United States

Evidence from nonclinical studies suggests a role of cannabinoid-1 receptor (CB1) in the development of diabetic nephropathy (DN). Kidney CB1 expression is upregulated in podocytes and tubular cells in murine models of obesity and diabetes mellitus (DM). Inhibition by CB1 inverse agonists has been shown to ameliorate diabetes-induced albuminuria, inhibit kidney fibrosis and inflammation, and prevent podocyte dysfunction. Mouse models of DM have shown that genetic deletion of CB1 in podocytes or proximal tubular cells protects against glomerular and tubular dysfunction.
GFB-024 is a recombinant humanized monoclonal antibody functioning as a CB1-specific inverse agonist. Pharmacodynamic studies demonstrated that GFB-024 protects human podocytes in vitro. Targeting the CB1 pathway with GFB-024 is a potentially novel approach to protecting podocytes from injury in patients with CB1-associated DN.
In a phase 1, randomized, double-blind, placebo-controlled single ascending dose (SAD) trial, we evaluated the safety, tolerability, pharmacokinetics (PK), and immunogenicity of GFB-024 in up to 56 healthy overweight and obese (BMI 25 – 40 kg/m2) participants.
A total of 38 subjects have completed 5 cohorts in the SAD, receiving single doses of 5 mg to 300 mg administered subcutaneously. GFB-024 was well tolerated, and there were no dose-limiting adverse effects.
PK exposures achieved in the trial exceeded the estimated efficacious exposure, based on four different in vitro activity assays. Absorption was prolonged with flat exposure over the first 7 days, with a Tmax ranging from approximately 4-7 days. Elimination half-life was approximately 12-13 days, with exposures remaining in the estimated efficacious range at 28 days for all but the lowest dose tested.
In summary, GFB-024 was safe and well tolerated following single doses of up to 300 mg. PK analysis demonstrated potentially efficacious exposure, a prolonged absorption phase, and elimination half-life compatible with once-monthly dosing.

ENDO 2022

Phase 1, Randomized, Controlled Trial of GFB-024, a Once-Monthly CB1 Inverse Agonist, in Healthy Overweight and Obese Participants and in Participants with Type 2 Diabetes Mellitus

**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?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

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. 

technical paper

IntroductionAdherence to lifestyle modifications can prevent diabetes; however, this poses a challenge for most individuals. CGM is an effective tool for glycemic control and lifestyle modifications in patients with diabetes on insulin therapy. We explored the impact of CGM use in individuals with prediabetes.HypothesisIndividuals using CGM in addition to diabetes education will show improved adherence to lifestyle modifications and greater reduction of HbA1c as compared to those receiving diabetes education alone.Design57 individuals with prediabetes were randomized to use CGM for 3 months along with diabetes education (CGM group) or receive diabetes education alone (EDU group). Food frequency (UKDDQ) and physical activity (IPAQ) questionnaires were completed by participants at the beginning and at the end of the study (6 months). Primary outcomes were lifestyle modifications and changes in HbA1c. Secondary outcomes were changes in weight and blood pressure.ResultsBoth groups reported an increase in healthy food choices; however, EDU group performed better (EDU: 12.4 vs 9.2, p<0.01, CGM: 10.6 vs 9.5, p 0.08). Unhealthy food choices were reduced in both groups. CGM group reported a greater increase in moderate physical activity levels (CGM: 230 vs 61 minutes/week, p 0.01, EDU: 78.5 vs 48 minutes/week, p 0.22). Vigorous physical activity was reported by more individuals in CGM group than EDU (35% vs 4%). Greater reduction in mean HbA1c was noted in CGM group (5.7 vs 5.9%, p 0.05) compared to EDU (5.8 vs 5.9%, p 0.01). 33% of participants in both groups had normalization of HbA1c at the end of study. CGM group had greater reduction in weight (3.5 vs 0.8 lb, p 0.1) and systolic BP (12 vs 2.4 mmHg, p 0.01) than EDU. Irrespective of group assignment, the study population had improvement in all outcomes despite the beginning of COVID-19 pandemic during 4th month of the study. Smaller number of participants in the CGM group reported negative impact of the pandemic on their lifestyle modifications than EDU (Diet: 68% vs 80%, Physical activity: 48% vs 64%). Majority of CGM group participants were able to maintain lifestyle changes after discontinuing CGM use (Diet: 72%, Physical activity: 84%). On comparing both interventions, 56% of CGM group participants felt CGM to be more helpful than education for lifestyle modifications, while 40% felt both interventions helped equally.ConclusionsCGM use in addition to diabetes education resulted in improvement in physical activity, food choices, HbA1c, weight, and BP in individuals with prediabetes. The significance of diabetes education was noted by greater improvement in food choices in EDU group. CGM can be an effective tool for diabetes prevention with lifestyle modifications in prediabetes. The long-term impact of CGM use and cost-benefit analysis in high-risk patients needs to be explored further.

Impact of Continuous Glucose Monitoring (CGM) on Lifestyle Modifications in Individuals with Prediabetes

Post-bariatric hypoglycemia (PBH) is a rare disease characterized by recurrent episodes of severe, symptomatic hyperinsulinemic hypoglycemia (HH). The predominant form occurs after Roux-en-Y gastric bypass (RYGB). However, severe cases also present after other upper-gastrointestinal procedures. The mechanisms mediating PBH have long been debated, though a critical role for excessive postprandial secretion of glucagon-like peptide-1 (GLP-1) has been established. Avexitide (exendin 9-39) is a first-in-class GLP-1 receptor antagonist in development for treatment of PBH. Prior studies with avexitide administration have shown normalization of postprandial insulin concentrations and reductions in the occurrence of symptomatic hypoglycemia. In the Phase 2 multicenter PREVENT trial, avexitide administered to post-RYGB patients at a total daily dose of 60 mg [30mg twice daily (BID) or 60mg once daily (QD)] for 28 days was well-tolerated and demonstrated significant reductions in the rates of hypoglycemia, symptoms, and percent time in hypoglycemia. Based on the favorable safety and tolerability profile and dose-response relationship observed, we sought to evaluate whether higher doses of avexitide may further enhance the pharmacodynamic response. In addition, we sought to extend the evaluation to patients with HH after vertical sleeve gastrectomy, esophagectomy, gastrectomy or Nissen fundoplication. In this Phase 2b, open label, cross over study (NCT04652479), eligible patients included males and females (>18 years of age; BMI ≤40 kg/m²) with a history of recurrent hypoglycemia refractory to medical nutrition therapy (MNT) and exhibiting at least two severe hypoglycemia events while adhering to MNT over a 14-day period. Metabolic and symptomatic parameters were assessed by self-monitoring of blood glucose (SMBG), e-Diary, and blinded continuous glucose monitor (CGM) during 14 days of MNT as compared to 14 days of MNT + avexitide administered by subcutaneous injection at a dose of 45mg BID and 90mg QD in crossover design and random order, for a total of 28-days of treatment. An interim analysis (mixed-effect model) at ~50% completion was conducted (n=8). The primary and secondary endpoints were met with statistical significance. Compared with MNT alone, avexitide 45mg BID and 90mg QD reduced the rate of Level 1 hypoglycemia (SMBG<70 mg/dL) by 66% (p=0.022) and 74% (p=0.010); Level 2 hypoglycemia (SMBG <54 mg/dL) by 70% (p=0.003) and 93% (p=0.001); and Level 3 hypoglycemia (severe event characterized by altered mental and/or physical function requiring assistance) by 77% (p=0.002) and 93% (p=0.001), respectively. Objective assessment by blinded CGM corroborated SMBG/e-Diary results, demonstrating significant reductions in the rates of hypoglycemia and improvements in percent time in hypoglycemia. Responses were comparable across surgical subtypes. Greater improvements were consistently observed with once daily (90 mg QD) than twice daily (45 mg BID) dosing and exceeded those reported in the PREVENT trial. Avexitide was well-tolerated, with no serious adverse events. Complete study results will be presented.

Efficacy and Safety of Avexitide for Treatment of Hypoglycemia after Gastrointestinal Surgery; Assessment of Novel Dosing Regimens in an Expanded Indication

Current markers of β-cell function poorly reflect glucose homeostasis after diabetes onset, mainly because of the lack of integration of parameters of insulin sensitivity and β-cell responsiveness to glucose. Recently, the widespread use of continuous glucose monitoring (CGM) helped stratify glucose control in patients with long-term diabetes in new clinically relevant glucotypes. In our DIATAG study, we investigated how CGM metrics may help to segregate patients with new-onset type 1 diabetes (T1D) entering or not in partial remission (PR). We collected data from 66 pediatric patients with T1D (10.4±4.7 yo) during the first year after diabetes onset (Δ). Clinical parameters (i.e. A1C, insulin daily dose, insulin-dose adjusted A1c [IDAA1C]) and CGM data were collected at Δ+3, +6, +9, +12 months (n=168, 57% remitters [IDAA1C <9]). A panel of 46 CGM metrics was calculated on an hourly and daily basis using iglu package (1). Using unsupervised hierarchical clustering based on daily CGM metrics and clinical parameters, we identified four clusters of glucose metrics that differed from each other (p<0.05). Cluster 1 was characterized by an increased glucose stability (coefficient of variation [CV] 32±5%) within time in target (TIT [63-140 mg/dL], 83±6%) with 9% of sensor values in 63-70 mg/dL range in the early morning period. Cluster 2 exhibited a progressive decrease of TIT (60±14%) and an increase of target above range (TAR [>180 mg/dL], 20±9%) especially during the day while target in range (TIR [70-180 mg/dL]) was nearly equivalent to Cluster 1 (80±8%). Notably, CV remained low (32±5%) with little time spent below range (TBR [<70 mg/dL], 5±2%). Cluster 3 demonstrated a net increase of CV during all the nycthemere (46±7%) concomitantly to an increase of TBR (14±7%). Interestingly, TIT and TAR remained close to values from Cluster 2 (55±11%; 23±10%). Finally, Cluster 4 demonstrated major hyperglycemias (TAR, 55±13%) with increased CV (47±9%) and decreased TBR (8±6%). Interestingly, clustering allowed clinically relevant segregation of intermediate values of IDAA1C (i.e. 7.5-10) between Cluster 2 and Cluster 3, independently of the remission status. Moreover, participants at Δ+3 months distributed across all clusters (with respectively 38% in Cluster 1, 26% in Cluster 2, 29% in Cluster 3 and 7% in Cluster 4) highlighting the usefulness of CGM in characterizing disease heterogeneity from diabetes onset. In our study, a combination of CGM metrics and clinical parameters unraveled key clinical milestones of glucose homeostasis and remission status during the first year of T1D. 1. Broll et al, 2021;16(4): e0248560.

Identification of Four New Glucotypes during the First Year after Type 1 Diabetes Onset Using Continuous Glucose Monitoring Metrics

VC-02 combination product (PEC‑Direct) is a clinical-stage regenerative medicine therapy being developed for patients with type 1 diabetes and hypoglycemia unawareness intended to restore homeostatic glycemic control.  It is comprised of stem cell-derived pancreatic progenitors (PEC‑01) loaded into a macroencapsulation device that facilitates the in-growth of blood vessels into the device lumen, supporting the maturation and differentiation of the graft into hormone-positive islet cells comprising glucose‑responsive beta cells. Initial data from one patient (2021, Keymeulen) demonstrated clinically-relevant levels of stimulated C-peptide and corresponding improvements in glycemic control within six months after subcutaneous implantation. These improvements now include a time-in-range consistently >90% and a persistent HbA1C <7%.Enrollment of additional patients with negligible C‑peptide levels in the ongoing VC02‑101 study implanted with the same device configuration has now resulted in multiple patients demonstrating C-peptide levels ≥0.3 ng/mL.  All patients are challenged with a mixed-meal tolerance test at baseline and post‑implantation to detect and monitor increases in plasma C‑peptide levels.  In some cases, the increased C‑peptide levels are observed to occur as soon as two months post‑implant with subsequent increases resulting as the patients progress further into the treatment period as high as 0.4 ng/mL by five months with additional time points at longer periods of exposure pending.  This progressive rise in beta cell function is consistent with preclinical findings showing time-dependent differentiation of the pancreatic progenitors into beta cells and subsequent increases in beta cell mass.The increases in plasma C‑peptide levels also correlate with trends toward improved glycemic control parameters.  Relative to baseline levels, improvements are observed in time‑in‑range data captured from continuous glucose monitoring, as well as substantial decreases in HbA1C by as much as 1.5%.  These positive trends also correlate with decreases in the amount of exogenous insulin required by as much as 70%.  Importantly, these data and observations have been obtained at separate clinical sites involving different surgeons, indicating that the achievement of functional engraftment of PEC‑Direct is reproducible with proper product handling, surgical training, and patient compliance with an adjunctive immunosuppression regimen.  These data provide further proof‑of‑concept that continued optimization of PEC‑Direct can result in a functional cure for T1D.

Further Clinical Evaluation of Stem Cell-Derived Islet Replacement Therapy (VC-02) Demonstrates Production of C-Peptide with Improvements in Glycemic Control Parameters in Multiple Patients with Type 1 Diabetes and Hypoglycemia Unawareness

The adrenal cortex and gonads are the two major endocrine hubs that participate in the hormonal regulation of vital aspects of mammalian homeostasis. Accordingly, their developmental aberrancies drive various congenital and adult-onset diseases. Lineage tracing studies in mice reveal that the adrenal cortex and gonads originate from a common progenitor, the adrenogonadal primordium. However, the specification pathways of the adrenal cortex and gonads and their lineage relationship remain poorly understood in humans. Here, we conduct the high resolution spatial and temporal lineage trajectory mapping of early human adrenal and gonadal lineages using single cell transcriptomics and histologic profiling on human embryos at 3-8 week post fertilization. We find that in humans, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to-mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages challenges the paradigm established in rodents and present evidence that the specification programs directing the formation of adrenal glands and gonads differ between rodents and humans. The molecular details of early lineage diversification of human adrenals and gonads will serve as both a framework for understanding the molecular pathology of human disorders.

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

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.

WNT2B Deficiency Results in Hyperreninemic Hypoaldosteronism in Humans and Mice

Introduction: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by bilateral adrenal macronodules responsible for adrenal Cushing. To date, two genetic causes of PBMAH are known: germline inactivating variants of the tumor suppressor genes ARMC5 identified in 2013 (Assié, N Eng J Med 2013), responsible for 20 to 25% of index cases, and KDM1A, identified recently (Vaczlavik, GIM 2021; Chasseloup, Lancet D&E 2021), responsible for the rare presentation associated with food-dependent Cushing's syndrome (FDCS) due to aberrant expression of the GIP receptor (GIPR) in adrenocortical cells. Multiple other illegitimate receptors are known to be responsible for abnormal cortisol response to various physiological stimuli in PBMAH. A recent multiomic analysis, identified three distinct molecular PBMAH groups: G1 with ARMC5-mutated tumors, G2 with KDM1A-mutated tumors from FDCS patients, and G3 with no identified genetic cause at present. We aimed to identify specific expression profiles of illegitimate receptors in the three groups.
Methods: Based on the transcriptome data obtained by RNA sequencing (Illumina) of the tumors from 31 patients (G1/ARMC5, 16 patients; G2/KDM1A, 6 patients; G3, 9 patients), expression of the following genes, encoding potential illegitimate receptors, were compared: ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AVPR1A, AVPR1B, AVPR2, GCGR, GIPR, HTR4, HTR7, LHCGR. Calculations were performed using R statistical software. The Bioconductor limma package was used to analyze mRNA differential expression.
Results: G1/ARMC5 tumors showed a relative overexpression of the vasopressin receptors AVPR1A and AVPR1B compared to the two other groups (fold-change [FC] =7.39, p<0.001 and 3.98, p<0.001, respectively) but a lower expression of AVPR2 (FC=0.43, p=0.015). G2/KDM1A tumors showed a dramatic overexpression of GIPR compared to the two other groups (FC=105.02, p<0.001) but also of the adrenergic receptors ADRA1D and ADRA2A (FC=2.93, p=0.027 and 9.99, p<0.001, respectively) and of the LH/hCG receptor (LHCGR) (FC=12.20, p<0.001). G3 tumors showed a slight overexpression of the adrenergic receptor ADRA1B (FC=3.49, p=0.001) and in few tumors ADRA1D, AVPR2 and LHCGR were highly expressed suggesting molecular heterogeneity in G3.
Conclusion: This study reveals specific expression profiles of illegitimate receptors related to the three molecular groups. ARMC5 tumors are associated with the overexpression of two vasopressin receptors, while, besides GIPR, KDM1A inactivation seems to drive the overexpression of the LH/hCG receptor, as previously suggested in patients with FDCS (Bertherat, JCE&M 2005), potentially responsible for Cushing's syndrome associated with pregnancy and menopause. These molecular patterns need to be corroborated by clinical data with a systematic testing of the aberrant cortisol responses. Additionally, further studies would be needed to investigate the clinical relevance and significance of moderate fold-changes in gene expression (e.g. <4).

Genetic Alterations of ARMC5 and KDM1A Are Associated With Different Expression Profiles of Illegitimate Receptors in Primary Bilateral Macronodular Adrenal Hyperplasia

Context: Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas.
Methods: We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France.
Results: 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss.
Discussion: We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors.

Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Adrenocortical carcinoma (ACC) is a rare aggressive cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. Here, we investigated the role of circulating cell-free DNA (ccfDNA)-related biomarkers for prognostication and monitoring of ACC.
We investigated 79 patients with ACC (29M/50F, 52±14yrs; 35 primary tumors [ACC-P] and 44 recurrences [ACC-R]); while 27 patients with adrenocortical adenomas [ACA] (9M/18F, 56±16yrs) and 19 healthy subjects (HS; 9M/10F, 37±9yrs) served as controls.
We extracted ccfDNA from 1-4 ml EDTA-plasma using Nonacus Cell3 Xtract or Qiagen QIAamp MinElute kit and quantified by fluorimeter (ccfDNA concentrations, Biomarker 1). Targeted next-generation sequencing (Illumina NextSeq500) was performed in a first subgroup of 52 baseline ccfDNA samples (23 ACC-P, 20 ACC-R, 8 ACA) using a customised panel of 30 ACC-specific genes (Cell3 Target Nonacus). Leucocyte DNA was sequenced to discriminate germline from somatic variants (Biomarker 2). Sequencing data from matched tumor DNA were available for 28 ACC (20 ACC-P, 8 R-ACC). A combined Biomarker score was calculated for prediction of clinical outcome. ACC-P had the highest ccfDNA concentrations (mean±SD 1.08±1.50 ng/µl) compared to ACC-R (0.29±0.23 ng/µl, P<0.05), ACA (0.17±0.13 ng/µl, P<0.005) and HS (0.11±0.07 ng/µl, P<0.005). Using a cutoff of 0.204 (median HS+2SD), 68% of ACC-P were postitive for Biomarker 1 (vs. ACC-R 55%, ACA 28%, HS 5%; P<0.0001 by Chi square test). At ccfDNA sequencing, 43% of ACC-P showed at least one somatic mutation (=positive Biomarker 2), vs. 15% in ACC-R and 0% in ACA. Mutational status at ccfDNA level matched with tumor DNA in 80% of cases. In 23 ACC-P with available sequencing data, the combined Biomarker score was strongly associated with both progression-free and overall survival (P<0.0001, HR 8.56, 95%CI 2.55-58.7, and P=0.0008, HR 13.3, 95%CI 3.77-47.1, respectively).
10 ACC-P were followed up for at least 6 months: 6 patients tumor-free at last CT scan were negative for Biomarker 1 whereas 3 out of 4 patients with early disease relapse were positive. In two recurrent cases, somatic mutations in ACC driver genes, i.e. MEN1 and ZNRF3, observed at baseline in both tumour and ccfDNA, remained detectable during monitoring.
In conclusion, ccfDNA-related biomarkers are frequently detected in patients with primary ACC. They may represent a promising, non-invasive tool to predict early disease progression and complement imaging in disease surveillance. Our findings on ccfDNA-based liquid biopsy will be validated in a larger cohort of ACCs with long-term follow up.

Circulating cell-free DNA-based biomarkers for prognostication and disease surveillance in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an aggressive cancer with a significant risk of recurrence and high mortality rates. ACC has limited response to currently used treatments and there is an unmet clinical need to identify new treatment options.  Disease rarity coupled with absence of preclinical models of disease heterogeneity contributed to limited therapeutic progress in ACC. We previously established new in vitro and in vivo ACC models (CUACC1, CUACC2, CUACC9) with variable genomic alterations commonly seen in ACC tumors. Prior work demonstrated that maternal leucine embryonic kinase (MELK) is an oncogenic kinase in ACC, and that it can be targeted with the MELK inhibitor OTSSP167. To further evaluate the anti-tumor efficacy and mode of action of OTSSP167, this study used genetically diverse ACC cell lines (CUACC1, CUACC2, H295R), patient derived xenografts (PDX) (CUACC1 and CUACC9) and a cell line xenograft model (H295R) of ACC. ACC PDXs treated with 10 mg/kg OTSSP167 had significantly reduced MELK expression, tumor growth inhibition of more than 80%, reduced mitosis, and increased tumor tissue necrosis. Transcriptomic and phospho-proteomic analysis at multiple treatment timepoints in in vitro models revealed that OTSSP167 induced wide transcriptomic and proteomic changes involving effectors of signal transduction, cell cycle, protein translation, and epigenetic regulation to exert its anti-tumorigenic effects. Evaluation of OTSSP167's role in protein synthesis via a puromycin based SUnSET (surface sensing of translation) and kinase assay established that OTSSP167 inhibits de-novo protein synthesis, and targets RSK1 and its substrate rpS6 (S235/S236). Varying the degree of MELK silencing in H295R cell lines using a doxycycline inducible shRNA construct, inhibited RSK1 phosphorylation at T573 and S380 indicating a MELK-RSK1 axis in ACC. Additionally, OTSSP167's effect on the DNA damage pathway was validated, which activated the G2/M checkpoint protein Wee1, increased inhibitory phosphorylation of CDK1, causing an accumulation of cyclin B1 and G2/M arrest independent of TP53 status. Combination treatment with the Wee1 inhibitor AZD1775 and OTSSP167 enhanced OTSSP167 mediated cytotoxic effects in in-vitro models with an increase in mitotic entry and caspase mediated apoptosis. In summary, OTSSP167 exerted its anti-tumorigenic effects via inhibition of the protein translation machinery, DNA damage and cell cycle dysregulation causing drug sensitization. Results from these studies are a first demonstration of the anti-cancer effects of OTSSP167 in sensitization to Wee1 inhibition in ACC. These data suggest that OTSSP167 is a potential therapeutic option for patients with ACC and could be combined with Wee1 inhibitors in a clinical trial using a synthetic lethal approach.

Phase 1, Randomized, Controlled Trial of GFB-024, a Once-Monthly CB1 Inverse Agonist, in Healthy Overweight and Obese Participants and in Participants with Type 2 Diabetes Mellitus

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