Showing papers by "David T. Breault published in 2023"

Telomerase reverse transcriptase (TERT)-expressing cells mark a novel stem cell population in the adult mouse brain

Abstract: Telomerase reverse transcriptase (TERT) is expressed by quiescent adult stem cells (ASC) in numerous adult murine and human tissues, but has never been explored in the adult brain. Here, we demonstrate that TERT+ cells in the adult mouse brain represent a novel population of multipotent ASCs that are localized to numerous classical neuro/gliogenic niches (including the ventricular-subventricular zone, hypothalamus, and olfactory bulb), as well as more recently described regions of adult brain plasticity such as the meninges and choroid plexus. Using a direct-reporter mouse line, we found that TERT+ cells expressed known neural stem cell markers such as Nestin and Sox2, but not markers of committed stem/progenitor cells, nor markers of mature neuronal or glial cells. TERT+ ASCs rarely expressed the proliferation marker Ki67, and in vitro TERT+ cells lost TERT expression when activated by growth factors, together indicating a quiescent phenotype similar to what has been observed in other tissues. When cultured, TERT+ cells behaved like neural stem cells by forming neurospheres, which could proliferate and become more metabolically active once stimulated by growth factors. TERT+ cells were observed in numerous brain niches, particularly near the ventricles and cerebrospinal fluid barriers, but notably, TERT+ cells were never observed in the hippocampus. Lineage tracing of TERT+ cells in adult transgenic mice (mTERTrtTA::oTET-Cre::RosamTmG) revealed large-scale expansion of TERT+ progeny and differentiation to diverse cell types in multiple brain regions. For example, lineage-traced cells expressed markers of mature neurons, oligodendrocytes, astrocytes, ependymal cells, and choroid epithelial cells, thus demonstrating the striking multipotency of this stem cell population in basal tissue turnover of the adult brain. Together, these data demonstrate that TERT+ cells represent a novel population of multipotent stem cells that contribute to basal plasticity and regeneration in the adult mouse brain. Graphical Abstract

Calcineurin regulates aldosterone production via dephosphorylation of NFATc4.

Abstract: The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (Calcineurin (Cn)) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis. Inhibition of Cn with tacrolimus abolished the potassium (K+)-stimulated expression of aldosterone synthase, encoded by CYP11B2, in the NCI-H295R human adrenocortical cell line as well as ex vivo in mouse and human adrenal tissue. ZG-specific deletion of the regulatory Cn subunit CnB1 diminished Cyp11b2 expression in vivo and disrupted K+-mediated aldosterone synthesis. Phosphoproteomic analysis identified Nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a target for Cn-mediated dephosphorylation. Deletion of NFATc4 impaired K+-dependent stimulation of CYP11B2 expression and aldosterone production while expression of a constitutively active form of NFATc4 increased expression of CYP11B2 in NCI-H295R cells. Chromatin immunoprecipitation revealed NFATc4 directly regulates CYP11B2 expression. Thus, calcineurin controls aldosterone production via the Cn-NFATc4 pathway. Inhibition of Cn-NFATc4 signaling may explain low plasma aldosterone levels and hyperkalemia in patients treated with tacrolimus and the Cn-NFATc4 pathway may provide novel molecular targets to treat primary aldosteronism.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans

Abstract: Background and aims WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine. WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

β-catenin-driven differentiation is a tissue-specific epigenetic vulnerability in adrenal cancer.

Abstract: Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.

Abstract 1501: Epigenetic dedifferentiation as a therapeutic strategy in adrenal cancer

Abstract: Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex without curative medical therapies. CIMP-high is an aggressive ACC molecular subtype defined by global CpG island hypermethylation with paradoxical activation of adrenal differentiation (driven by master transcription factor SF1) and stemness (driven by β-catenin). We show DNA hypermethylation redistributes histone methyltransferase EZH2 and its mark, H3K27me3. EZH2 inhibition remains lethal to CIMP-high ACC cells, erasing transcriptional programs without altering DNA methylation. We reconcile this phenomenon by discovery of two nuclear complexes, SF1/β-catenin and EZH2/β-catenin, present in physiology and persistent through advanced ACC. We find SF1/β-catenin is a chromatin-bound complex that controls the ACC super-enhancer landscape, while EZH2/β-catenin is restricted to off-chromatin pools. EZH2 inhibition purges SF1/β-catenin from chromatin, sparing EZH2/β-catenin, inducing dedifferentiation and restraining ACC growth in vitro and in vivo. Our studies illustrate how cell-of-origin programs dictate cancer evolution, exposing differentiation as an therapeutic vulnerability. Citation Format: Dipika R. Mohan, Kleiton S. Borges, Isabella Finco, Christopher R. LaPensee, Juilee Rege, Donald W. Little, Tobias Else, Madson Q. Almeida, Derek Dang, James Haggerty-Skeans, Ana Claudia Latronico, Berenice B. Mendonca, Richard J. Auchus, William E. Rainey, Suely K. Marie, Thomas J. Giordano, Sriram Venneti, Maria Candida B. Fragoso, David T. Breault, Antonio M. Lerario, Gary D. Hammer. Epigenetic dedifferentiation as a therapeutic strategy in adrenal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1501.