United States

Adrenal homeostasis relies on the centripetal differentiation and migration of peripheral adrenocortical progenitors to replenish the steroidogenic cells in the cortex. This homeostatic process is essential for steroidogenic functions and its dysregulation has been implicated in many adrenal diseases. Steroidogenic Factor 1(SF1) is a nuclear receptor which functions as a transcriptional factor to regulate global steroidogenic gene expression and adrenal development. However, the regulation of SF1 in the processes of differentiation (steroidogenesis) versus self-renewal of progenitors necessary for adult adrenal homeostasis remains largely unknown. Transcriptional activity of SF1 is regulated by post-translational modifications including phosphorylation (S203) and SUMOylation (K119 and K194). SUMOylation inhibits CDK7-mediated SF1 phosphorylation and SUMO-defective SF1 preferentially activates &quot;SUMO-sensitive&quot; target genes including Shh, rather than many well-characterized &quot;SUMO-insensitive&quot; target genes mediating differentiation. Based on these findings, together with data that CDK7 uniquely links cell cycle to transcriptional programs engaged in proliferating cells and the marked stem cell failure reported in multiple tissues of CDK7-KO mice, we hypothesized that CDK7-mediated SF1 phosphorylation in Shh-expressing adrenocortical progenitors regulates a unique transcriptional program to maintain their undifferentiated proliferative state during adrenal homeostasis. We engineered SF1-S203A/D mouse models which contain biallelic non-phosphorylatable/phosphomimetic germline mutations of SF1 at Serine 203. We crossed S203D mice with a) Shh-LacZ reporter strain to visualize Shh-expressing progenitors locked in an SF1 phosphomimic state and b) Shh-CreERT2;mTmG strain to GFP label Shh progenitors for lineage tracing. We observed increased beta-galactosidase staining in the zG of SF1-S203D reporter mice compared to controls, indicating an expanded Shh progenitor pool when SF1 is phosphorylated at S203. After 14 days of lineage tracing, GFP was co-stained with Cyp11B2, a marker for zG steroidogenic lineage, to quantify the percentage of Shh-expressing progenitors that differentiated into Cyp11B2-expressing zG cells. SF1-S203D mice exhibited a markedly lower differentiation rate of Shh-expressing progenitors compared to wild type, suggesting that SF1 phosphorylation preserved their undifferentiated state. Moreover, SF1-S203A mutation leads to a decreased adrenal/body weight ratio as well as downregulation of Shh. For insights into the role of CDK7 in SF1 phosphorylation during adrenocortical homeostasis, we generated conditional CDK7-KO mice. Adrenocortical-restricted CDK7-KO mice had decreased adrenal size, proliferation rate and Shh expression. Lineage tracing revealed increased differentiation of Shh-expressing progenitors when CDK7 was deleted specifically and temporally in Shh-expressing progenitors. Finally, we compared the differentiation rate in WT, CDK7-KO and CDK7-KO on an S203D background. The rapid differentiation induced by CDK7 loss was partially rescued by SF1-S203D, confirming the contribution of SF1 phosphorylation in mediating the functions of CDK7 in adrenocortical progenitors. Based on these findings, we propose a novel model in which CDK7-mediated SF1 phosphorylation regulates a unique transcriptional program that contributes to maintaining the undifferentiated proliferative state of adrenocortical progenitors.


ENDO 2022

Unravelling the Role of CDK7-mediated SF1 Serine 203 Phosphorylation in Adrenal Homeostasis

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
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Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Adrenal homeostasis relies on the centripetal differentiation and migration of peripheral adrenocortical progenitors to replenish the steroidogenic cells in the cortex. This homeostatic process is essential for steroidogenic functions and its dysregulation has been implicated in many adrenal diseases. Steroidogenic Factor 1(SF1) is a nuclear receptor which functions as a transcriptional factor to regulate global steroidogenic gene expression and adrenal development. However, the regulation of SF1 in the processes of differentiation (steroidogenesis) versus self-renewal of progenitors necessary for adult adrenal homeostasis remains largely unknown. Transcriptional activity of SF1 is regulated by post-translational modifications including phosphorylation (S203) and SUMOylation (K119 and K194). SUMOylation inhibits CDK7-mediated SF1 phosphorylation and SUMO-defective SF1 preferentially activates "SUMO-sensitive" target genes including Shh, rather than many well-characterized "SUMO-insensitive" target genes mediating differentiation. Based on these findings, together with data that CDK7 uniquely links cell cycle to transcriptional programs engaged in proliferating cells and the marked stem cell failure reported in multiple tissues of CDK7-KO mice, we hypothesized that CDK7-mediated SF1 phosphorylation in Shh-expressing adrenocortical progenitors regulates a unique transcriptional program to maintain their undifferentiated proliferative state during adrenal homeostasis. We engineered SF1-S203A/D mouse models which contain biallelic non-phosphorylatable/phosphomimetic germline mutations of SF1 at Serine 203. We crossed S203D mice with a) Shh-LacZ reporter strain to visualize Shh-expressing progenitors locked in an SF1 phosphomimic state and b) Shh-CreERT2;mTmG strain to GFP label Shh progenitors for lineage tracing. We observed increased beta-galactosidase staining in the zG of SF1-S203D reporter mice compared to controls, indicating an expanded Shh progenitor pool when SF1 is phosphorylated at S203. After 14 days of lineage tracing, GFP was co-stained with Cyp11B2, a marker for zG steroidogenic lineage, to quantify the percentage of Shh-expressing progenitors that differentiated into Cyp11B2-expressing zG cells. SF1-S203D mice exhibited a markedly lower differentiation rate of Shh-expressing progenitors compared to wild type, suggesting that SF1 phosphorylation preserved their undifferentiated state. Moreover, SF1-S203A mutation leads to a decreased adrenal/body weight ratio as well as downregulation of Shh. For insights into the role of CDK7 in SF1 phosphorylation during adrenocortical homeostasis, we generated conditional CDK7-KO mice. Adrenocortical-restricted CDK7-KO mice had decreased adrenal size, proliferation rate and Shh expression. Lineage tracing revealed increased differentiation of Shh-expressing progenitors when CDK7 was deleted specifically and temporally in Shh-expressing progenitors. Finally, we compared the differentiation rate in WT, CDK7-KO and CDK7-KO on an S203D background. The rapid differentiation induced by CDK7 loss was partially rescued by SF1-S203D, confirming the contribution of SF1 phosphorylation in mediating the functions of CDK7 in adrenocortical progenitors. Based on these findings, we propose a novel model in which CDK7-mediated SF1 phosphorylation regulates a unique transcriptional program that contributes to maintaining the undifferentiated proliferative state of adrenocortical progenitors.


technical paper

Introduction
Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications.
Methods
24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ and 39 during MTP) from 34 CS patients. In addition, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment.
Results:
Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ >400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83 – 1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (>25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were >50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p <0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001).
Conclusion:
KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of ULN.

Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

Glucocorticoids (GC) are an integral part of anti-inflammatory therapy. However, their therapeutic effectiveness is limited by their deleterious adverse effects such as the development of obesity, diabetes, and cardiometabolic disease. GC-induced obesity arises from excessive fat accumulation in white adipose tissue (WAT) as well as suppressed thermogenic function of brown adipose tissue (BAT). The sympathetic nervous system (SNS) plays a key role in the regulation of both WAT as well as BAT biology. As part of this project, we investigate the interaction between SNS and GC signaling in the adipose organ.
To that end, we modulated the SNS activity in adipose tissues of mice by altering the housing temperature of corticosterone-treated mice and respective controls to either 29°C, 22°C, or 13°C. Following the four-week treatment course, GC-treated mice housed at 29°C and 22°C gained more weight and accumulated more visceral and subcutaneous white fat than their temperature-matched controls. Interestingly, maintaining mice in the 13°C environment protected animals from GC-induced weight gain and obesity. Additionally, cold adaptation effectively prevented the development of GC-driven hyperglycemia, hyperinsulinemia and hyperleptinemia, all of which were readily observed at 29°C and 22°C. Furthermore, the sympathetic innervation as well as the thermogenic capacity of BAT were preserved in cold-acclimated mice in spite of corticosterone treatment, whereas mice housed at higher temperatures showed marked suppression of the thermogenic function in BAT following GC exposure. To further test whether sympathetic activation is responsible for the prevention of GC-induced dysmetabolism by prolonged adaption to cold, we investigated the effect of a pharmacological β3-AR agonist, CL316,243 during GC excess. Similar to cold exposure, selective activation of the β3-AR not only averted the development of GC-induced fat accumulation and hyperinsulinemia, but also preserved the thermogenic capacity in BAT despite exposure to supra-physiological levels of corticosterone.
Taken together, our data indicate that both cold-acclimation as well as β3-AR activation counteract GC-induced obesity, adipose tissues dysfunction, and related metabolic comorbidities. Thus, targeting the β3-AR may represent a potential therapeutic approach to alleviate GC-induced metabolic abnormalities.

The beta3-Adrenergic Receptor as a Target for the Prevention of Glucocorticoid-induced Adipose Tissue Dysfunction in Rodents

Context:  About 5-10% of  adrenocortical cancer (ACC) arise in patients with genetic predisposition syndromes, including Li-Fraumeni, Lynch, MEN1, or Familial Adenomatous Polyposis (APC). Recent European (2018) and American guidelines (2020) recommend that all adults with ACC should be offered clinical genetic counseling. There is a paucity of data regarding systematic germline testing in adults with ACC and the extent of the genetic evaluation is unclear.

  Objective: To describe the germline genetic characteristics in a cohort of adult patients with ACC  evaluated in  a tertiary clinical care center.

  Methods: Data including demographics, personal and family history of neoplasia, pathology reports, clinical features and genetic testing were retrospectively collected from charts of patients treated at Centre hospitalier de l'Université de Montréal (CHUM). After genetic counseling, genetic testing was proposed to patients with a pathologic diagnosis of ACC. From 2005 to 2015, TP53 gene analysis was performed using direct sequencing and MLPA. Since 2016, multigene testing was performed for a progressively increasing number of oncogenic genes using a custom next-generation panel for germline leucocyte DNA including  at least the  TP53, MSH2, MSH6, MLH1,PMS2, EPCAM, MEN1, BRCA1 and BRCA2 genes (Invitae, CA). An extended panel, based on past medical and familial history, was performed at the discretion of the geneticist. Patient data was retrospectively investigated.

  Results: We analyzed data from 44 patients with available germline genetic results. Median age of the patients was 46 years (ranging from 22 to 79 years), including 11 males (25.0%) and 33 females (75.0%). Ten patients (22.7%)  underwent only TP53 gene analysis and 32 patients (72.7%) were studied using the larger oncogenic genetic panel.
Germline pathogenic or likely pathogenic variants were identified in 5 of the 44 patients (11.4%) while genetic variants of unknown significance (VUS) were found in 7 of the 44 patients (15.9%). Among patients with pathogenic variants, one had a family history and known germline mutation in the BRCA2 gene (8765delAG, p.Glu2846GlyfsX23). One patient had a personal and familial medical history suggesting a MEN1 syndrome that was confirmed with the finding of a germline MEN1 mutation (c.1556delC, p.Pro519Leu fs40). Unsuspected germline pathogenic variants were found in three patients: 1) TP53 (c.425delC, p.Pro142fs), 2) MUTYH (c.536A>G, p.Tyr179Cys) and 3) MSH6 (c.3649-3655, p.Arg1217Leu fs9) genes. Variants of unknown significance (VUS) were found in the following genes: POT1, MSH3, PALB2, RAD51d, APC, ATM and BRCA2.
  Conclusions: Germline pathogenic variants were found in 11.4% of our cohort of patients with ACC. VUS were found in 15.9% of patients but their significance remains to be determined. Genetic counseling and germline genetic testing should be offered to all patients with ACC; however the optimal use and extent of oncogenic gene panels need to be better defined.

Germline Genetic Testing in a Cohort of Adults with Adrenocortical Carcinoma : Insights From a Clinical Care Setting

Congenital adrenal hyperplasia (CAH) is a set of defects in cortisol synthesis due to mutations in genes encoding steroidogenic enzymes. Consequently, the loss of glucocorticoid-mediated negative feedback on the HPA axis leads to chronically elevated ACTH (Adrenocorticotropin Hormone) secretion. The sustained ACTH elevation, in turn, stimulates proliferation and hypertrophy of the adrenal cortex and leads to overproduction of androgens.
To study the complexity of the ACTH-responsive cell population in the adrenal cortex, we performed single-cell RNA sequencing of the steroidogenic lineage in the adult mouse adrenal. We identified the transcription factor HHEX as a gene with restricted expression in the ACTH-responsive zona fasciculata. Although the role of HHEX in adrenal biology is completely unknown, a meta-analysis identified a germ-line variant of uncertain significance near the gene HHEX associated with increased DHEAS (adrenal-specific androgen) in human serum samples, which led us to hypothesize that HHEX contributes to the unique function of the ACTH-responsive inner cortex.
To investigate the mechanisms by which HHEX controls adrenal differentiation and homeostasis, we generated genetically modified mice harboring a deletion of the Hhex gene in the adrenal cortex. Hhex KO mice exhibited progressive adrenomegaly by 15 weeks of age, accompanied by hypertrophy prominent in the inner cortex and disruption of the endothelial organization. Expression of the transcription factor Nr5a1/Sf‑1 and steroidogenic enzymes involved in corticosterone production were significantly upregulated at 6 weeks old, prior to adenomegaly, suggesting a precocious increase in cell production of glucocorticoids. For insights into the signaling pathways controlled by HHEX, we analyzed global transcriptional changes during loss of Hhex and found dramatic downregulation of members of the membranous progesterone receptor family (PAQR). PAQR signaling has been implicated in the downregulation of cAMP, a primary mediator of ACTH signaling, and thereby could, in part, drive the increase in steroidogenesis in Hhex KO mice.
These findings suggest that the inner cortex contains a progesterone-responsive cell population in which HHEX provides additional fine-tuning of ACTH-driven cAMP signaling by progesterone and derivatives. Since we observed an increase in Hhex expression in a mouse model of CAH, experiments are now ongoing to assess the implication of HHEX and autocrine progesterone signaling in contributing to the hypertrophy and steroidogenic phenotype observed in CAH.

ROLE OF THE TRANSCRIPTION FACTOR HHEX IN INNER ADRENAL CORTEX HOMEOSTASIS AND RESPONSE TO PROGESTERONE SIGNALING

Final height (FH) in congenital adrenal hyperplasia (CAH) patients is frequently under -1.5 SDS in relation to the normal population, even in those with good compliance, chronic androgen exposure or supraphysiological glucocorticoid doses could corroborate to this finding. Recombinant human growth hormone (GH) has been used as adjuvant therapy to improve FH in many disorders, but there are scarce data in CAH patients. Objective: to analyze the effect of GH therapy on final height of CAH patients with compromised height prediction. Patients and Methods: data of 26 patients (7F/19M) were retrospectively evaluated: 10 salt wasting-SW, 11 simple virilizing-SV and 5 with nonclassical form-NC. The SW patients were diagnosed at the neonatal period, SV and NC patients at mean chronological age of 3.6±2 and 7.2±3.8 yrs, respectively. All patients received cortisone acetate, hydrocortisone equivalent dose of 12.6±6.6 mg/m²/day in classical and 8.24±4.7 mg/m²/day in NC form; SW patients also received fludrocortisone 36±9.6mcg/m²/day. Near final height (NFH) was considered as BA of 13yrs for girls and 15yrs for boys; height and target height (TH) were described as SDS. All patients presented hormonal/molecular CAH diagnosis. The t test was used in the statistical analysis. Results: Mean chronological and bone age (BA) at the beginning of GH therapy was 9.0±2.4 and 12.3±2yrs, respectively, mean duration of GH therapy was 4.1±1.9yrs and daily dose was 0.15 UI/kg/day. All patients were submitted to pubertal blockage with leuprorelin acetate 11.25 mg each 3 months during 3.9±1.1yrs or cyproterone acetate, mean dose 70±19 mg/m²/d during 4.7±2.5yrs. At the last evaluation, 19 patients (14M/5F) achieved FH: at the beginning of GH therapy mean H/BA in males was -2.1±0.38 and after -0.65±1 SDS (p<0.001). Mean FH in males was 165.7±5.9 cm and mean TH 170.7±8 cm (p>0.05); the TH-FH was -0.6±1.2 SDS. Among females, mean H/BA at the beginning of GH therapy was -2.2±0.7 and after -0.84±0.9 SDS (p=0.01). Mean FH in females was 156±8 cm, mean TH was 160±5.8cm (p>0.05); the TH-FH was -0.6±0.7 SDS. Considering patients with FH and NFH, the FH-TH in males was 0.4±0.2 SDS and -0.15±0.9 SDS in females. An improvement of approximately 12.7cm in males and 12.4cm in females was observed. No patient presented hypertension and 7/26 patients presented insulin resistance, which normalized after GH withdrawal. Conclusion: we observed that GH therapy associated with puberty blockers allowed the achievement of final height in the expected range of target height, without significant side effects.

Improvement of final height in congenital adrenal hyperplasia patients treated with recombinant growth hormone

Background: HSD-1, an intracellular enzyme, converts cortisone to cortisol in target tissues in which cortisol excess is associated with morbidity including liver, adipose, bone, brain, muscle, skin, and eye. SPI-62 is a potent HSD-1 inhibitor in Phase 2 development for treatment of Cushing's syndrome and autonomous cortisol secretion, and as adjunctive therapy to prednisolone in polymyalgia rheumatica. In Phase 1 clinical trials SPI-62 was generally well tolerated and associated with maximal liver and brain HSD-1 inhibition.
Methods: We analyzed data from a multiple ascending dose trial (1) to characterize the contribution of HSD-1 to intracellular cortisol that can bind to intracellular receptors as well as HPA and HPG axis modulation by SPI-62. Data from all non-elderly subjects who received 10-50 mg SPI-62 (doses that achieved maximal liver HSD-1 inhibition) were combined for analysis. ANCOVA models with a treatment effect and baseline covariate were conducted; reported statistics are least squares means and standard errors.
Results: Compared to placebo (n=10), single doses of SPI-62 (n=40) were associated with 24-hour urinary tetrahydrocortisol (2.27[0.134] v 4.44[0.269] mmol) and allotetrahydrocortisol (2.98[0.146] v 4.80[0.291] mmol) decreases, and 24-hour urinary tetrahydrocortisone (32.71[1.149] v 9.19[2.300] mmol) increase. Serum cortisol was decreased at 2-hours (152.2[11.64] v 226.6[24.00] nM) but not at 4- or 12-hours post-dose. ACTH was increased at 4- and 12-hours (45.6[1.64] v 32.1[3.29]; 38.5[1.67] v 26.3[3.33] pg/mL) but not at 2-hours post-dose. After 14 daily doses, SPI-62 was associated with 24-hour urinary tetrahydrocortisol (2.05[0.154] v 4.36[0.321] mmol), allotetrahydrocortisol (2.75[0.181] v 4.13[0.377] mmol), and tetrahydrocortisone (42.73[1.968] v 8.51[0.410] mmol) changes. ACTH was increased at pre-dose and 2-, 4-, and 12-hours post-dose (47.3[2.13] v 30.4[4.45]; 45.6[1.71] v 27.0[3.61]; 45.3[1.79] v 35.2[3.72]; 34.4[1.71] v 18.8[3.57] pg/mL). No differences on urinary cortisol or cortisone, serum cortisone, or CRH were observed after single or multiple doses. After multiple doses, SPI-62 was associated with increased DHEA-S (342.2[9.94] v 155.0[20.52] mg/dL) and, in females, testosterone (2.1[0.11] v 1.4[0.25] nM). No differences on aldosterone, estradiol, FSH, LH, progesterone, or SHBG were observed.
Discussion: Single SPI-62 doses resulted in ~40-50% decreases of urinary cortisol metabolites which indicate similar decrease of hepatocellular cortisol. Following a corresponding decrease, circulating cortisol homeostasis was restored rapidly by ACTH increase. Urinary cortisol was not affected. As other HSD-1 inhibitors, SPI-62 is associated with moderate androgen increases that, to date, appear not to be associated with adverse effects. As HSD-1 contributes much of the intracellular cortisol that can access intracellular receptors in target tissues for cortisol morbidity, we hypothesize that HSD-1 inhibitors have potential as treatments for conditions of cortisol excess such as Cushing's syndrome and autonomous cortisol secretion.
Reference: (1) Bellaire et al., Clin. Transl. Sci. 2019 May;12(3): 291-301.

HPA Axis Modulation by a Potent Inhibitor Indicates 11Beta-Hydroxysteroid Dehydrogenase Type 1 (HSD-1) Is a Significant Contributor to Cortisol Levels

The Sonic Hedgehog (Shh) gene expressed in the subcapsular cortical region of the adrenal gland has been found to play a role in adrenal gland development. The Shh(+) cell population at the fetal stages contributes to different cortical layers in the adrenal gland. However, the (1) capability of these cells after weaning and (2) how soon they can renew the adrenal cortex in the postnatal stages is not fully understood. Here, we conducted a lineage tracing experiment to track Shh(+) cells and cells descended from them in post-weaning mice to ultimately better understand the processes of adrenal cortex renewal and remodeling over time in young adult mice.
  This experiment used the NuTRAP; Shh-Cre-ERT2 mice as the Shh-reporter mouse model. This tamoxifen-inducible mouse model allows us to specifically target and label Shh-expressing cells and all descendant cells with green fluorescence. Tamoxifen was given at postnatal days (P) 22, P24, and P26 to enable the Cre recombinase activity driven by the Shh promoter. Adrenal glands were then analyzed after two and four months. This lineage tracing experiment found that Shh(+) cells and their descendant cells reached the margin of the Cyp2f2(+) cortical zone in 2 months and the cortical-medullary boundary in 4 months. This finding indicates that the Shh(+) cell population in post-weaning mice can proliferate, differentiate, and eventually renew the entire adrenal cortex over a four-month period of time.
  Understanding the adrenal cortex's renewal rate helps us design our follow-up study to test the capability of Shh(+) cells in adult mice at different ages and how their potency changes overtime at the 'omic' level. Because this NuTRAP;Shh-Cre-ERT2 mouse model also allows us to isolate cell-type-specific DNA/RNA, we can further decipher the underlying gene/pathways which control this progenitor cell population of the adrenal gland cortex.

Lineage Tracing Sonic Hedgehog-Expressing Cells in Adrenal Glands in Post-Weaning Mice

Background: Within a spectrum of cancers implicated by VHL germline pathogenic variants, one rare cancer which may present in patients are pheochromocytomas (PHEOs). These tumors occur in the adrenal glands and predispose patients to a variety of morbid clinical symptoms due to the excess secretion of catecholamines. As a part of these patients' management, imaging is important to localize the tumors of which there are both morphologic (CT and MRI) and molecular (PET/CT with ¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA) imaging modalities.
Purpose: To determine which imaging modalities provided the optimal detection of VHL-related PHEO.
Methods: The study compared detection of 19 PHEOs (18 histologically proven and 1 is a contralateral tumor awaiting resection) in 12 patients. The CT and MRI were evaluated by a body radiologist and the PET/CT of¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA were evaluated by a nuclear medicine physician in a blinded fashion. Both experts graded using a five-point Likert scale (1 – not PHEO and 5 – PHEO). For analysis, the Likert scale was dichotomized with 1-3 designating negative for PHEO and 4-5 positive for PHEO. The findings were reported as detection rates of positive PHEOs found on imaging to the gold standard of pathologically (aside from one contralateral PHEO awaiting surgery) proven PHEOs. The McNemar test was performed to compare the statistical relevance of detection rates between histology and the imaging modalities. Two-sided p values <0.05 were considered significant.
Results: The detection rates were CT 17/19 [89.5%, 95% CI = 66.9%-98.7%], MRI 17/17 [100%, 95% CI = 80.5%-100%], ¹⁸F-FDG PET/CT 16/17 [94.1%, 95% CI = 80.5%-100%], 68Ga-DOTATATE 5/9 [55.6%, 95% CI = 21.2%-86.3%], ¹⁸F-DOPA 12/14 [85.7%, 95% CI = 57.2%-98.2%], and ¹⁸F-DA 4/6 [66.7%, 95% CI = 22.3%–95.7%]. All McNemar tests showed p-values greater than 0.05, which were not statistically significant.
Conclusion: The study was performed in a small patient set with VHL-related PHEO showed MRI, with the highest detection percent followed by ¹⁸F-FDG PET/CT. More trials with a larger patient set are needed to support the statistical relevance as it was not seen in this study. Furthermore, performing a study on a patient cohort with additional VHL associated cancers could prove clinically beneficial for patients.

Morphologic or Molecular Imaging - Diagnostic Capabilities in von Hippel-Lindau related pheochromocytoma

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.

Epigenetic Programs Stabilize a Differentiated Cell State Required for Sustained Proliferation in Adrenocortical Carcinoma

Bisphenol A (BPA) is known to produce adverse effects on fertility, cardiovascular, and metabolic health. The constraints placed on BPA production led companies to switch to BPA's structural analogs, Bisphenol S (BPS) and Bisphenol F (BPF). These chemicals are used in various equipment, food and drink cans, and paper, permitting widespread exposure. We recently found that prenatal exposure to low levels of these chemicals can increase blood pressure in the offspring.  However, the underlying neurobiological mechanism is unclear. We hypothesized that these chemicals could alter the levels of monoamines, specifically in brain regions that regulate cardiovascular function such as the paraventricular nucleus (PVN) of the hypothalamus. Pregnant Sprague Dawley rats were exposed to saline, 5 µg/kg BW of BPA or BPS, or 1 µg/kg BW of BPF from gestation day 6 to 21. Their adult Male and Female offspring were implanted with a radiotelemetry device to measure blood pressure and heart rate levels. Following telemetry, animals were sacrificed, and the brains were collected, frozen and sectioned. The PVN was microdissected, and analyzed for monoamine levels using HPLC-EC. Males exposed to BPA had significant increases in the levels of serotonin (5HT) in the PVN compared to the control group, while a marked increase in 5HT levels were only observed in females exposed to BPF and not BPA (p = 0.0002). No significant changes were observed in Dopamine (DA) or norepinephrine (NE) levels. There were significant sex differences (p<0.0001) and treatment effects (p=0.0003) in the DOPAC/DA turnover ratio with males exposed to BPA, BPS, and BPF having markedly less turnover rates compared to control. No changes in DOPAC/DA turnover was apparent in females. There were significant sex (p<0.0001) and treatment effects (p<0.0001) in the 5HIAA/5HT turnover ratio, with males in the BPA and BPS groups having significantly less turnover compared to controls. In contrast, Females exposed to BPS and BPF had significantly lower 5HIAA/5HT turnover ratios but higher turnover ratios in the groups exposed to BPA. These results indicate that 5-HT production increases markedly in males exposed to BPA and in females exposed to BPF.  This is associated with reduced metabolism of 5-HT.  This suggests that the significant increases in 5-HT activity in the PVN could possibly contribute to the development of hypertension in these animals.

Unravelling the Role of CDK7-mediated SF1 Serine 203 Phosphorylation in Adrenal Homeostasis

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