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Background Osilodrostat, a potent oral 11β-hydroxylase inhibitor, provided rapid and sustained normalization of mean urinary free cortisol (mUFC) in patients with Cushing's disease (CD) during the 48-week (W) core period of LINC 4 (NCT02697734), and was well tolerated. We report long-term efficacy and safety results from the LINC 4 core and extension phases combined. Methods 73 adults with CD and mUFC >1.3× the upper limit of normal (ULN) were enrolled. LINC 4 comprised a 12W, randomized, double-blind, placebo-controlled period followed by 36W of open-label osilodrostat. At W48, patients could enter an optional extension. Dose adjustments were permitted based on efficacy/tolerability (range during open-label treatment 1–30 mg bid). LINC 4 ended when all patients transitioned to a separate long-term safety study (not reported) or discontinued treatment. Efficacy/safety are reported for all patients unless otherwise stated, and excludes data collected during W1–12 for placebo recipients. Results Of the 65 patients who completed the core phase, 60 entered the extension. Median (range) osilodrostat exposure from core baseline to study end was 87.1 (2–127) W; median average (IQR) dose was 4.6 (3.7–9.2) mg/day. 15 patients discontinued osilodrostat; 7 after W48 (6 because of adverse events [AEs]). The proportion of patients with normal mUFC (≤138 nmol/24h [50 µg/24h]) was 68.5% (n=50/73) at W48, 61.5% (n=40/65) at W72 and 72.4% (n=42/58) at the end-of-treatment extension (EOT) visit. Median mUFC decreased from 2.5×ULN (core baseline) to 0.5×ULN (W48 and W72), to 0.4×ULN (EOT). Median late-night salivary cortisol decreased from 2.8×ULN (core baseline) to 1.2×ULN (W48 and W72), to 1.1×ULN (EOT).The most common AEs during the entire study were decreased appetite (46.6%), arthralgia (45.2%), fatigue (39.7%), nausea (37.0%), headache (34.2%) and dizziness (30.1%). Hypocortisolism- and adrenal-hormone-precursor-accumulation-related AEs occurred in 28.8% (21/73) and 61.6% (45/73) of patients during the entire study, less frequently in the extension than the core. Most were grade 1/2 and resolved with dose reduction/interruption and/or concomitant medication. During the extension, hirsutism and acne were reported as AEs by 1 and 0 patients, respectively.Median ACTH increased from 1.1×ULN (core baseline) to 3.0×ULN (W48), to 3.6×ULN (W72), to 3.5×ULN (EOT). Median change (95% CI) in pituitary tumor volume (assessed by MRI) from core baseline to last available assessment was 4.0 mm3 (−24.1–169.8); pituitary-tumor-enlargement-related AEs led to drug discontinuation in 2 and 0 patients during the core and extension, respectively. No trend was observed between tumor volume change and osilodrostat dose. Conclusion Osilodrostat provided long-term control of cortisol production during LINC 4. Fewer AEs related to hypocortisolism and accumulation of adrenal-hormone-precursors occurred during the extension than during the core. Osilodrostat is an effective and well-tolerated long-term treatment option for CD patients. Presentation: Tuesday, June 14, 2022 10:15 a.m. - 10:30 a.m.

Background Cushing's disease (CD) is a serious disorder associated with increased cardiovascular morbidity and mortality, and reduced patient quality of life (QoL), because of hypercortisolism. We report long-term effects of osilodrostat, a potent 11β-hydroxylase inhibitor, on cardiovascular/metabolic-related risk factors, physical features of hypercortisolism and QoL in CD patients following the core and extension phases of the LINC 4 study (NCT02697734). Methods LINC 4 comprised a 12-week (W), randomized, double-blind, placebo-controlled period, 36W of open-label osilodrostat, and an optional extension phase in adults with CD and mUFC >1.3xULN. Dose adjustments were permitted based on efficacy/tolerability (range during open-label treatment 1–30 mg bid). LINC 4 ended when all patients transitioned to a separate long-term safety study or discontinued treatment. Cardiovascular/metabolic-related parameters, physical features of hypercortisolism (rating: 0=absent; 1=mild; 2=moderate; 3=severe), and CushingQoL score were evaluated at core baseline, every 2, 4, 12 or 24W (depending on study phase/parameter) and at the end-of-treatment extension (EOT) visit. Change from baseline is provided for patients with assessments at core baseline, W48 and EOT. Results Of the 65 patients completing W48, 60 entered the extension. Median (range) osilodrostat exposure from core baseline to study end: 87.1 (2–127) W; median (IQR) average dose: 4.6 (3.7‍–‍9.2) mg/day. Mean changes (95% CI) in cardiovascular/metabolic-related parameters from core baseline to W48 and EOT, respectively, included decreases in systolic (−9.7 [−14.9, −4.6] mmHg; −12.4 [−17.4, −7.4] mmHg; baseline: 131.5 mmHg) and diastolic (−4.2 [−7.3, −1.2] mmHg; −5.6 [−8.9, −2.4] mmHg; baseline 87.5 mmHg) blood pressure, fasting plasma glucose (−3.1 [−6.8, 0.6] mg/dL; −3.5 [−8.5, 1.4] mg/dL; baseline: 95.3 mg/dL) and cholesterol (−0.5 [−0.8, −0.2] mmol/L; −0.6 [−0.9, −0.3] mmol/L; baseline: 5.5 mmol/L). Improvements (mean change [95% CI]) from core baseline to W48 and EOT also occurred for weight (–4.3 [–5.9, –2.6] kg; –6.8 [–8.8, –4.8] kg; baseline: 78.3 kg) and waist circumference (–4.5 [–6.0, –3.1] cm; –7.6 [–9.6, –5.6] cm; baseline: 102.8 cm). Physical features of hypercortisolism improved (severity reduced) or remained stable from core baseline to EOT in most patients (respectively): ecchymosis (21% [n=10/48]; 79% [n=38/48]), striae (26%; n=12/46; 72% [n=33/46]), hirsutism in females (29% [n=11/38]; 61% [n=23/38]), muscle weakness (33% [n=16/49]; 61% [n=30/49]), facial rubor (48% [n=23/48]; 46% [n=22/48]), central obesity (55% [n=27/49]; 37% [n=18/49]), and fat pads (dorsal: 58% [n=28/48]; 31% [n=15/48]; supraclavicular: 65% [n=32/49]; 35% [n=17/49]). CushingQoL score improved from core baseline to W48 and EOT (mean change [95% CI]: 12.0 [8.2, 15.9]; 17.1 [12.5, 21.7]; baseline: 51.8). Conclusion Most cardiovascular/metabolic-related parameters continued to improve during long-term osilodrostat treatment. Additionally, most physical features of hypercortisolism, including hirsutism, either improved or remained stable, and CushingQoL score improved. Osilodrostat is an effective treatment that may alleviate disease burden for many patients with CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Serotonin (5-HT) is an important hormonal modulator and neurotransmitter, and 5-HT has been demonstrated in pituitary tissue from several species. Previous responses to 5-HT antagonists have been reported in some patients with Cushing disease although this effect was unsustained, and ACTH and cortisol levels actually increased in some patients. To better understand the role of serotonin in the regulation of corticotroph tumor growth and ACTH secretion, we first measured serotonin levels in supernatants (SNs) derived from murine corticotroph tumor AtT20 cells. We demonstrated that AtT20 cells secrete serotonin (2±0.05ng/105cells/24h) which was ~50% of the levels secreted by the serotonin secreting mid-gut carcinoid BON-1 cell-line (4.2±0.05ng/105cells/24h). In contrast, serotonin secretion was not detected in rat pituitary tumor lactotroph (GH3) or human embryonic kidney (HEK293) cell SNs, or in our Ham’s F12 medium control. Immunocytochemical staining using serotonin specific antibodies demonstrated that serotonin was diffusively present in AtT20 cell cytoplasm, suggesting that serotonin was endogenously generated in the AtT20 cells. Using real-time PCR, we demonstrated that both serotonin synthesis enzymes, tyrosine hydroxylase-1 (THP1) and -2 (THP2) are expressed in AtT20 cells with higher relative THP1 expression, confirming endogenous corticotroph pituitary tumor serotonin production. We further demonstrated that the synthetic glucocorticoid Dexamethasone (100nM x 24h) suppressed TPH1 expression and inhibited corticotroph tumor serotonin secretion. We next evaluated the actions of serotonin and various serotonin antagonists on corticotroph tumor cell proliferation and ACTH secretion. Ritanserin (10-5M) inhibited murine corticotroph tumor proliferation by 5% and inhibited ACTH secretion by ~25%. In contrast, metergoline (10-5M) and the orally administered TPH inhibitor, telotristat etiprate (10-5M for 1-3 days), inhibited ACTH secretion by 50% and 30% respectively but did not reduce cell proliferation, suggesting that Metergoline and telotristat may regulate ACTH secretion independently of their anti-proliferative effect. Addition of serotonin (10-4~10-5M) to corticotroph tumor cells cultured in reduced serum (OptiMEM) or no serum conditions, resulted in an 80% increase in cell proliferation but had little effect on ACTH secretion with a 1-4% increase. In summary, we have demonstrated that serotonin is synthesized in and secreted from corticotroph tumor cells and that serotonin plays a role in regulation of corticotroph tumor proliferation and ACTH secretion in vitro. Our findings using inhibitors of serotonin action indicate potential for targeting this pathway as a novel treatment for patients with CD.

Neuroendocrine tumors (NETs) comprise a group of complex heterogeneous and increasingly prevalent neoplasms. They arise from diverse body regions, share derivation from primitive neuronal stem cells, secrete various bioactive peptides and exhibit a wide spectrum of tumor behavior from relatively indolent to rapidly progressive. Pituitary tumors represent 20% of intracranial tumors and share some features of NET histo-pathologic morphology, their excess hormone production and some have proposed re-naming pituitary tumors as PitNETs. However there has been no precise cell type composition comparison at the whole genome level in these two types of tumors. To explore intratumoral heterogeneity at a single cell resolution in pituitary and pancreatic NETs, we employed single cell RNA sequencing (scRNA-seq) to analyze in parallel cell populations from surgically resected pituitary tumors (n=4) and pancreatic NETs (pNET, n=2) using a 10x Genomics platform (v3). Using Cell Ranger pipeline for alignment and mapping, we obtained an average of 8,306±2,519 cells/sample with 1,516±822 genes/cell (mean reads/cell 67.9±30K). Seurat v3 was then used for read pre-filtering, normalization, and cluster identification. We identified 5 genes commonly expressed in both pNET and pituitary tumor populations, namely CALY (clathrin light chain binding GO:0032051), SPINT (peptidase inhibitor activity GO:0030414), CHGB (hormone activity GO:0005179), SCG5 (GTP binding GO:0005525) and SEZ6L2. As proof of their oral epidermal embryonic origin, the commonly expressed genes in 4 pituitary tumor samples include pituitary tissue restricted transcription factors (POU1F1, BEX1/2 GO:0033613), GNAS (G-protein beta/gamma-subunit complex binding GO:0031683), NLRP1 (peptidase activator activity involved in apoptotic process GO:0016505), PTN (protein phosphatase regulator activity GO:0019888) and ATP6V1G1 (ATPase binding GO:0051117). In parallel, 71 genes were present at high levels in pNET and involved in molecular functions such as calcium ion binding (HSPA5; ENPP2; C2CD4B; CALR; HSP90B1 GO:0005509) and endopeptidase inhibitor activity (PCSK1N; WFDC2 GO:0004866). Although our exploratory findings are limited to 6 samples, the robust commonly expressed genes within each tumor type clearly separated pituitary and pancreatic neuroendocrine tumors into distinct entities. We did observe conservation of chromogranins and prohormone convertases in these two tumor types, but it only represented a very small portion of overlap in transcriptome in pNETs. In summary, our findings suggest that pituitary and neuroendocrine tumors have only limited common molecular features, and by and large they stand as separate biologic populations. Our observations may begin to provide insight into the differences in tumor progression that are encountered clinically between pituitary tumors and pancreatic NETs.

Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p

Silent pituitary corticotroph tumors derive from the Tpit (aka TBX19) pituitary lineage. Accounting for ~ 30% of corticotroph tumors, they are not infrequently clinically aggressive and invade locally into adjacent sellar structures, making complete surgical resection challenging and contributing to their higher recurrence rates. How silent and active corticotroph tumor subtypes differ is not clear although some studies reported that silent corticotroph tumors exhibit reduced PC1 expression causing impaired POMC processing. We used single cell RNAseq to compare the transcriptome between silent (n = 2) and active (n = 4) corticotroph tumors at the single cell level. We obtained an average of 265 million reads, and 24,682 genes per patient with an average of 1,240 genes expressed and 3,5442 unique molecular identifiers (UMIs) detected per cell. We further defined 5 distinct cell populations from a total of 23,269 cells, namely tumor cells (62%), stromal cells (25%), immune cells (7%), progenitor cells (5%), and a minor population of erythrocytes (1%). Tumor cells clustered in an origin-dependent manner and all expressed POMC and TBX19. However, the gene signatures of silent and active corticotroph tumors differed in 3 major aspects. Firstly, and supporting prior studies, a series of hormone processing peptidase genes, including PC1 (aka PCSK1), PDIA3, SEC11C, SPCS1 and CTSB, were reduced in silent corticotroph tumors (p=5.54e-5) compared to active corticotroph tumors. Secondly, genes involved in organization of secretory vesicles such as SCG5, TIMP1, VGF, SYT17, LGALS3 and CALY were also reduced in silent corticotroph tumors, which could further compound their inability to secrete ACTH. Thirdly, the silent corticotroph tumors exhibited several features of endothelial-to-mesenchymal transition (EMT), including increased expression of the mesenchymal genes CDH2 (aka NCAD), COL1A1, PCDH9, FGF5, ID2 (p=8.4e-3), and loss of EPCAM, which regulate cell migration and movement. Upstream analysis suggested that aberrant STAT3 activation may be related to these changes. Consequently, we noted that the stromal content was higher in silent corticotroph tumors (47.5% vs. 18.13%), concordant with the observed EMT de-differentiation of tumor cells. In summary, our scRNAseq analysis provides an unprecedented precise investigation of the transcriptomic features of thousands of heterogenous corticotroph tumor cells simultaneously. We demonstrate that although silent corticotroph tumor cells still express the pituitary lineage markers PITX1 and TBX19, they exhibit EMT, potentially affording increased migratory capacity at the cost of reduced neuroendocrine function with inability to produce and secrete mature ACTH. Our findings provide novel insights into the pathogenesis of silent versus active corticotroph tumor, but may reveal novel molecular targets for treatment of these challenging tumors.

Background: In a prior Phase III, randomized-withdrawal study, osilodrostat, a potent oral 11β-hydroxylase inhibitor, provided rapid and sustained normalization of mean urinary free cortisol (mUFC) in most patients (pts) with CD. Now, we report efficacy and safety results from another Phase III study of osilodrostat in pts with CD that included an upfront, double-blind, randomized, placebo-controlled phase (LINC 4: NCT02697734). Methods: Adults with CD with mUFC >1.3 x ULN were randomized 2:1 to osilodrostat 2 mg bid or matching placebo for a 12-week (W) double-blind period, with dose adjustments at W2, 5 and 8 (range 1-20 mg bid) based on efficacy and tolerability; dose matching and adjustments were managed by independent endocrinologists. From W12 to W48, all pts received open-label osilodrostat, with dose adjustments permitted (range 1-30 mg bid). At W48, pts could enter an optional extension. Primary endpoint: proportion of randomized pts in each arm who received ≥1 treatment dose with mUFC ≤ULN at W12. Results: 73 pts were randomized and received osilodrostat (n=48) or matching placebo (n=25; baseline median [range] mUFC 2.5 x ULN [0.7-12.5] vs 2.2 x ULN [0.2-18.9]). 77% of osilodrostat recipients achieved mUFC ≤ULN at W12 vs 8% of placebo recipients (OR 43.4; 95% CI 7.1-343.2; P Conclusion: Osilodrostat was superior to placebo at normalizing mUFC levels at W12 (77% vs 8%). Improvements in mUFC levels were sustained at W36. Few pts discontinued because of AEs; hypocortisolism-related AEs were infrequent and manageable. We conclude that osilodrostat is a highly effective and well-tolerated treatment for pts with CD.

Cushing Disease (CD), caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, leads to excess production of adrenal-derived cortisol. Current medical treatments for CD include adrenal steroidogenesis inhibitors (Ketoconazole, Metyrapone), the somatostatin receptor ligand Pasireotide, and the glucocorticoid receptor antagonist (Korlym). Although Pasireotide is tumor directed, its effect on tumor growth is modest, and it frequently causes hyperglycemia. Additionally, long-term compliance with these drugs is < 30% - either due to escape from control caused by increased tumor-derived ACTH or drug side effects. Clearly there is a large unmet need for improved medical treatments for CD. We hypothesize that an ideal drug would act on the tumor itself, potently and selectively inhibit corticotroph tumor-derived ACTH production to attain sustained eucorticolism and inhibit corticotroph tumor growth. Using our recently developed ACTH quantification assay (ACTH AlphaLISA), we screened an annotated kinase inhibitor library (KIL, n=430) at 10μM, 1μM and 100nM doses. The KIL contains inhibitors of PI3K/AKT/mTOR (n=95), protein tyrosine kinases (n=87), MAPK (n=45), angiogenesis (n=44), cell cycle (n=44), JAK/STAT (n=26), and others (n=89). Out of 430 compounds screened, 6, 20 and 115 compounds exhibited >50% ACTH AlphaLISA inhibition and 36, 105 and 263 compounds exhibited >50% nuclei inhibition in murine corticotroph tumor AtT20 cells at 100nM, 1μM and 10μM respectively. Three out of the 6 compounds that exhibited efficacy >50% at 100nM are currently being evaluated in Phase II clinical trials in a variety of solid tumors. Given the involvement of PI3K/AKT pathway in pituitary tumor pathogenesis and recent findings of aberrant USP8 mutant-associated EGFR activation in corticotroph tumors, we have thus far performed dose-response curves for one of the PI3K inhibitors using two separate assays of ACTH secretion (AlphaLISA and ELISA) and cell proliferation (Hoechst nuclei staining and CellTilter Glow assay). Both demonstrated comparable inhibition rates with an EC50 of 246nM for ACTH inhibition determined by AlphaLISA assay, and LC50 of 18 nM for cytotoxicity. Experiments to elucidate the mode of action of the PI3K inhibitor on ACTH synthesis and/or secretion, demonstrated it inhibited POMC mRNA expression by 40% at 300nM and 87% at 1μM (p

Lack of an immortalized or long-term hormone-secreting human pituitary tumor in vitro cell line has significantly hampered studies of etiopathogenesis and development of targeted therapies for pituitary tumors. Various media, vessels, substrates and additives have been explored to optimize primary cultures. Some methods have prolonged cell survival time but maintaining hormone secretion is still a major challenge. We have recently developed methodology to maintain robust hormone secretion in human prolactinoma, somatotroph and corticotroph tumors in vitro for up to 9 months. To gain insight into the changes in gene expression profiles that occurred prior to/coincident with loss of hormone secretion during in vitro human pituitary tumor cultures, we compared the global transcriptiome landscape in serial passages from two human prolactinoma primary cultures. We obtained 27.1 million quality filtered reads per sample of which ~90% were exonic reads detecting an average of 16,000 genes. 3D principal component analysis identified 3 distinct clusters, with close similarity between cell passages 1-2, those beyond passage 4-5 and a stand-alone passage 3. Analysis of differential gene expression (DEG) over time using DAVID knowledgebase and hierarchical clustering demonstrated that most changes in gene expression occurred in the first 1-2 weeks in in vitro culture. During the first week of in vitro culture, although the cells maintained robust PRL secretion, reduced activation of angiogenesis and growth signal pathways (PI-3K, MAPK pathways, cell adhesion, GTPase activity, cell proliferation and cell-cell junction assembly) was observed. In the following 1-2 weeks, the cell population maintained PRL secretion but exhibited continued loss of survival signals, with increased expression of extracellular matrix (ECM) and surface adhesion molecules consistent with development of a fibroblastic phenotype that dominated later stage cultures. Based on our findings, we propose that the molecular events underpinning loss of hormone production in primary pituitary tumor cultures represent transition from endocrine cells to pituitary specific fibroblastic folliculo-stellate cell likely selected by the in vitro culture conditions. Our data further reveal that underexpression of several GPCRs and resultant downregulation of the PKA pathway may lead to loss of hormone production in parallel with loss of cell survival signals. In summary, our study defines a temporal progressive transcriptome transition signature to explain loss of hormone secretion and morphological alterations observed during human pituitary tumor in vitro culture and may provide molecular targets to further optimize our culture methodology to attain “immortalized” hormone-producing human pituitary tumor cell-lines.