Corticotroph Cells Research Articles

8738 Unveiling Potential Therapeutic Candidates for ACTH-Silent Pituitary Neuroendocrine Tumors Through High Throughput Drug Screening

Abstract Disclosure: J.M. Marques: None. N.D. D' Alessandre: None. G.D. Guardia: None. F.L. Craveiro: None. C. Grutzmacher: None. G.O. Silva: None. B.B. Mendonca: None. L.R. Carvalho: None. F. Fattahi: None. P.A. Galante: None. R.L. Batista: None. Introduction: ACTH-silent pituitary neuroendocrine tumours (PitNETs) are a subtype of PitNETs that arise from corticotroph cells of the anterior pituitary gland. They can show positive immunoreactivity for adrenocorticotropic hormone (ACTH) but with no clinical or laboratory evidence of hypercortisolism. ACTH-silent PitNETs display notably more aggressive behavior and treatment resistance. Currently, there is no established pharmacological therapy for this specific tumor subtype. Aim: To use high throughput drug screening to identify compounds that modulate ACTH secretion and tumor progression in ACTH-silent PitNETs. Methods: WTC11 cells (GM25256) were differentiated into pituitary using E6 with SB431542 10µM, SHH 200ng/mL, FGF8 100ng/mL and FGF10 50ng/mL until day 30. Pituitary cells were passaged in 384 well plates and treated with FDA-approved compound library with 3113 drugs at 1μM for 72h (#L1300, Selleckchem). Cells were stained with ACTH and imaged on ImageXpress® HCS. ACTH intensity was normalized to total cell number, measured by DAPI, using MetaXpress Software and compounds z-score was calculated, considering -2.5 for analysis. Gene and pathway enrichment analysis were performed and PitNET RNAseq data, publicly available (GSE207937), was used to determine target drugs based on similar target pathways and gene expression. To validate hits, we collected tumor samples from a patient with an aggressive silent ACTH PitNET. PitNET was histologically confirmed and hypercortisolism was excluded clinically and laboratory. Samples were cultured on DMEN/F12 with 10% FBS and 1% antibiotic and pituitary hormones were measured at days 3, 6 and 8. Cells were treated with 1uM of Letrozole, Linsitinib and Raloxifene for 48h and sample for RNA and hormone dosage were collected. Results: We identified 8 drugs that decreased ACTH in induced pituitary cells and selected 3 of them for validation. PitNET was successfully cultured and treated with selected drugs and ACTH secretion was detected at all periods (972.1 pg/mL to 10490 pg/mL), gonadotropins were below the detected level. Letrozole significantly reduced ACTH in the PitNET culture at day 8 (600.9 ± 25.8pg/mL, p<0,001). ACTH was also decreased in Linsitinib treated cells (932 ± 12.7pg/mL, p<0.05), while Raloxifene did not change ACTH secretion (980 ± 95.1pg/mL, p=0.061). Microscopically, PitNET cells aggregate in spheres and this seems to be affected by letrozole treatment, not influencing cell viability. Conclusion: We performed a high throughput drug screen using induced pituitary cells that led to the discovery of 3 compounds that might be used as an alternative for PitNET treatment. Pathway and gene expression analysis from target drugs and PitNET RNAseq may help in the understanding of drug effect in the PitNET context and improve the understanding of tumorigenesis and pharmacological treatment. Presentation: 6/1/2024

Captopril inhibits the overproduction of proopiomelanocortin and adrenocorticotropic hormone in the pituitary gland of male diabetic mice in close relationship with an increase in glucocorticoid receptor expression.

Prior investigation shows that diabetic patients present hypothalamus-pituitary-adrenal (HPA) axis hyperactivity related to impaired negative feedback. This study investigates the effect of Captopril on the overproduction of adrenocorticotropic hormone (ACTH) and its precursor proopiomelanocortin (POMC) in the pituitary gland of male diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice, and the animals were treated with Captopril for 14 consecutive days, starting 7 days post-diabetes induction. Plasma corticosterone levels were evaluated by ELISA, while pituitary gland expressions of angiotensin-II type 1 receptor (AT1), angiotensin-II type 2 receptor (AT2), ACTH, Bax, Bcl-2, KI-67, POMC, and glucocorticoid receptor (GR) were evaluated using immunohistochemistry or western blot. Diabetic mice showed pituitary gland overexpression of AT1, without altering AT2 levels, which were sensitive to Captopril treatment. Furthermore, diabetic mice presented hypercortisolism, along with an increase in the number of corticotroph cells, POMC and ACTH expression, and number of proliferative cells, and a decrease of GR expression in the pituitary gland. In addition, treatment with Captopril reduced systemic corticosterone levels, corticotroph and proliferative cell numbers, and Bcl-2, POMC, and ACTH expression in the pituitary gland of diabetic mice, besides increasing the expression of Bax and GR. In conclusion, these findings show that Captopril is a promising therapy for treating complications associated with HPA axis hyperactivity in diabetic patients, in a mechanism probably related to the downregulation of POMC production in the pituitary gland and subsequent reduction of systemic corticosterone levels.

PCSK1N as a tumor size marker and an ER stress response protein in corticotroph pituitary adenomas.

Silent corticotroph adenoma (SCA) exhibits more tumor aggressiveness features than functioning adenomas (FCA). We aimed to investigate PCSK1N expression in CA and examine if ER stress-induced responses affect cell survival in a corticotroph tumor cell model. Clinical and imaging characteristics were recorded in 33 patients with FCA (20 women, 11 macroadenomas) and 18 SCA (8 women, all macroadenomas). Gene expression of proopiomelanocortin (POMC), T-box transcription factor 19(TBX19)/TPIT, proprotein convertase subtilisin/kexin type 1(PCSK1)/PC1/3, and its inhibitor PCSK1N, was measured by RT-qPCR in adenoma tissue.Mouse pituitary corticotroph tumor (AtT-20) cells were treated with tanespimycin (17-AAG), a HSP90 chaperone inhibitor, to induce ER stress, followed by gene and protein analyses. POMC, TPIT, and PCSK1 expression were higher, whereas PCSK1N was lower in FCA compared to SCA. PCSK1N correlated with POMC (rs= -0.514, p <0.001), TPIT (rs= -0.386, p = 0.005), PCSK1 (rs= -0.3691, p = 0.008), and tumor largest diameter (rs= 0.645, p <0.001), in all CA. Induction of ER stress by 17-AAG in AtT-20 cells led to a decrease of POMC and an increase of PCSK1N gene expression at 24h. Moreover, a downregulation of cell cycle, apoptosis, and senescence pathways, and alterations in cell adhesion and cytoskeleton were observed at the protein level. PCSK1N is higher in SCA compared with FCA, and associated with corticotroph cell markers and tumor size. PCSK1N is likely to be part of the adaptive response to ER stress, potentially conferring a survival advantage to the corticotroph tumor cell in conjunction with other proteins.

Bone Morphogenetic Protein Signaling Agonist SB4 (BMPSB4) Inhibits Corticotroph Pituitary Neuroendocrine Tumors by Activation of Autophagy via a BMP4/SMADs-Dependent Pathway.

Corticotroph pituitary neuroendocrine tumors (PitNETs), associated with Cushing's disease (CD), have limited treatment options other than surgical resection. Bone morphogenetic protein 4 (BMP4), a potential therapeutic target, is decreased in patients with CD. Previous studies have identified BMPSB4 as a potent agonist of the BMP4 signaling pathway. Here, we investigated the effect of BMPSB4 on the corticotroph PitNET cell line AtT20/D16v-F2 and explored the underlying mechanisms and therapeutic potential. We verified the low expression patterns of BMP4 and downstream p-SMAD1/5/9 in CD samples at the transcriptional and protein levels. In addition, BMPSB4 activated SMAD1/5/9 in a time- and concentration-dependent manner, with concomitant inhibitory effects on AtT20/D16v-F2 cells. Further RNA sequencing, transmission electron microscopy (TEM), and transfection with the mRFP-EGFP-LC3 adenoviral vector revealed that BMPSB4 induced cellular autophagy, which was the basis for the inhibitory effect of BMPSB4. Moreover, we demonstrated that autophagy induced by BMPSB4 was achieved through the SMADs-dependent pathway. In vivo, BMPSB4 inhibited tumor growth and significantly reduced adrenocorticotrophin (ACTH) and corticosterone (CORT) secretion, thereby alleviating the CD phenotype. In conclusion, this study identified BMPSB4 as an effective therapeutic agent for CD. BMPSB4 activates autophagy through a SMADs-dependent pathway, which in turn promotes autophagy-mediated cell death. Our work further elucidates the mechanism of the BMP4 signaling pathway in CD and suggests broad prospects for the development and application of BMPSB4 in CD therapy.

PIT-EASY survey: validation of the European Pituitary Pathology Group proposal for reporting pituitary neuroendocrine tumors.

The aim of this multicenter prospective survey called PIT-EASY was to assess the relevance of the European Pituitary Pathology Group (EPPG) diagnostic tools for pituitary neuroendocrine tumors (PitNETs) to improve the quality of their histological diagnosis. Each center performed at least 30 histological cases of PitNETs using the EPPG tools and assessed their value using a scorecard with 10 questions. For each center, the histological cases were carried out by pathologists with varying levels of expertise in pituitary pathology defined as junior, intermediate, and expert. Two hundred and ninety histological cases were collected from six French and Italian centers. The three EPPG tools were validated and regarded as helpful for a more accurate and time-efficient diagnosis. The usefulness of level 2 and level 3 of the "EPPG's multi-step approach for immunohistochemistry" including pituitary transcription factors (PIT1, TPIT, and SF1) and chromogranin, SSTRs, and P53 respectively was higher in "other non-functioning" (silent plurihormonal PIT1, silent corticotroph, and null cell): 88% vs 32%, p < 10-6 and 42% vs 14%, p = 0.002, respectively. The diagnostic algorithm proved more useful for junior pathologists (p = 0.0001) and those with intermediate experience. PIT-EASY survey confirmed the importance of a standardized approach to PitNETs for an accurate and reproducible diagnosis and served as validation of the EPPG proposal. The tool appeared to be of practical value to junior participants and staff with intermediate experience for safe routine diagnostic reporting.

Circulating levels of mir-375 in cushing's disease patients and evaluation of its role in the regulation of sstr2 expression in murine pituitary corticotroph tumor cell model

Circulating levels of mir-375 in cushing's disease patients and evaluation of its role in the regulation of sstr2 expression in murine pituitary corticotroph tumor cell model

ER stress causes variable ACTH production and secretion in corticotroph tumour cells

ER stress causes variable ACTH production and secretion in corticotroph tumour cells

Long-term exposure of corticotroph pituitary tumor cells to glucocorticoid results in resistance to its anti-proliferative effect

Long-term exposure of corticotroph pituitary tumor cells to glucocorticoid results in resistance to its anti-proliferative effect

18F]FET PET/MRI: An Accurate Technique for Detection of Small Functional Pituitary Tumors.

Small functional pituitary tumors can cause severely disabling symptoms and early death. The gold standard diagnostic approach includes laboratory tests and MRI, with or without inferior petrosal sinus sampling (IPSS). In up to 40% of patients, however, the source of excess hormone production remains unidentified or uncertain. This excludes patients from surgical, Gamma Knife, and CyberKnife therapy and adversely affects overall cure rates. We here assess the diagnostic yield of O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET) PET/MRI for detection of small functional pituitary tumors in these patients. Methods: This retrospective analysis included patients with Cushing disease (CD) but prior negative or inconclusive MRI results who underwent [18F]FET PET/MRI between February 1, 2021, and December 1, 2022. PET/MR images and MR images alone were evaluated by experienced nuclear radiologists, neuroradiologists, or radiologists. Postoperative tissue analysis (when performed) was used as a reference standard to assess diagnostic metrics (i.e., sensitivity and positive predictive value). Results were also compared with previously obtained MR images, preceding IPSS, and clinical or biochemical follow-up. Results: Twenty-two patients (68% female; mean age ± SD, 48 ± 15 y; range, 24-68 y) were scanned. All patients showed a clear metabolic focus on [18F]FET PET, whereas reading of the MRI alone yielded a suspected lesion in only 50%. Fifteen patients underwent surgery directed at the [18F]FET-positive focus. Tissue analysis confirmed a pituitary adenoma/pituitary neuroendocrine tumor of the corticotroph cell type (TPIT lineage) in 10 of 15 and a pituicytoma in 1 of 15, rendering a sensitivity of 100% and a positive predictive value of 73%. Lateralization was more accurate with [18F]FET PET/MRI than with IPSS in 33%. Twelve of 16 (75%) patients who received surgical, Gamma Knife, or CyberKnife therapy after [18F]FET PET/MRI reached short-term remission. Conclusion: [18F]FET PET/MRI shows a high diagnostic yield for localizing small functional pituitary tumors. This multimodal imaging technique provides a welcome improvement for diagnosis, planning of surgery, and clinical outcome in patients with Cushing disease, particularly those with repeated negative or inconclusive MRI results with or without IPSS.

CSIG-28. R17 INDUCES A PRO-ONCOGENIC PROTEIN HYPERPHOSPHORYLATION PROGRAM IN CUSHING’S DISEASE

Abstract OBJECTIVES to investigate molecular drivers of CD. METHODS We performed single-nucleus assays for transposase-accessible chromatin (snATACseq) and single-nucleus RNAseq (snRNAseq) in 4 CD adenomas including two adenoma-normal pairs. To assess proteins bound to the R17 promoter, we performed reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin and R17 promoter gRNAs followed by mass spectrometry (LC-MS). Mechanistic studies were performed in murine corticotroph cells (mCort) stably overexpressing R17 versus GFP. Using LC-MS, we quantified total and phospho-peptides from human CD adenomas versus adjacent normal pituitary glands (n = 3/group) and from mCort-R17 versus mCort-GFP cells (n = 5/group). RESULTS We found increase in R17 chromatin accessibility and gene expression in CD versus normal corticotrophs. rChIP identified R17 promoter enrichment of SUB1 and histone variant H1.4 indicating active remodeling. In CD specimens and in mCort-R17 cells, PP2A targets EGFR and ERK were hyperphosphorylated. LC-MS identified 30 peptides differentially phosphorylated in both CD adenomas and mCort-R17 cells. mCort-R17 cells demonstrated accelerated cell cycle progression with increased proliferation (P &amp;lt; 0.001). In mCort-R17 cells, the PP2A agonist Fingolimod partially rescued AKT phosphorylation and induced apoptosis. CONCLUSIONS R17 is activated in CD due to upstream chromatin remodeling events. Aberrant R17 signaling leads to protein hyperphosphorylation and cell cycle acceleration, which is partially reversible by the PP2A agonist Fingolimod. Fingolimod may represent a novel therapeutic option for patients with CD.

SAT618 Uncovering The Role Of O-glcnacylation In Pituitary Adenoma Pathogenesis: Insights From Corticotroph Tumors

Abstract Disclosure: L.J. Massman: None. R. Gonzalez: None. B. Laing: None. D. Coss: None. A.G. Ioachimescu: None. N. Zwagerman: None. S. Olivier-Van Stichelen: None. Functioning corticotroph pituitary adenomas are associated with high mortality, morbidity, and a high propensity of biochemical recurrence postoperatively. Silent corticotroph adenomas cause mass-effect manifestations and have an increased risk for tumor regrowth. The pathogenetic mechanisms responsible for the clinical course of corticotroph PA are poorly understood. In this study, we investigated the role of the O-GlcNAc posttranslational modification in their pathophysiology. O-GlcNAcylation is involved in protein signaling pathways and deregulated in cancers and diabetes. First, we performed RNA-sequencing in functioning and silent corticotroph and gonadotroph adenomas (n=25). We discovered that O-GlcNAc enzymes were upregulated in ACTH-secreting corticotroph tumors and correlated with larger, more invasive tumors. Also, an in vitro mechanistic analysis of corticotroph tumor cells (ATt-20) demonstrated that O-GlcNAcylation was essential for their proliferation and secretory ability. Specifically, O-GlcNAcylation affected Proopiomelanocortin mRNA splicing and stability, suggesting a new mechanism of fast hormonal response in corticotropes. Overall, our study indicates a key role for O-GlcNAcylation in corticotroph adenomas’ cellular proliferation and hypersecretion. Presentation: Saturday, June 17, 2023

OR01-02 Epigenomic Dysregulation Of PPP1R17 Drives The Cell Cycle In Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. S. Stoica: None. D. Mandal: None. D. Maric: None. A. Elkahloun: None. B. Sisay: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. Previously, with syngeneic, pairwise transcriptomic analysis, we found a robust upregulation of PPP1R17 in canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ CD adenomas. PPP1R17 is an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we investigated the epigenomic dysregulation of PPP1R17, its interaction with PP2A, and downstream effects on cell cycle regulation. Methods: We performed single nucleus assays for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs along with parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300). Fixed human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). Results: We mapped the chromatin accessibility landscape of CD adenomas compared to normal corticotrophs. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus exclusively within adenomatous corticotrophs. Using rChIP, we identified enrichment at the R17 promoter of the RNA polymerase II transcriptional coactivator p15 (SUB1) and the histone variant H1.4, indicating active chromatin remodeling. PPP1R17 protein and its target PP2A were overexpressed in the adenoma compartment but not in the adjacent normal gland. PLA confirmed robust interaction between R17 and PP2Ac within the CD adenoma compartment. Mechanistically, mCortR17 cells showed inhibition of PP2A with phosphorylation its known targets - EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P &amp;lt; 0.001) with increased proliferation (two-way ANOVA P &amp;lt; 0.001). PP2A agonists Fingolimod, DT061 and ABL127 led to proliferation arrest in mCortR17 cells. Conclusions: We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. PP2A agonists reversed the effects of PPP1R17 overexpression in-vitro. Our findings uncover a novel therapeutic phosphatase target for patients with CD. Presentation: Thursday, June 15, 2023

FRI324 Pituitary Macroadenoma With Carcinomatous Transformation And Multiple Cranial Masses And Spinal Leptomeningeal Carcinomatosis: A Case Report

Abstract Disclosure: C. Cristancho: None. G. Szabo: None. Background: Aggressive pituitary tumors are invasive tumors not responding to standard therapies and presenting with multiple local recurrences; if metastases occur, the tumors are defined as pituitary carcinoma (PC) Clinical Case: 51- year-old Hispanic woman with history of pituitary macroadenoma diagnosed at the age of 41, when presented with weight gain and decrease in visual acuity. Per records initial Brain MRI reported hyperintense sellar lesion of 2.1x2.3x2.6 cm with supra-sella extension and shift of optic chiasm. Patient had 4 pituitary surgeries between 2014-2018 due to growth and had radiation and Temozolomide between 2018-2020. Pathology was diffusely positive for ACTH, PRL, and Ki-67 was 10-20%, P 55 positive staining was 25-30%. As a result from pituitary surgeries patient developed central hypothyroidism, and central AI. Subsequent mornings cortisol were nl between 16-18. No UFC or LNSC were available. MEN1 negative. Brain MRIs done between 2019 and 2020 showed stable 13x8 mm residual pituitary mass with postoperative changes. Patient denied history of diabetes, fragility fractures, HTN, DVTs. She had regular menstrual periods until age of 42. Had left adrenal mass resection in 2019 with benign pathology results. During her initial endocrinology visit her ROS was positive for lower back pain and mild proximal muscle weakness. Patient denied visual changes, headaches, skin changes, polydipsia or polyuria. Physical exam was remarkable for small buffalo hump, and 5-/5 proximal muscle weakness. Negative for moon face, purple striae, acanthosis nigricans, hirsutism, acne. Initial tests were consistent with ACTH-dependent CS: elevated cortisol PM: 18.2 (n &amp;lt;10ug/dL) and LNSC 0.58 and 0.64 mcg/dl (n &amp;lt;0.09), 24-hr UFC 264 (n &amp;lt;50 mcg/dl), abnormal ACTH 150 (n &amp;lt;50 pg/ml).Brain MRI revealing extra-axial mass of 1.6 x1.1 cm overlying posterior aspect of right cerebellar hemisphere and focal lesion in left prepontine cistern of 0.8 cm. Leptomeningeal Enhancement. No sellar mass. Spine MRI showed scattered nodules lining the cord throughout the cervical and upper thoracic spine concerning for metastatic disease. Right cerebellar mass biopsy positive for metastatic pituitary neuroendocrine tumor corticotroph cell subtype, ACTH, chromogranin A, synaptophysin positive; PRL, TSH, GnRH, alpha subunit staining was negative; Ki-67 of 2%. Temozolomide, Capecitabin, and Ketoconazole 200 mg TID were started. Subsequent LNSC &amp;lt;0.03 and &amp;lt;0.03 mcg/dl, UFC at 3 different times were 12.8, 11.2 and 8.1 mcg/dl showing rapid decrease of cortisol levels in a 4-week period. A block-replace approach was decided with Dexamethasone 1 mg daily and a lower dose of Ketoconazole 200 mg BID. Patient reports no side effects. Presentation: Friday, June 16, 2023

THU108 Pituitary Carcinoma Diagnosed After Ten Years Of Smoldering Ectopic Nasopharyngeal Cushing’s

Abstract Disclosure: L. Carafone: None. E. Vates: None. M. Johnson: None. I. Shafiq: None. Introduction: Pituitary carcinomas are rare and typically carry a poor prognosis. Diagnosis of pituitary carcinoma is made when there is metastasis to one or more sites distant from the pituitary, including craniospinal or systemic metastases. Clinical Case: A 54-year-old man presented to the endocrine clinic in 2011 with recurrent urinary tract infections, thoracic compression fractures, uncontrolled hypertension with hypokalemia, and weight gain. Examination revealed facial plethora, supraclavicular fat pad, central obesity, multiple ecchymoses, proximal muscle weakness and muscle atrophy of the upper and lower extremities. Workup demonstrated abnormal 1 mg overnight dexamethasone suppression test (cortisol after 1 mg dexamethasone 25.7 mcg/dL, n≤1.8 mcg/dL), elevated salivary cortisol (4340 and 2950 ng/dL, n≤99 ng/dL), elevated 24 hr urinary-free cortisol (271.7 mcg/24hr, n≤60 mcg/24 hr), and elevated ACTH (240 pg/mL, n≤46 pg/mL). MRI brain showed a large, lobulated mass in the sphenoid sinus adjacent to the inferior left lateral aspect of the pituitary fossa. He underwent surgical resection of the mass and pathology was consistent with pituitary adenoma, corticotroph cell type. Following surgery, laboratory studies showed normal salivary cortisol (&amp;lt;50 ng/dL, n≤99 ng/dL) and 24 hr urinary-free cortisol (22.4 mcg/24hr, n≤60 mcg/24 hr). After two years of sustained remission, he experienced local recurrence for which he underwent repeat surgery with subsequent remission. One year later, this was again followed by recurrence for which he was treated with intensity-modulated radiation therapy and medical therapies including cabergoline, ketoconazole and eventually metyrapone. Six years after the second surgery, MRI showed increasing heterogeneous enhancement at the postoperative site, and he underwent a third surgery with tumor resection and the same pathology results as the initial resection. He was subsequently treated with fractionated proton therapy. He continued to require metyrapone to manage hypercortisolism despite reassuring serial MRI scans. A right neck mass was palpated in 2022 and CT demonstrated a right level 2b lymph node measuring 2.3 x 1.8 x 2.3 cm. FNA revealed neoplastic cells and he underwent right neck dissection with excision of the lymph node. Pathology was consistent with pituitary tumor, corticotroph cell type. Given metastasis of the pituitary tumor to a cervical lymph node, a diagnosis of pituitary carcinoma was made. Conclusion: The time course in pituitary carcinoma is highly variable from the initial diagnosis of the pituitary tumor to the diagnosis of metastatic disease. In this case, the diagnosis of pituitary carcinoma was made nearly one decade after the initial identification of a pituitary tumor. Metastatic disease should be considered in aggressive pituitary tumors when biochemical and radiologic findings are discordant. Presentation: Thursday, June 15, 2023

THU478 Unchecked Kinase Activation Underlies Pituitary Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. Y. Li: None. D. Mandal: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Common mechanisms underlying canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ pituitary adenomas causing Cushing’s disease remain unknown. Using syngeneic, pairwise transcriptomic analysis at the single cell/nucleus resolution, we found a ubiquitous and robust overexpression of PPP1R17 in CD adenoma corticotrophs. The role of PPP1R17, an endogenous inhibitor of tumor-suppressor phosphatase PP2A in the embryonic context remains unknown in the pituitary. Here, we elucidated mechanistically, the effect of PPP1R17 on the phopshoproteome of CD adenomas. Methods: We quantitatively profiled the proteome and phosphoproteome of three pairs of CD adenomas and their syngeneic adjacent normal pituitary glands with TMT labeling and mass spectrometry (LC-MS/MS). We validated the mechanisms with murine corticotroph cells (mCort) overexpressing R17 (mCortR17) or GFP (mCortGFP). Elevated total and phosphprotein expression was assessed using R packages by performing cross sample normalization, noise filtering and differential protein abundance or phosphorylation analyses. Pathway enrichment analysis was performed on the KEGG database using ShinyGo 0.76.2. Results: We identified 37 proteins differentially phosphorylated in CD adenomas compared to adjacent normal pituitary glands, including the AP2-associated protein kinase 1 (Aak1) and the apoptotic chromatin condensation inducer Acin1. Of these 37 proteins, 30 (81%) were also differentially phosphorylated in mCortR17 versus mCortGFP cells, suggesting that differential phosphorylation in CD is driven by R17 hyperactivity. Of the 35 proteins overabundant in CD adenomas, 15 (43%) overlapped with mCortR17, including the Myc-associated protein (Max) and Presenilin 1 (Psen1). Proteins enriched in both human CD and mCort datasets were involved in NOTCH signaling and neurotransmitter release, while phospho-proteome profiling highlighted R17-mediated perturbation of RNA polymerase II transcription termination and mRNA processing. Conclusions:We first discovered a heavily dysregulated phosphoproteome in CD adenomas compared to syngeneic normal glands. We found that we could replicate the signature phosphoproteomic profile by overexpressing PPP1R17 in-vitro. These data indicate a central role for phosphatase (PP2A) inhibition in CD adenomas, and suggest potential novel anti-tumor targets for patients with CD. Presentation: Thursday, June 15, 2023

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

OR2802 A Novel CABLES1 Missense Variant Associated With Cushing's Disease Disrupts Protein Structure And Stability

Abstract Disclosure: A.L. Franco-Álvarez: None. M. Torres Morán: None. R.G. Rebollar-Vega: None. N. Pankratz: None. J. Lane: None. F.R. Faucz: None. P. Chittiboina: None. D.M. Kay: None. J.L. Mills: None. C.A. Stratakis: None. L.C. Hernández-Ramírez: None. Introduction. The adrenal-pituitary feedback loop is disrupted in Cushing's disease (CD). One of the glucocorticoid-responsive genes participating in this regulatory pathway is the negative cell cycle regulator CABLES1. We have previously reported five germline missense CABLES1 variants associated with CD and macroadenomas: two in young adults (p.E178K and p.L240F), two in children (p.G312D and p.D463G), and one in a middle-aged adult (p.P31L). The first four variants resulted in loss of the cell growth inhibition activity, but the fifth one has not been functionally validated. Aims. To report in detail a case of CD associated with the CABLES1 p.P31L variant and to characterize the molecular consequences of this variant using in silico and in vitro approaches. Methods. Variant detection was done via exome sequencing. Immunohistochemistry for CDKN1B and CABLES1 was performed on pituitary tumor slides. Loss of heterozygosity for CABLES1 p.P31L and common somatic variants were investigated in the tumor tissue. Protein stability studies were done for the CABLES1 variants p.P31L, p.L240F, p.G312D and p.D463G transiently expressed in HEK293 cells, using the cycloheximide chase method. The interaction of the CABLES1 variants with CDK2 was analyzed by transient expression in HEK293 cells and co-immunoprecipitation. Results. The p.P31L variant was found in a 53-year-old woman with CD. She achieved remission after resection of a macroadenoma (reported as a null-cell tumor). Recurrence one year later prompted a reintervention, and this a 60 mm Crooke cell adenoma was resected, again with postoperative remission. The p.P31L variant is of uncertain significance according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. The affected residue (P31) is part of a PPP motif that lies within a proline-rich region of CABLES1 (residues 30-52) within the N-terminal disorder, with unknown functional role. By immunohistochemistry, the corticotropinoma retained CABLES1 expression, but had lost 70% of CDKN1B expression. Loss of heterozgosity for CABLES1 p.P31L, as well as and hotspot BRAF, USP8, and USP48 were negative in the tumor. The p.P31L variant had an increased degradation constant (0.13±0.04) and a reduced half-life (5.80±1.93 hours), compared with wild type CABLES1 (degradation constant of 0.06± 0.02 and half-life of 13.52 ±5.11 hours; P=0.0036 for degradation constant and P=0.0091 for half-life). The rest of variants showed no significant differences. All variants retained their ability to interact with CDK2. Conclusions. The CABLES1 p.P31L variant encodes an unstable protein with a reduced half-life, which accordingly may result into a reduced tumor suppressor effect. Further studies are required to fully understand how CABLES1 loss of function impacts corticotroph cell growth. Presentation: Sunday, June 18, 2023

Altered glucocorticoid reactivity and behavioral phenotype in rx3-/- larval zebrafish.

The transcription factor rx3 is important for the formation of the pituitary and parts of the hypothalamus. Mutant animals lacking rx3 function have been well characterized in developmental studies, but relatively little is known about their behavioral phenotypes. We used cell type staining to reveal differences in stress axis architecture, and performed cortisol measurements and behavior analysis to study both hormonal and behavioral stress responses in rx3 mutants. Consistent with the role of rx3 in hypothalamus and pituitary development, we show a distinct loss of corticotrope cells involved in stress regulation, severe reduction of pituitary innervation by hypothalamic cells, and lack of stress-induced cortisol release in rx3 mutants. Interestingly, despite these deficits, we report that rx3-/- larval zebrafish can still display nominal behavioral responses to both stressful and non-stressful stimuli. However, unlike wildtypes, mutants lacking proper pituitary-interrenal function do not show enhanced behavioral performance under moderate stress level, supporting the view that corticotroph cells are not required for behavioral responses to some types of stressful stimuli but modulate subtle behavioral adjustments under moderate stress.

Somatostatin receptor type 2 (SSTR2) expression regulation by miR-375 in a murine pituitary corticotroph tumor cell model

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

519 Single Nucleus ATAC Sequencing Reveals Increased PPP1R17 Chromatin Accessibility Driving Cushing’s Disease

INTRODUCTION: Epigenetic drivers of pituitary adenomas causing Cushing’s disease (CD) are virtually unknown. Here, we investigate the epigenetic mechanisms underlying gene dysregulation in CD. METHODS: We isolated single nuclei from 3 CD adenoma – normal gland pairs and performed single nucleus RNA sequencing (snRNAseq) and assays for transposase-accessible chromatin (snATACseq, 10x Genomics). We integrated these data with prior paired single cell data (scRNAseq, n = 3) and bulk RNAseq (CD = 3, non-CD = 3). We performed bulk DNA methylation assays on independent adenomas (CD = 3, non-CD = 3; Illumina Infinium MethylationEPIC). CD adenomas were examined by multiplexed immunohistochemistry (mIHC). We generated murine corticotroph (mCort) cell lines stably transfected with lentiviral expression vectors (Origene) and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). RESULTS: snATACseq and snRNAseq data identified increased chromatin accessibility with upregulated PPP1R17 expression in single nuclei from CD adenomas, in agreement with our prior scRNAseq and bulk RNAseq data (16-fold in paired analysis, FDR &lt; 0.01). DNA methylation failed to identify global or local differential methylation at the PPP1R17 locus. mIHC confirmed PPP1R17 protein overexpression in human CD tumors. PPP1R17 overexpression in mCort cells accelerated the cell cycle with fewer cells accumulating in S-phase (16.4% vs 24.1%, P &lt; 0.01), and increased cell proliferation (two-way ANOVA P &lt; 0.01). CONCLUSIONS: We identified coordinated chromatin accessibility, transcriptional activation and protein expression of PPP1R17 in human CD adenomas compared to surrounding normal tissues, and recapitulated hyperproliferation in-vitro by stable PPP1R17 overexpression. Our findings uncover targetable epigenetic mechanisms in CD pathogenesis.