaminolysis. However, it remains unclear whether androgen receptor (AR) signaling modifies glutamine metabolism to suppress Th17 cell-mediated airway inflammation. We show that Th17 cells from male humans and mice had decreased glutaminolysis compared to females, and that AR signaling attenuated Th17 cell mitochondrial respiration and glutaminolysis in mice. Using allergen-induced airway inflammation mouse models, we determined females had a selective reliance upon glutaminolysis for Th17-mediated airway inflammation, and AR signaling attenuated glutamine uptake in CD4+ T cells by reducing expression of glutamine transporters. Minimal reliance on glutamine uptake in male Th17 cells compared to female Th17 cells was also found in circulating T cells from patients with asthma. AR signaling thus attenuates glutaminolysis, demonstrating sex-specific metabolic regulation of Th17 cells with implications for Th17 or glutaminolysis targeted therapeutics.

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Subset-specific mitochondrial stress and DNA damage shape T cell responses to fever and inflammation

September 2024

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39 Reads

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4 Citations

Science Immunology

Heat is a cardinal feature of inflammation, yet its impacts on immune cells remain uncertain. We show that moderate-grade fever temperatures (39°C) increased murine CD4 T cell metabolism, proliferation, and inflammatory effector activity while decreasing regulatory T cell suppressive capacity. However, heat-exposed T helper 1 (T H 1) cells selectively developed mitochondrial stress and DNA damage that activated Trp53 and stimulator of interferon genes pathways. Although many T H 1 cells subjected to such temperatures died, surviving T H 1 cells exhibited increased mitochondrial mass and enhanced activity. Electron transport chain complex 1 (ETC1) was rapidly impaired under fever-range temperatures, a phenomenon that was specifically detrimental to T H 1 cells. T H 1 cells with elevated DNA damage and ETC1 signatures were also detected in human chronic inflammation. Thus, fever-relevant temperatures disrupt ETC1 to selectively drive apoptosis or adaptation of T H 1 cells to maintain genomic integrity and enhance effector functions.


Mitochondrial Fatty Acid Synthesis and Mecr Regulate CD4+ T Cell Function and Oxidative Metabolism

July 2024

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10 Reads

Lipid metabolism is fundamental to CD4+ T cell metabolism yet remains poorly understood across subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid-associated genes essential for T cell subset functions. These screens established mitochondrial fatty acid synthesis (mtFAS) genes Mecr, Mcat and Oxsm as highly impactful. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Effector and memory T cells were reduced in Mecrfl/fl; Cd4cre mice, and MECR was required for activated CD4+ T cells to efficiently proliferate, differentiate, and survive. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced TCA intermediates, and accumulated intracellular iron, which contributed to cell death and sensitivity to ferroptosis. Importantly, Mecr-deficient T cells exhibited fitness disadvantages in inflammatory, tumor, and infection models. mtFAS and MECR thus play important roles in activated T cells and may provide targets to modulate immune functions in inflammatory diseases. The immunological state of MECR- and mtFAS-deficient patients may also be compromised.


Harnessing temperature-dependent STING signaling to reprogram regulatory T cells and enhance effector T cell immunity.

May 2024

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3 Reads

The Journal of Immunology

The cGAS-STING pathway plays a critical role in immune surveillance through its capacity to sense aberrantly localized DNA in the cytosol caused by infection, cancer cell death, or cell stress-induced self-DNA leakage. STING pathway activation ultimately results in the production of Type I Interferons (IFN-I) and other inflammatory cytokines that stimulate T cell immunity. However, the T cell-intrinsic effects of STING agonists have been underexplored, especially in regulatory T cells (Tregs), which have an opposing but crucial role in suppressing excessive inflammation. Unexpectedly, we have found that STING activation repolarizes Tregs to IFN-I producing Th1/Th9-like cells with enhanced antitumor function and reduced oxidative metabolism and suppressive capacity. Inflammation can also coincide with heat (i.e. fever-range temperature, FRT, 39°C), which can cause nuclear or mitochondrial self-DNA leakage, yet the effects of FRT on STING in Tregs are unknown. We were surprised to find that FRT strongly potentiates the effects of STING activation in Tregs and that FRT appears to prime cGAS/STING signaling. Together, our initial data suggest a novel axis linking FRT to STING signaling in inhibiting Treg immunosuppressive function and in promoting Treg transdifferentiation to inflammatory effector-like T cells. These exciting findings motivate further investigation to dissect the mechanisms intertwining STING activation and FRT and their effect on Treg function and metabolism.


Tissue-Specific Dependence of Th1 Cells on the Amino Acid Transporter SLC38A1 in Inflammation

September 2023

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48 Reads

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5 Citations

Amino acid (AA) uptake is essential for T cell metabolism and function, but how tissue sites and inflammation affect CD4 ⁺ T cell subset requirements for specific AA remains uncertain. Here we tested CD4 ⁺ T cell AA demands with in vitro and multiple in vivo CRISPR screens and identify subset- and tissue-specific dependencies on the AA transporter SLC38A1 (SNAT1). While dispensable for T cell persistence and expansion over time in vitro and in vivo lung inflammation, SLC38A1 was critical for Th1 but not Th17 cell-driven Experimental Autoimmune Encephalomyelitis (EAE) and contributed to Th1 cell-driven inflammatory bowel disease. SLC38A1 deficiency reduced mTORC1 signaling and glycolytic activity in Th1 cells, in part by reducing intracellular glutamine and disrupting hexosamine biosynthesis and redox regulation. Similarly, pharmacological inhibition of SLC38 transporters delayed EAE but did not affect lung inflammation. Subset- and tissue-specific dependencies of CD4 ⁺ T cells on AA transporters may guide selective immunotherapies. HIGHLIGHTS T cells dynamically regulate glutamine amino acid transporters when activated SLC38A1 supports Th1 cell mTORC1 and proliferation by redox and hexosamine pathways Targeting SLC38A1 does not affect lung inflammation but delays IBD and EAE Nutrient transporter needs of T cell subsets vary based on disease and tissue site


RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

February 2023

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43 Reads

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11 Citations

Cell Reports

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.


Subset-specific mitochondrial and DNA damage shapes T cell responses to fever and inflammation

November 2022

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69 Reads

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1 Citation

Heat is a cardinal feature of inflammation. Despite temperature variability and dependence of enzymes and complexes, how heat and fever affect immune cells remains uncertain. We found that heat broadly increased inflammatory activity of CD4 ⁺ T cell subsets and decreased Treg suppressive function. Th1 cells, however, also selectively developed mitochondrial dysfunction with high levels of ROS production and DNA damage. This led Th1 cells to undergo Tp53 -dependent death, which was required to minimize the accumulation of mutations in heat and inflammation. Th1 cells with similar DNA damage signatures were also detected in Crohn’s disease and rheumatoid arthritis. Fever and inflammation-associated heat thus selectively induce mitochondrial stress and DNA damage in activated Th1 cells that requires p53 to maintain genomic integrity of the T cell repertoire. One Sentence Summary Fever temperatures augment CD4 ⁺ T cell-mediated inflammation but induce differential metabolic stress and DNA damage in T cell subsets, with Th1 cells selectively sensitive and dependent on p53 to induce apoptosis and maintain genomic integrity.


RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

January 2022

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9 Reads

Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.


Fig. 2 Obesity leads to both systemic and local changes to T cell microenvironments. Obesity or weight loss can have striking effects on T cell metabolism in the obesity paradox in which tumors are promoted yet sensitized to anti-PD1
Microenvironmental influences on T cell immunity in cancer and inflammation

January 2022

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179 Reads

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65 Citations

Cellular & Molecular Immunology

T cell metabolism is dynamic and highly regulated. While the intrinsic metabolic programs of T cell subsets are integral to their distinct differentiation and functional patterns, the ability of cells to acquire nutrients and cope with hostile microenvironments can limit these pathways. T cells must function in a wide variety of tissue settings, and how T cells interpret these signals to maintain an appropriate metabolic program for their demands or if metabolic mechanisms of immune suppression restrain immunity is an area of growing importance. Both in inflamed and cancer tissues, a wide range of changes in physical conditions and nutrient availability are now acknowledged to shape immunity. These include fever and increased temperatures, depletion of critical micro and macro-nutrients, and accumulation of inhibitory waste products. Here we review several of these factors and how the tissue microenvironment both shapes and constrains immunity.


Citations (8)


... In this issue of the JCI, Chowdhury et al. address this knowledge gap head on, investigating how biological sex shapes T cell metabolism and function in the context of allergic airway inflammation (10). The study offers compelling evidence that androgen signaling fundamentally alters T helper 17 (Th17) cell metabolism, providing mechanistic insight into sex differences in allergic responses and potentially broader implications for autoimmune disease susceptibility. ...

Reference:

Sex, cells, and metabolism: Androgens temper Th17-mediated immunity
Androgen signaling restricts glutaminolysis to drive sex-specific Th17 metabolism in allergic airway inflammation

The Journal of clinical investigation

... Additionally, altered expression of genes related to axon guidance (AG) and synaptic plasticity (SP), particularly involving CREB signalling, has been highlighted via meta-analysis [53]. The transporter protein, SLC38A, involved in AAT, may modulate IRs by altering amino acid (AA) availability, which in turn influences immune cell function [54]. CXCL10, a chemokine, plays a critical role in MS by recruiting T cells to the CNS, contributing to the breakdown of the blood-brain barrier (BBB) and amplifying the inflammatory response [55]. ...

Tissue-Specific Dependence of Th1 Cells on the Amino Acid Transporter SLC38A1 in Inflammation

... Since this assay system is located~3 kb downstream of the origin, initiation should not contribute to the observed effects. These results indicate that Mcm10 promotes replication through the (GAA) n repeat and likely other hard-to-replicate genome regions, as was previously observed for replication termination in Xenopus egg extracts 105 . ...

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

Cell Reports

... These data provide evidence that heat benefits pro-inflammatory CD4 + T cell responses. However, this study also showed that Th1 cells were particularly sensitive to increased temperatures, as they accumulated high levels of mitochondrial ROS, resulting in DNA damage and cell death (208). These preclinical studies provide insight into how systemic factors, such as the organismal temperature, might affect the GVL immunity. ...

Subset-specific mitochondrial and DNA damage shapes T cell responses to fever and inflammation

... Unlike RA and SLE, which are typically associated with a more pronounced autoimmune antibody response, the production of autoantibodies is not a core feature of the pathological process in AS. Changes in methylation may influence the type and intensity of immune responses via gene expression regulation [24], potentially mediating differences in immune regulatory mechanisms across different diseases. The significant differences in CXCR5 circulating methylation observed between RA and SLE relative to spondyloarthritis could impact CXCR5 and its associated immune processes, leading to different disease manifestations. ...

Microenvironmental influences on T cell immunity in cancer and inflammation

Cellular & Molecular Immunology

... Briefly, GPMVs were made from HeLa cells expressing PMP22. We employed the N41Q (glycosylation deficient) variant of PMP22 since it traffics to the plasma membrane with greater efficiency than wild type but exhibits the same ordered domain preference (18,24). The disordered phase was labeled with the lipophilic stain DiI while PMP22 was immunolabeled with AlexaFluor 647. ...

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

Journal of Biological Chemistry

... This includes the activities of OGG1-initiated BER, MutY-initiated BER, mismatch repair, nucleotide excision repair (NER), and accurate TLS bypass by DNA polymerase η. We therefore compared spontaneous and KBrO 3induced mutation spectra among WT human cell lines and those lacking OGG1 8 , MUTYH 8 , Pol η 50 , or HMCES, a recently identified replication-associated factor that participates in bypass of ssDNA lesions [51][52][53][54][55] and protects cells from cytotoxicity associated with KBrO 3 exposure 51,54 . Loss of OGG1, MUTYH, and HMCES resulted in moderatẽ 2 to 3-fold increases in the amount of spontaneously acquired mutations per genome compared to corresponding WT lines, while Pol η-deficiency failed to increase spontaneous mutagenesis (Supplementary Fig. 12A). ...

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation

Cell Reports

... Replication termination is a highly regulated process that is critical for accurate genome maintenance 4 . To initiate termination, forks must merge, a process that depends critically on topoisomerase activity 5,6 . When converging CMGs meet, they pass each other, leading to disengagement of the lagging strand template from the outer face of CMG. ...

Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination

Cell Reports