Glucocorticoid-producing cells of the adrenal cortex (i.e. zona fasciculata, zF) constitute the critical effectors of the hypothalamic-pituitary-adrenal axis, mediating the mammalian stress response. With glucocorticoids being essential for life, zF dysfunction perturbs multiple organs that participate in optimizing cardiometabolic fitness. The zF forms a dynamic and heterogenous cell population endowed with the capacity to remodel through the engagement of both proliferative and differentiation programs that enable the adrenal to adapt and respond to diverse stressors. However, the mechanisms that sustain such differential responsiveness remain poorly understood. In this study, we resolve the transcriptome of the steroidogenic lineage by scRNA-seq using Sf1-Crehigh; RosamT/mG reporter mice. We identify HHEX, a homeodomain protein, as the most enriched transcription factor in glucocorticoid-producing cells. We utilize genetic mouse models to demonstrate that Hhex deletion causes glucocorticoid deficiency in male animals. Molecularly, we demonstrate that HHEX is an androgen receptor (AR) target gene, shaping the sexual dimorphism of the adrenal gland by repressing the female transcriptional program at puberty, while also maintaining zF cholesterol ester content by protecting lipid droplets from androgen-induced-lipophagy. Moreover, our study reveals that, in both sexes, HHEX is crucial for maintaining the identity of the innermost adrenocortical cell subpopulation. Specifically, loss of HHEX impairs the expression of Abcb1b (P-glycoprotein/MDR1), an efflux pump regulating steroid export and cellular levels of xenobiotics. Together, these data demonstrate that HHEX serves as a multi-functional regulator of post-natal adrenal maturation that is potentiated by androgens. In the adrenal cortex, cholesterol used for steroid production is stored in lipid droplets. The authors demonstrate here the importance of the transcription factor HHEX in maintaining glucocorticoid levels and protecting lipid droplets from androgen-induced lipid depletion.

Supplementary Information:Supplementary Figures 1-6 with Figure Legends, Supplementary Table 1 (reference only), Supplementary Table 2, Supplementary Methods, Supplementary References

Supplementary Table 1: NCI-H295R RNA-seq FPKM table, Related to Figures 2-4, 6.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The steroid hormone aldosterone, produced by the zona glomerulosa (zG) of the adrenal gland, is a master regulator of plasma electrolytes and blood pressure. While aldosterone control by the renin-angiotensin system is well understood, other key regulatory factors have remained elusive. Here, we replicated a prior association between a non-coding variant in WNT2B and an increased risk of primary aldosteronism, a prevalent and debilitating disease caused by excessive aldosterone production. We further show that in both mice and humans, WNT2B is expressed in the mesenchymal capsule surrounding the adrenal cortex, in close proximity to the zG. Global loss of Wnt2b in the mouse results in a dysmorphic and hypocellular zG, with impaired aldosterone production. Similarly, humans harboring WNT2B loss-of-function mutations develop a novel form of Familial Hyperreninemic Hypoaldosteronism, designated here as Type 4. Additionally, we demonstrate that WNT2B signals by activating the non-canonical Wnt/planar cell polarity pathway. Our findings identify WNT2B as a key regulator of zG function and aldosterone production with important clinical implications.

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. Significance: Oncogenic β-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for β-catenin–driven cancers.

Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/β-catenin signaling are frequently observed, the β-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/β-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/β-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/β-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/β-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/β-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this β-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/β-catenin inhibitor. These results show promise for the further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.

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.

Journal Article Targeting of a New Node in Lipid Metabolism as a Potential Treatment Strategy for ACC Get access Christopher R LaPensee, Christopher R LaPensee Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48106, USA Correspondence: Christopher R LaPensee, PhD, Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, 109 Zina Pitcher Pl. Ann Arbor, MI 48109, USA. Email: lapensee@med.umich.edu. https://orcid.org/0000-0003-3946-1835 Search for other works by this author on: Oxford Academic Google Scholar Gary D Hammer Gary D Hammer Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48106, USA Search for other works by this author on: Oxford Academic Google Scholar Endocrinology, Volume 164, Issue 3, March 2023, bqad003, https://doi.org/10.1210/endocr/bqad003 Published: 13 January 2023 Article history Received: 29 August 2022 Editorial decision: 06 January 2023 Published: 13 January 2023 Corrected and typeset: 27 January 2023

Abstract Proper development and zonation of the adrenal cortex is intimately connected with its lifelong capacity to produce steroid hormones. Both adrenocortical development and homeostasis are mediated in part by paracrine WNT/β-catenin signaling, an essential pathway for zonal maintenance and aldosterone production. Patients harboring homozygous or compound heterozygous loss-of-function (LOF) mutations in the WNT ligand WNT2B present with congenital diarrhea and require parenteral nutrition. Despite evidence for euvolemia, WNT2B-null patients exhibit markedly elevated plasma renin levels with no corresponding increase in aldosterone, suggesting a primary adrenal defect in aldosterone synthesis. To test this hypothesis, we generated both global and conditional Wnt2b knockout mouse models. In the mouse adrenal, Wnt2b is expressed exclusively in the capsule, the outer compartment of mesenchymal cells that overlay and signal to the subcapsular zona glomerulosa (zG), the adrenocortical zone that harbors aldosterone-producing cells and critical long-term progenitor cells. Global loss of Wnt2b from early mouse development results in a near-complete absence of the histological zG. Moreover, Wnt2b-null mice exhibit significantly increased plasma renin but normal aldosterone levels compared to wild-type controls. These data suggest that WNT2B-null patients indeed have an adrenocortical phenotype caused by apparent zG hypofunction. To further define the mechanism(s) by which Wnt2b LOF leads to zG hypofunction, we temporally deleted Wnt2b in the adrenal capsule of adult mice. Wnt2b conditional knockout (cKO) adrenals exhibit a zG-restricted decrease in Wnt/β-catenin signaling as determined by decreased β-catenin activity and target gene expression. Moreover, Wnt2b cKO mice demonstrated a disorganized zG and disrupted adrenocortical zonation. To determine the contribution of WNT2B to aldosterone production, we administered a sodium-deficient diet to control and Wnt2b cKO mice to activate the renin-angiotensin-aldosterone system. Indeed, while control mice exhibited the expected increase in both plasma renin and aldosterone, Wnt2b cKO mice showed marked elevation of renin despite normal aldosterone compared to controls, consistent with hyperreninemic hypoaldosteronism. Together, our work supports a WNT2B-dependent mechanism for adrenal zG development and maintenance in mice and humans and provides relevant models to study the consequences of WNT2B deficiency within the zG. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

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 demonstrate that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin binds master adrenal transcription factor SF1 and hijacks the adrenocortical super-enhancer landscape to maintain differentiation. Off chromatin, β-catenin binds histone methyltransferase EZH2, which is redistributed by the CIMP-high DNA methylation signature. SF1/β-catenin and EZH2/β-catenin complexes exist in normal adrenals and are selected for through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC favors EZH2/β-catenin assembly and purges SF1/β-catenin from chromatin, erasing differentiation and restraining cancer growth in vitro and in vivo . Our studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive, and poorly understood cancer of the adrenal cortex. Abnormal epigenetic patterning is a major predictor of dismal disease outcomes. Patients with genome-wide CpG island hypermethylation (CIMP-high) experience rapidly recurrent and routinely fatal disease. A deeper understanding of how epigenetic programs coordinate tumorigenesis is essential to develop improved medical therapies. We identify that DNA hypermethylation is directed to targets of a histone modifying complex, PRC2, suggesting cross-talk between DNA and histone methylation in CIMP-high ACC. In contrast to our expectations, we discovered that DNA methylation and PRC2-directed histone methylation are mutually exclusive. Additionally, we identified that EZH2 (a canonical member of the PRC2) forms a separate complex with tissue-specific proteins to coordinate sustained proliferation and steroidogenic differentiation in vitro and in vivo. These complexes persist through advanced stages of human malignancy and are conserved in mouse models of adrenal carcinogenesis. Taken together, our studies illustrate how CpG island hypermethylation exposes a pharmacologically targetable tissue-specific therapeutic vulnerability, and stabilizes a differentiation state required for sustained proliferation. Ultimately, we hope this work illuminates novel strategies for tissue-specific disruption of the aberrant epigenetic wiring supporting this devastating disease. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 1:24 p.m. - 1:30 p.m.

Abstract Dysregulation of normal adrenal structure and function contributes to a spectrum of diseases from hypoplasia to cancer. Peripheral adrenocortical progenitor cells in the zona glomerulosa (zG) centripetally migrate and differentiate to replenish steroidogenic cells of the zG and the inner cortex over time. Both the fate of progenitor cells and aldosterone production by steroidogenic cells in the zG are regulated by Wnt/β-catenin signaling, but the cell-specific effects of individual WNT ligands in the adrenal cortex are not fully understood. To further characterize Wnt signaling components crucial for progenitor cell fate and zG identity, we analyzed mouse adrenals using single molecule in situ hybridization, which revealed the previously unknown expression of Wnt2b exclusively in the adrenal capsule. Wnt2b is co-expressed in the capsule with the Wnt signaling potentiator Rspo3, the loss of which causes zG depletion and reduced adrenal size in mice. Therefore, we hypothesized that capsular WNT2B activates Wnt signaling in the underlying zG to maintain the undifferentiated state of progenitor cells. To define the role of WNT2B in these processes, we first generated whole body Wnt2b knockout (KO) mice, which exhibit complete zG loss, as defined by known markers of zG identity (β-catenin and DAB2). To more fully determine the mechanism by which Wnt2b deletion results in zG loss, we crossed Wnt2b-floxed and capsule-specific Gli1-CreERT2 mice to generate a Wnt2b conditional knockout (cKO) model and study the effects of Wnt2b loss on the zG during homeostasis of the adult adrenal cortex. Gli1-CreERT2 activation by tamoxifen in 6-week-old mice significantly decreased Wnt2b expression and resulted in a lower adrenal-to-body weight ratio in Wnt2b cKOs compared to controls four weeks later. Adrenocortical proliferation (Ki67) was also significantly decreased in Wnt2b cKO mice, suggesting that WNT2B may promote progenitor cell self-renewal. To characterize the consequences of WNT2B loss on canonical Wnt signaling, we assessed activation of β-catenin, the primary Wnt signaling effector. High β-catenin activity in the zG observed in wild-type mice was disrupted in Wnt2b cKO mice, together with markedly reduced expression of adrenocortical Wnt target genes Axin2 and Wnt4. In addition, Wnt2b loss resulted in downregulation of steroidogenic genes Cyp11b2 and Hsd3b6. Together, these data reveal that capsule-derived WNT2B is required for zG differentiation and maintenance, potentially through activating adrenocortical Wnt/β-catenin signaling and downstream target gene expression involved in both progenitor cell fate and steroid-producing cell function. Studies to more fully elucidate the dynamic effects of WNT2B on the adrenal zG are ongoing as they have important implications for adrenal homeostasis and disease, including both primary adrenal failure and neoplasia.

The Wnt signaling pathway is a critical mediator of the development and maintenance of several tissues. The adrenal cortex is highly dependent upon Wnt/β-catenin signaling for proper zonation and endocrine function. Adrenocortical cells emerge in the peripheral capsule and subcapsular cortex of the gland as progenitor cells that centripetally differentiate into steroid hormone-producing cells of three functionally distinct concentric zones that respond robustly to various endocrine stimuli. Wnt/β-catenin signaling mediates adrenocortical progenitor cell fate and tissue renewal to maintain the gland throughout life. Aberrant Wnt/β-catenin signaling contributes to various adrenal disorders of steroid production and growth that range from hypofunction and hypoplasia to hyperfunction, hyperplasia, benign adrenocortical adenomas, and malignant adrenocortical carcinomas. Great strides have been made in defining the molecular underpinnings of adrenocortical homeostasis and disease, including the interplay between the capsule and cortex, critical components involved in maintaining the adrenocortical Wnt/β-catenin signaling gradient, and new targets in adrenal cancer. This review seeks to examine these and other recent advancements in understanding adrenocortical Wnt/β-catenin signaling and how this knowledge can inform therapeutic options for adrenal disease.

Abstract The adrenal cortex is comprised of distinct concentric zones that produce hormones essential for life - the outermost zona glomerulosa (zG) produces aldosterone, and the innermost zona fasciculata (zF) produces cortisol. Adrenal zonation is maintained by a balance between paracrine (Wnt/β-catenin) and endocrine (ACTH/PKA) signaling. Wnt/β-catenin signaling is maintained in the outer cortex (zG, upper zF) by a gradient of Wnt ligands that diminish centripetally, and PKA signaling is maintained in the inner cortex (zF) by ACTH. Recent studies in vivo suggest that sustained PKA signaling promotes zF proliferation, enabling lineage conversion towards zF by inhibiting Wnt/β-catenin transcriptional programming. While these studies were crucial in elucidating mechanisms supporting adrenal zonation, it remains unclear if PKA-mediated repression of Wnt/β-catenin is carried out at the chromatin level or if it is secondary to ligand-dependent modulation of paracrine signaling as may happen in vivo. To address this question, we utilized the adrenocortical cell line (NCI-H295R), which harbors a mutation in CTNNB1 rendering Wnt/β-catenin signaling constitutively active. We stimulated PKA in NCI-H295R using forskolin, and assessed genome-wide chromatin accessibility by ATAC-seq and transcriptome changes by RNA-seq. Motif analysis of ATAC-seq from baseline NCI-H295R revealed that chromatin accessibility is dominated by transcription factors SF1 (master regulator of the adrenal cortex and steroidogenesis), AP1 (effector of PKA) and LEF1 (effector of Wnt/β-catenin). Following forskolin administration, we observed decreased accessibility in chromatin containing LEF1 binding motifs, and increased accessibility in chromatin bearing AP1 and SF1 motifs, suggesting that PKA activation drives AP1/SF1-dependent transcription and inhibits Wnt/β-catenin-dependent transcription at the chromatin level. Indeed, RNA-seq revealed that forskolin administration decreased the expression of zG and Wnt/β-catenin target genes, while simultaneously increasing expression of AP1/SF1 target genes. Collectively, these data demonstrate that PKA activation leads to profound chromatin remodeling that enables zF identity even in the setting of constitutive Wnt/β-catenin signaling. Ongoing studies are aimed at elucidating how chromatin modifiers and transcriptional machinery coordinate the dynamic regulation of differentiation programs required for adrenocortical homeostasis and zonation.

Abstract Adrenocortical carcinoma (ACC) is an aggressive cancer that affects 1-2 people per million in the United States annually. The only option for cure is surgical resection, and the 5-year survival rate for ACC remains low at 35%. Improved understanding of this disease is needed to develop rational therapies. Genomic alterations activating Wnt/Beta-catenin signaling occur in approximately 40% of ACCs and are associated with poor prognosis. However, the biologic consequences of constitutive Wnt/Beta-catenin activation in ACC are poorly understood. Treatment with Beta-catenin inhibitors BC2059 and PKF115-584 decreases viability in the NCI-H295R ACC cell line, which harbors an activating CTNNB1 (Beta-catenin) mutation. In addition, BC2059 significantly inhibits NCI-H295R tumor growth in an orthotopic xenograft model. Our data supports the hypothesis that Wnt/Beta-catenin regulates ACC cell growth and survival. To better characterize transcriptional programs engaged by Wnt/Beta-catenin signaling in ACC, we performed independent component analysis on The Cancer Genome Atlas (TCGA) ACC transcriptome dataset to identify components of coordinately expressed genes. One component is significantly enriched for Wnt signaling activity and is strongly associated with somatic CTNNB1 (Beta-catenin) mutation. The Wnt-enriched component is also enriched for extracellular matrix (ECM)-receptor interaction, including (but not restricted to) expression of COL11A1 (Collagen alpha-1(XI)), COL26A1 (Collagen alpha-1(XXVI)), LAMC3 (Laminin, gamma 3), and ITGA2 (Integrin, alpha 2), suggesting that Wnt/Beta-catenin is regulating composition of the ACC microenvironment. Current literature supports that ACC has relatively low contribution from stromal cells, suggesting that tumor-cell-derived ECM may have substantial biologic effect in the tumor microenvironment. To follow up on these observations, we performed immunohistochemical staining on tissue microarrays containing 97 ACC samples. We observed a strong correlation of COL11A1 expression and nuclear Beta-catenin localization, suggesting COL11A1 may be regulated by Wnt/Beta-catenin. Furthermore, COL11A1 expression is significantly associated with decreased overall survival and decreased event-free survival. Cell culture studies confirm that expression of COL11A1, COL26A1, and LAMC3 is significantly reduced in NCI-H295R cells treated with PKF115-584 or BC2059, consistent with our hypothesis that Wnt/Beta-catenin regulates expression of ECM components in ACC. These results illustrate a novel role for Wnt/Beta-catenin activity in ACC, and suggest that Beta-catenin-associated expression of ECM components may contribute to cancer cell survival and disease progression. Citation Format: Morgan K. Penny, Antonio M. Lerario, Chris LaPensee, Thomas J. Giordano, Ruolan Han, Erika A. Newman, Gary D. Hammer. The role of the Wnt/Beta-catenin pathway in adrenocortical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3955.

Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and >50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.

Adrenocortical carcinoma (ACC) is a rare and often aggressive cancer that affects 1-2 people per million in the United States annually. Genomic alterations activating canonical Wnt signaling occur in approximately 40% of ACCs and are associated with poor prognosis. However, the biological consequences of constitutive canonical Wnt activation in ACC are poorly understood. To better characterize the transcriptional programs that are engaged in ACC, we performed independent component analysis on The Cancer Genome Atlas (TCGA) ACC transcriptome dataset to identify components of coordinately expressed genes. One of the components identified was significantly enriched for Wnt signaling (p = 0.001) and was strongly associated with somatic CTNNB1 (β-catenin) mutations (p = 2.276e-07). Interestingly, this Wnt-enriched component also showed enrichment for extracellular matrix (ECM)-receptor interaction (p = 0.00139), including (but not restricted to) expression of COL11A1 (Collagen alpha-1(XI)), LAMC3 (Laminin, gamma 3), and ITGA2 (Integrin, alpha 2), suggesting that Wnt signaling is regulating cell-ECM interactions and ECM composition in ACC. To follow up on this observation, we performed immunohistochemical staining on tissue microarrays (TMAs) containing 97 ACC samples. We observed a strong correlation of COL11A1 expression and nuclear β-catenin localization (p = 0.0088), suggesting COL11A1 expression may be regulated by canonical Wnt signaling. Furthermore, COL11A1 expression was associated with decreased overall survival (p = 0.0003) and decreased event-free survival (p = 0.0075). To determine whether ECM and ECM-receptor genes are regulated by canonical Wnt activity, the NCI-H295R human ACC cell line, harboring an activating CTNNB1 mutation, was used. Cells were treated with PKF115-584, an inhibitor of canonical Wnt signaling that disrupts β-catenin interaction with TCF/LEF transcription factors. PKF115-584 treatment significantly reduced expression of COL11A1, LAMC3, and ITGA2 in NCI-H295R cells at early timepoints (p≤0.05), consistent with our hypothesis that canonical Wnt signaling regulates expression of ECM components in ACC. At late timepoints following PKF115-584 treatment, NCI-H295R cell viability decreased, indicating that canonical Wnt activity may regulate cell survival in ACC. These results illustrate a novel role for canonical Wnt activity in ACC, and suggest that β-catenin-regulated transcription of ECM and ECM-receptor components may promote cancer cell survival and aggressive disease. Future studies are aimed at characterizing the contribution of extracellular proteins to ACC phenotypes.Citation Format: Morgan K. Penny, Antonio M. Lerario, Chris LaPensee, Thomas J. Giordano, Gary D. Hammer. Canonical Wnt-associated extracellular matrix in adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1894.

The adrenal cortex is characterized by three histologically and functionally distinct zones: the outermost zona glomerulosa (zG), the intermediate zona fasciculata, and the innermost zona reticularis. Important aspects of the physiology and maintenance of the adrenocortical stem/progenitor cells have emerged in the last few years. Studies have shown that the adrenocortical cells descend from a pool of progenitors that are localized in the subcapsular region of the zG. These cells continually undergo a process of centripetal displacement and differentiation, which is orchestrated by several paracrine and endocrine cues, including the pituitary-derived adrenocorticotrophic hormone, and angiotensin II. However, while several roles of the endocrine axes on adrenocortical function are well established, the mechanisms coordinating the maintenance of an undifferentiated progenitor cell pool with self-renewal capacity are poorly understood. Local factors, such as the composition of the extracellular matrix ( ECM) with embedded signaling molecules, and the activity of major paracrine effectors, including ligands of the sonic hedgehog and Wnt signaling pathways, are thought to play a major role. Particularly, the composition of the ECM, which exhibits substantial differences within each of the three histologically distinct concentric zones, has been shown to influence the differentiation status of adrenocortical cells. New data from other organ systems and different experimental paradigms strongly support the conclusion that the interactions of ECM components with cell-surface receptors and secreted factors are key determinants of cell fate. In this review, we summarize established and emerging data on the paracrine and autocrine regulatory loops that regulate the biology of the progenitor cell niche and propose a role for bioengineered ECM models in further elucidating this biology in the adrenal.

ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.