Secretase Inhibitors Research Articles

Secretase promotes AD progression: simultaneously cleave Notch and APP.

Alzheimer's disease (AD) involves complex pathological mechanisms. Secretases include membrane protein extracellular structural domain proteases and intramembrane proteases that cleave the topology to type I or type II. Secretases can effectively regulate the activation of Notch and amyloid precursor protein (APP), key factors in the progression of AD and cancer. This article systematically summarizes the intracellular localization, cleavage sites and products, and biological functions of six subtypes of secretases (α-secretase, β-secretase, γ-secretase, δ-secretase, ε-secretase, and η-secretase), and for the first time, elucidates the commonalities and differences between these subtypes of secretases. We found that each subtype of secretase primarily cleaves APP and Notch as substrates, regulating Aβ levels through APP cleavage to impact the progression of AD, while also cleaving Notch receptors to affect cancer progression. Finally, we review the chemical structures, indications, and research stages of various secretase inhibitors, emphasizing the promising development of secretase inhibitors in the fields of cancer and AD.

Gamma Secretase Inhibition May Mitigate the Impact of Low BCMA Target Density in Relapsed/Refractory Multiple Myeloma: Results from a Comparative Analysis of Two Phase 1 Clinical Trials

Gamma Secretase Inhibition May Mitigate the Impact of Low BCMA Target Density in Relapsed/Refractory Multiple Myeloma: Results from a Comparative Analysis of Two Phase 1 Clinical Trials

Gamma secretase inhibitors, in combination with cytotoxic agents, decrease migration and invasion of uterine leiomyosarcoma

Gamma secretase inhibitors, in combination with cytotoxic agents, decrease migration and invasion of uterine leiomyosarcoma

A real-world study on the clinicopathological profile, treatment outcomes and health-related quality of life, anxiety and depression among patients with desmoid tumor at two tertiary care centers in India.

The medical management of DT comprises tyrosine kinase inhibitors (TKIs), hormonal agents, anti-inflammatory drugs with the recently approved gamma secretase inhibitor nirogacestat being the current standard of care. Real-world data on evolving treatment landscapes of DT remains scarce. This is a retrospective study of patients with DT registered between 1995 and 2020 at All India Institute of Medical Sciences, New Delhi and Tata Medical Center, Kolkata. Baseline characteristics were analyzed in form of median values and interquartile range. Categorical and continuous variables were compared by chi square and independent samples T- tests respectively. Anxiety, depression and QoL were prospectively measured among 30 patients using Hospital Anxiety and Depression (HADS) and Functional Assessment of Cancer Therapy-General (FACT-G) scales respectively between 2022 to 2023. 200 patients were included with a male-predominant (n=111, 55.5%) population and median age 26.5 (2.5-75) years. Extremity (n=100, 50%) and abdomen (n=65, 32.5%) were commonest primary sites and median of 2 (1-4) lines of treatment were received. First-line included surgery (n=116, 58%), systemic therapy (n=67, 33.5%), radiotherapy (10, n=5%) and active surveillance (n=7, 3.5%). First-line systemic agents included tamoxifen (n=55, 27.5%), imatinib (n=7, 3.5%), sorafenib (n=1, 0.5%) and chemotherapy (n=4, 2%). 2019 onward, 3% and 63% underwent active surveillance and surgery respectively. Best radiological response obtained with tamoxifen was stable disease (SD) (n=76, 59%) and partial response (PR) (n=31, 24.2%). Best radiological response obtained with sorafenib was PR (n=17, 60.7%) and SD (n=9, 32.1%). Thirty patients underwent HADS and FACT-G scale assessment. Mean HADS-Anxiety subscale score was 3.6 (+/-3.9 SD) and HADS-Depression sub-scale score was 2.6 (+/-3.5 SD) with clinically significant anxiety and depression in 2 (6.7%) patients each. The overall mean FACT-G score was 87.5 (+/-12.6 SD) and lower mean physical well-being (p=0.006) and emotional well-being (0.017) scores were significantly associated with higher HADS-anxiety (>/=8) scores. Assessment of anxiety, depression and QoL are paramount to gauge the psychological impact of DT. This study gives an overview of clinical and management profile of patients with DT in India, with limitations of selection bias, heterogeneous population and small sample size for QoL assessment.

Impact of soluble BCMA and non–T-cell factors on refractoriness to BCMA targeting T-cell engagers in multiple myeloma

AbstractAdoptive T-cell therapy is a promising therapy for multiple myeloma (MM), but its efficacy hinges on understanding the relevant biologic and predictive markers of response. B-cell maturation antigen (BCMA) is a key target antigen in MM with active development of multiple anti-BCMA T-cell engagers (TCEs) and chimeric antigen receptor T-cell therapies. The regulation of surface BCMA expression by MM cells, which leads to shedding of soluble BCMA (sBCMA), has triggered debate about the significance of sBCMA as a predictive marker and its potential impact on treatment outcomes. To address this, we leveraged whole-genome sequencing and in vitro assays to demonstrate that sBCMA may independently predict primary refractoriness to anti-BCMA therapies. In addition to sBCMA, tumor burden and surface BCMA antigen density collectively influenced the anti-BCMA TCE cytotoxic efficacy. Correlative analyses of 163 patients treated with the anti-BCMA TCE teclistamab validated and further underscored the association between elevated baseline sBCMA (>400 ng/mL) and refractoriness. Importantly, increasing the TCE dose, using TCE against alternative targets (eg, GPRC5D), and gamma secretase inhibitors were able to overcome the high sBCMA levels. These findings highlight the importance of taking into account the baseline sBCMA levels, disease burden, and TCE dose intensity when administering anti-BCMA TCEs, thereby offering critical insights for optimizing therapeutic strategies to overcome specific high-risk features and primary anti-BCMA TCE refractoriness.

Quercetagetin 3,7 dimethyl ether polymorphs as multi-targeted anti-amyloid agents: Target to cognitive impairment in Alzheimer's disease

The extraction of biologically active natural products from plants and the assessment of their medicinal properties play a crucial role in drug development. Taking that into consideration, the current research work was designed. Bioassay-guided isolation from Parthenium hysterophorus led to the isolation of two antioxidant compounds: polymorphs A (1) and B (2) of Quercetagetin 3,7-dimethyl ether which demonstrated IC50 values 13.00 ± 0.63 μM and 16.56 ± 0.82 μM, respectively in the DPPH assay. These polymorphs were further evaluated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. In silico studies, including molecular docking, and molecular dynamic simulations were conducted using β-secretase, γ-secretase, AChE, and BChE enzymes. Interestingly, compound 1 demonstrated excellent antioxidant activity and showed the better binding affinities -9.0 kcal/mol (BChE), −8.5 kcal/mol (β-secretase), -8.2 kcal/mol (γ-secretase) and -7.7 kcal/mol (AChE) as compared to compound 2. Due to these promising results, compound 1 was selected for molecular dynamic simulations to further evaluate the stability and flexibility of its complexes. Overall, both in silico and in vitro results confirmed that compound 1 and compound 2 exhibit distinct behavior, making them promising candidates as antioxidants and inhibitors of AChE, BChE, and secretases, potentially targeting cognitive impairment in Alzheimer's disease (AD).

Combined Treatment of Uterine Leiomyosarcoma with Gamma Secretase Inhibitor MK-0752 and Chemotherapeutic Agents Decreases Cellular Invasion and Increases Apoptosis.

Due to limited effective therapeutics for uterine leiomyosarcoma (uLMS), the impact of the gamma secretase inhibitor (GSI) MK-0752 with common chemotherapeutics was explored in uLMS. MTT assays were performed on two human uLMS cell lines, SK-UT-1B and SK-LMS-1, using MK-0752, docetaxel, doxorubicin, and gemcitabine, individually and in combination, to determine cell viability after treatment. Synergistic combinations were used in transwell invasion assays, cell cycle flow cytometry, proliferation assays, and RNA sequencing. In SK-UT-1B, MK-0752 was synergistic with doxorubicin and gemcitabine plus docetaxel. In SK-LMS-1, MK-0752 was synergistic with all individual agents and with the combination of gemcitabine plus docetaxel. MK-0752, gemcitabine, and docetaxel decreased invasion in SK-UT-1B 2.1-fold* and in SK-LMS-1 1.7-fold*. In SK-LMS-1, invasion decreased 1.2-fold* after treatment with MK-0752 and docetaxel and 2.2-fold* after treatment with MK-0752 and doxorubicin. Cell cycle analysis demonstrated increases in the apoptotic sub-G1 population with MK-0752 alone in SK-UT-1B (1.4-fold*) and SK-LMS-1 (2.7-fold**), along with increases with all combinations in both cell lines. The combination treatments had limited effects on proliferation, while MK-0752 alone decreased proliferation in SK-LMS-1 (0.63-fold**). Both MK-0752 alone and in combination altered gene expression and KEGG pathways. In conclusion, the combinations of MK-0752 with either doxorubicin, docetaxel, or gemcitabine plus docetaxel are potential novel therapeutic approaches for uLMS. (* p < 0.05, ** p < 0.01).

Efficacy and safety of nirogacestat in patients with desmoid tumor and adenomatous polyposis coli (APC) mutation: Phase 3 DeFi analyses.

11558 Background: Nirogacestat (niro) is a targeted and selective gamma secretase inhibitor approved in the US for adults with progressing desmoid tumors (DT). In the phase 3 DeFi study, niro demonstrated significant improvement vs placebo (pbo) in progression-free survival (PFS: HR, 0.29 [95% CI: 0.15-0.55]; P&lt;.001), objective response rate (ORR: 41% vs 8%; P&lt;.001); and patient-reported outcomes (PROs) of pain, DT-specific symptom burden, physical and role functioning, and overall quality of life ( P≤.01, all). DT are driven by Wnt/β-catenin signaling pathway alterations and about 10-20% of DT are associated with mutations in the APC tumor suppressor gene, which may confer more aggressive DT behavior. Because patients with APC mutations may do worse, regardless of specific therapy, a post hoc analysis was conducted to assess effects of niro in patients with progressing DT and APC mutations. Methods: DeFi was a global, double-blind study that evaluated the efficacy, safety, and tolerability of niro in adults with progressing DT. Patients were randomized to oral niro (150 mg) or pbo twice-daily in continuous 28-day cycles. Descriptive post hoc analyses were conducted to assess effects of niro in patients with somatic and/or germline APC mutations. Results: Among 29 patients in DeFi with APC mutations (niro=13; pbo=16), 19 (66%) were female, 16 (55%) were aged ≤30 years, and 22 (76%) were refractory to prior treatment (with a median of 3 prior lines of treatment). In patients with APC mutation, PFS was improved with niro vs pbo (HR, 0.21 [95% CI: 0.05-1.00], P=.016). Confirmed ORR was 38% (5/13) for niro vs 13% (2/16) for pbo. Niro-treated patients also had greater reduction in median best percent change from baseline compared with pbo-treated patients in target tumor size (-29.5 vs +2.2), volumetric MRI (-71.0 vs +5.4), and T2 hyperintensity (-74.1 vs -21.0). Across all PROs assessed — Brief Pain Inventory, GODDESS DT Symptom Scale and DT Impact Scale, and European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 — patients treated with niro had numerically greater improvement from baseline at cycle 10 than pbo. Analyses were limited due to the small sample size. Adverse events in &gt;50% of niro-treated patients with APC mutation were diarrhea, nausea, rash maculopapular, and fatigue; ovarian toxicity occurred in 9 of 10 of females of reproductive potential (6 resolved, 2 lost to follow-up, 1 ongoing and receiving niro). Conclusions: Improvement in PFS, ORR, tumor imaging characteristics and PROs was observed with niro compared to pbo in patients with DT harboring APC mutations. Efficacy and safety of niro in patients with APC mutations were generally consistent with findings for the overall DeFi population, suggesting that niro can provide clinically meaningful benefit to patients with progressing DT and APCmutations. Clinical trial information: NCT03785964 .

Onset and resolution of ovarian toxicity with nirogacestat treatment in females with desmoid tumors: Updated safety analyses from the DeFi phase 3 study.

Nirogacestat is a targeted gamma secretase inhibitor approved in the United States for adults with progressing desmoid tumors. In the phase 3 DeFi study (NCT03785964) of nirogacestat, ovarian toxicity (OT) was identified as a safety signal among females of reproductive potential (FORP). This analysis further describes the incidence, presentation, and resolution of OT. Patients were randomized to twice-daily oral nirogacestat (150 mg) or placebo, taken in continuous 28-day cycles. Investigator-identified OT in FORP was based on abnormal reproductive hormone values or perimenopausal symptoms (or both). Adverse event follow-up was conducted to assess OT resolution. Post hoc analyses included return of menstruation and return of follicle-stimulating hormone (FSH) to within normal limits (WNL) (≤20.4 mIU/mL). Of 92 randomized females, 73 in the safety population were FORP (n=36 nirogacestat, n=37 placebo). OT was identified in 75% (27 of 36) receiving nirogacestat and 0% (0 of 37) receiving placebo. As of October 24, 2022, investigators reported OT resolution in 78% (21 of 27) of patients, with median OT duration of 19.1 weeks. Off-treatment resolution was reported in all 11 patients (100%) who stopped nirogacestat treatment; of these, all nine with available menstruation information experienced return of menstruation and eight had FSH WNL at last reported assessment. Resolution was reported in 10 of 14 (71%) while on nirogacestat; of these, all 10 experienced return of menstruation and seven had FSH WNL. Two patients were lost to follow-up. Most FORP treated with nirogacestat experienced OT, with the majority resolving, including all who stopped treatment, suggesting that OT is transient.

Analysis for a model incorporating [formula omitted]secretase inhibitor to explore the mechanism of ‘Amyloid-[formula omitted] rise’

The abnormal accumulation of amyloid-β (Aβ) is a key factor in the advancement of Alzheimer’s disease (AD), prompting the development of numerous strategies to reduce Aβ levels in the brain. Aβ is produced through the hydrolysis of amyloid precursor protein (APP), beginning with its cleavage by β-secretase to yield a C-terminal fragment, C99, which is further processed by γ−secretase to release Aβ. The crucial role of γ−secretase in Aβ synthesis has led to the creation of various γ−secretase inhibitors aimed at regulating Aβ concentrations. Nonetheless, experimental observations have reported a paradoxical ‘Aβ rise’ phenomenon, where lower concentrations of γ−secretase inhibitor paradoxically lead to increased Aβ levels, thereby complicating the development of these inhibitors. To elucidate the underlying mechanisms of ‘Aβ rise’, we constructed and analyzed a mathematical model incorporating γ−secretase inhibitor. Our analysis reveals bistable behavior driven by saddle–node bifurcation within the model. Through comprehensive analysis, we identified the conditions precipitating ‘Aβ rise’ and discovered that varying concentrations of γ−secretase inhibitor result in three distinct Aβ trends: (i) a consistent decrease, (ii) an initial increase followed by a decrease (‘Aβ rise’), and (iii) a decrease, subsequent increase, and final decrease. These trends are consistent with empirical findings. Our study further reveals that the emergence of ‘Aβ rise’ is primarily attributed to both lower η cleaving rate for APP and degradation rate of C99. Therefore, inhibiting ‘Aβ rise’ can be achieved by enhancing the η cleaving rate for APP and degradation rate of C99. These findings give new insights into the underlying mechanism of ‘Aβ rise’ and potential therapeutic strategies for treating AD.

Drugs targeting TGF-β/Notch interaction attenuate hypertrophic scar formation by optic atrophy 1-mediated mitochondrial fusion.

Hypertrophic scar (HS) formation is a cutaneous fibroproliferative disease that occurs after skin injuries and results in severe functional and esthetic disability. To date, few drugs have shown satisfactory outcomes for the treatment of HS formation. Transforming growth factor-beta (TGF-β)/Notch interaction via small mothers against decapentaplegic 3 (Smad3) could facilitate HS formation; therefore, targeting TGF-β/ Notch interaction via Smad3 is a potential therapeutic strategy to attenuate HS formation. In addition, optic atrophy 1 (OPA1)-mediated mitochondrial fusion contributes to fibroblast proliferation, and TGF-β/Smad3 axis and the Notch1 pathway facilitate OPA1-mediated mitochondrial fusion. Thus, the aim of this study was to investigate whether drugs targeting TGF-β/Notch interaction via Smad3 suppressed fibroblast proliferation to attenuate HS formation through OPA1-mediated mitochondrial fusion. We found that the TGF-β pathway, Notch pathway, and TGF-β/Notch interaction via Smad3 were inhibited by pirfenidone, the gamma- secretase inhibitor DAPT, and SIS3 in human keloid fibroblasts (HKF) and an HS rat model, respectively. Protein interaction was detected by co-immunoprecipitation, and mitochondrial morphology was determined by electron microscopy. Our results indicated that pirfenidone, DAPT, and SIS3 suppressed the proliferation of HKFs and attenuated HS formation in the HS rat model by inhibiting TGF-β/Notch interaction via Smad3. Moreover, pirfenidone, DAPT, and SIS3 hindered OPA1-mediated mitochondrial fusion through inhibiting TGF-β/Notch interaction, thereby suppressing the proliferation of HS fibroblasts and HS formation. In summary, these findings investigating the effects of drugs targeting TGF-β/Notch interaction on HS formation might lead to novel drugs for the treatment of HS formation.

Beneficial Fruit-Derived Phytochemicals in Treating Alzheimer’s Disease – A Review

Fruits, as a major source of nutrition have been investigated for their emerging evidence of several neuroprotective activities beneficial to Alzheimer’s disease (AD). This paper discusses recent evidence suggesting several fruit-derived bioactive substances that possess anti-AD activity. The original articles from 2011–2022 were collected from Pubmed and Scopus with related keywords and filtered. Several fruit constituents have shown significant inhibitory action against β-amyloid and tau hyperphosphorylation, either directly or indirectly via secretase inhibition. Some were also found to be cholinesterase inhibitors with specific antioxidative roles, disruptors of the neuroinflammation system, and promoters of neurogenesis and the neurogenic process. Many fruit phytochemicals remarkably alter the capability defects shown in AD animal models, such as catechin, nobiletin, and alpha-mangostin. Therefore, further investigations exploring a certain bioactive isolate or formulated dosage form and its mechanism of action are necessary to provide effective prevention and supplementary treatment for AD.

Modeling neuropathology of Alzheimer’s disease in aged patient‐derived neurons through 3D direct neuronal reprogramming

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of adult‐onset neurodegenerative diseases characterized by three hallmarks of neuropathology including the extracellular deposition of amyloid β (Aβ), intracellular tau tangles, and neurodegeneration. The mechanisms that underlying the late‐onset AD (LOAD), the most common form of AD, remain largely unresolved due to the complexity of risk factors underlying the disease. Currently, endogenously recapitulating these events in patients‐derived neurons remains a major challenge, limiting our ability to understand the biology of the disease.MethodBy optimizing the potency of microRNA‐mediated direct neuronal conversion of patient fibroblasts, we developed a matrigel‐based three‐dimensional (3D) culture system for generating cortical neurons, a subtype known to be susceptible to develop AD pathologies. For characterizing AD‐associated phenotypes such as amyloid β(Aβ) plaques, tau pathology, and neurodegeneration in the reprogrammed cortical neurons, we carried out immunofluorescence staining, RNA‐sequencing and other biochemistry‐based assays.ResultsThe 3D reprogrammed neurons derived from familial AD and LOAD patients endogenously displayed all three AD hallmark pathologies, including increased extracellular Aβ deposition, phosphorylated tau, dystrophic (beaded) neurites characterized by the colocalization between Ubquitin‐K63 and pTAU, as well as spontaneous neuronal cell death in culture without additional cellular insult. By using this 3D reprograming model, we found that treating AD neurons with β or γ secretase inhibitors at pre‐Aβ aggregation phase, significantly ameliorated Aβ formation and subsequent tauopathy and neuronal cell death, whereas treating during the post‐Aβ aggregation phase only had a mild effect. Additionally, comparative transcriptomic analysis between AD neurons and age‐matched healthy control neurons showed enrichment of genes involved in inflammatory pathways.ConclusionWe established a novel patient derived neuronal 3D direct reprogramming model which robustly recapitulates key AD hallmarks including the extracellular deposition of Aβ plaques, formation of intracellular Tau tangles, and neurodegeneration. By using this 3D platform, we further demonstrated that inhibition of Amyloid β formation reduces tauopathy and neuronal cell death. Our unique patient specific 3D model system provides opportunities to investigate the interplay between AD pathological events and could serve as a platform for screening compounds and developing personalized therapeutics for AD.

Brain volume changes caused by anti‐amyloid beta drugs and association with ARIA

AbstractBackgroundThe potential benefit of anti‐Aβ drugs to cognition in Alzheimer’s disease (AD) remains under active debate. Anti‐Aβ drugs also frequently cause accelerated changes to brain volume. This is concerning because loss of brain tissue is the proximate cause of cognitive dysfunction in AD and volume changes are supportive and objective evidence of disease progression.MethodPubMed, Embase and Clinicaltrial.gov databases were searched to evaluate brain volume changes caused by different sub‐classes of anti‐amyloid beta (Aβ) drugs trialed in patients with Alzheimer’s disease. The inclusion criteria for this systematic review and meta‐analysis were: (1) randomized controlled trials of patients treated with anti‐Aβ drugs that have demonstrated to favorably change at least one biomarker of pathological Aβ; and (2) detailed MRI data sufficient to assess the volumetric changes in at least one brain region. Amyloid‐Related Imaging Abnormalities (ARIA) were investigated when reported in clinical trials. Of the 145 trials reviewed, 31 were included in the final analyses (n = 8062 to 10279).ResultA meta‐analysis on the highest dose of each trial on hippocampus, ventricle, and whole brain revealed drug‐induced acceleration of volume changes that varied by anti‐Aβ drug class. Secretase inhibitors accelerated atrophy to the hippocampus (mean difference: ‐37.1 µL [‐19.6% relative to change in placebo]; 95% confidence interval: ‐47.0 to ‐27.1) and whole brain (‐3.3mL [‐21.8% relative to change in placebo]; 95% confidence interval: ‐4.1 to 2.5). Conversely, ARIA‐inducing monoclonal antibodies accelerated ventricular enlargement (mean difference: +2.1mL [+38.7% relative to change in placebo]; 95% confidence interval: 1.5 to 2.8) where a striking correlation between ventricular volume and ARIA frequency was observed (r = 0.86, p = 6.22×10−7).ConclusionThese findings reveal the potential for anti‐Aβ therapies to compromise long‐term brain health by accelerating brain atrophy and provide new insight into the adverse impact of ARIA. Six recommendations emerge from these findings.

Gamma Secretase Inhibition Sensitizes Pancreatic Adenocarcinoma Tumors to RT In Vivo

Pancreatic adenocarcinoma (PDAC) is an extremely aggressive cancer that lacks curative treatment options. Almost half of patients present with unresectable disease limiting treatment to non-curative options. Patients treated with neoadjuvant radiation therapy (RT) exhibit increases in fibrosis and epithelial-to-mesenchymal transition (EMT). ADAM10, an extracellular sheddase, can stimulate stromal fibrosis, EMT, and radioresistance. ADAM10 also mediates EMT through Notch signaling by cleaving its extracellular domain. Further cleavage by gamma secretase produces the Notch intracellular domain (NICD), which translocates to the nucleus and activates downstream transcriptional targets. Here, we explore whether inhibition of Notch cleavage by gamma secretase radiosensitizes PDAC tumors. Bilateral flank subcutaneous PDAC isografts were produced in 40 mice using PK5L1940 KPC cells. Intraperitoneal injections of the gamma secretase inhibitor, DAPT (5 mg/kg), were delivered daily for 7 days, starting 3 days prior to RT. A single dose of 20 Gy was administered to each flank tumor, and volumes were measured twice weekly. Colony formation assays of KPC cells were performed after RT, in the presence or absence of DAPT. Since stromal fibrosis can mediate radio-resistance in the tumor microenvironment (TME), the effect of tumor cells on Notch pathway activation in mouse fibroblasts (3T3 cells) was investigated using a luciferase reporter assay. Thus, 3T3 cells transfected with a Notch pathway luciferase reporter were incubated with PDAC cells for 48 h, followed by measurement of luciferase activity. In vivo, the combination of DAPT and RT significantly delayed tumor growth, and some tumors were completely eradicated. Mean tumor size for the combination at 21 days was 21 mm3 (range = 0-53, p = 0.005), while tumor size was 577 mm3 (range = 217-955, p = 0.69) for DAPT alone, 435 mm3 for RT alone (range = 51-932, p = 0.79), and 367 mm3 for untreated vehicle (range = 97-1144). Surprisingly, DAPT did not reduce clonogenic survival in vitro. Both ADAM10 knockout and DAPT decreased NICD cleavage and transcription of the downstream target Hes1 in vivo and in vitro. Co-culture with PDAC cells increased Notch luciferase reporter activity in fibroblasts. This effect was not mimicked by PDAC-conditioned media, suggesting a requirement for intercellular contact. Notch pathway inhibition sensitizes PDAC tumors to RT in vivo, but not in vitro, suggesting involvement of the TME. Indeed, co-culture with PDAC cells stimulates notch signaling in fibroblasts, suggesting non-cell autonomous mechanisms mediating fibrosis in the TME driving radioresistance. Future studies will determine if ADAM10 inhibition targeting PDAC cells and/or gamma secretase inhibition targeting the TME enhances radiation sensitivity in vivo by blocking fibroblast Notch signaling.

Evaluation of the anticancer activity of RIN-1, a Notch signaling modulator, in head and neck squamous cell carcinoma

Notch signalling is one of the key molecular pathways involved in cell-to-cell signal transduction. Although the mechanisms of action of the NOTCH receptors are already relatively well known, their biological implications remain unclear, especially during the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Here, we present the growth- and differentiation-modulating effects of various “next generation” small molecule Notch modulators represented by RIN-1, and CB-103, on HNSCC, compared to gamma secretase inhibitors as “conventional” NOTCH interfering compounds, like DAPT. These molecules were tested in different cell- and tissue culture conditions represented by 2D monolayer, non-adherent or spheroid culture, 3D organoid cultures, and zebrafish in vivo model. The most pronounced, pleiotropic effects were observed for the NOTCH modulator RIN-1. At the molecular level, RIN-1-dependent activation of Notch signalling led to characteristic changes in the expression of NOTCH-regulated targets, i.e., the transcriptional suppressors HES1 and HEY1, p21 (CDKN1A) cell cycle inhibitor, and pro-apoptotic BAX markers. These changes led to restriction of proliferation, growth, and reduced motility of HNSCC cells in 2D cultures. Consequently, cell cycle arrest in the G2-M phase and induction of apoptosis were observed. Similar anticancer effects were observed in 3D cultures and in the zebrafish model. In contrast, RIN-1 treatment resulted in inhibition of Notch signalling and the growth of HNSCC spheroids under non-adherent cell culture conditions. Our results suggest that modulation of Notch signalling could be used as a chemotherapeutic agent in selected patients with intact NOTCH signaling.

Treatments with the specific δ-secretase inhibitor, compound 11, promote the regeneration of motor and sensory axons after peripheral nerve injury.

Limited axon regeneration following peripheral nerve injury may be related to activation of the lysosomal protease, asparaginyl endopeptidase (AEP, δ-secretase) and its degradation of the microtubule associated protein, Tau. Activity of AEP was increased at the site of sciatic nerve transection and repair but blocked in mice treated systemically with a specific AEP inhibitor, compound 11 (CP11). Treatments with CP11 enhanced axon regeneration in vivo. Amplitudes of compound muscle action potentials recorded 4 weeks after nerve transection and repair and 2 weeks after daily treatments with CP11 were double those of vehicle-treated mice. At that time after injury, axons of significantly more motor and sensory neurons had regenerated successfully and reinnervated the tibialis anterior and gastrocnemius muscles in CP11-treated mice than vehicle-treated controls. In cultured adult dorsal root ganglion neurons derived from wild type mice that were treated in vitro for 24 h with CP11, neurites were nearly 50% longer than in vehicle-treated controls and similar to neurite lengths in cultures treated with the TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF). Combined treatment with CP11 and 7,8-DHF did not enhance outgrowth more than treatments with either one alone. Enhanced neurite outgrowth produced by CP11 was found also in the presence of the TrkB inhibitor, ANA-12, indicating that the enhancement was independent of TrkB signalling. Longer neurites were found after CP11 treatment in both TrkB+ and TrkB- neurons. Delta secretase inhibition by CP11 is a treatment for peripheral nerve injury with great potential.

RINGSIDE phase 2/3 trial of AL102 for treatment of desmoid tumors (DT): Phase 2 results.

11515 Background: For patients with desmoid tumors (DT, aggressive fibromatosis), systemic therapy that results in tumor regression, symptom improvement and durable tolerability is needed. Gamma secretase inhibitors (GSIs) have demonstrated antitumor activity against DT. AL102 is a potent, orally available, selective GSI under investigation for treatment of DT. Methods: RINGSIDE (AL-DES-01) is a Phase 2/3 study for patients with progressing DT. In the open-label Phase 2 study (Part A), adults with progressing DT (≥10% unidimensional growth within 18 months or DT-related pain requiring non-opioid medication) were randomized to three dosing regimens: 1.2 mg QD, 2 mg intermittent BIW (2 days on 5 days off), or 4 mg intermittent BIW. Patients who complete Phase 2 roll over into an open-label extension (OLE). RINGSIDE Phase 3 (Part B) is a double-blind, placebo-controlled study evaluating the chosen dose regimen from Phase 2 (1.2 mg once daily) utilizing PFS as the primary endpoint. We report updated efficacy and safety results from RINGSIDE Phase 2. Results: Enrollment of all 42 patients into Phase 2 was completed as of March 2022. As of January 3, 2023, median time on study was 10.5 months (range 0.8 – 14.7) and 30 patients (71.4%) were still on study, 10 (23.8%) of whom rolled over to the OLE. Mean age was 39.9 years, 73.8% were women and 69% had received prior desmoid cancer therapy. The best response in the evaluable population as assessed by blinded independent central review (BICR) was partial response (PR) in 6/12 patients (50%) for 1.2 mg QD, 3/13 patients (23.1%) for 4 mg BIW, and 5/11 patients (45.5%) for 2 mg BIW. Disease control rate was 100%, 91%, and 97% in these groups, respectively. A consistent pattern of deeper, more rapid and maintained response was observed with 1.2 mg QD. Median volume change (BICR) from baseline was -51.9% for 1.2 mg QD, -9.5% for 4 mg BIW, and -15.2% for 2 mg BIW at Week 16 and -76.4%, -35.5%, and -51.2%, respectively, at Week 28. Similar patterns were observed for % changes from baseline in T2 signal intensity, suggesting reduction of tumor cellularity. Consistent with the mechanism of action of GSIs, the five most common Grade 1-2 treatment-emergent adverse events (TEAEs) were diarrhea, nausea, fatigue, alopecia, and dry skin. Grade 3 drug-related TEAEs were reported in 26.2% of patients across all tested doses. There were no Grade 4, Grade 5, or serious TEAEs related to AL102 per investigator assessment. There were no new safety signals. Conclusions: In this Phase 2 study, AL102 was safe and generally well tolerated across all tested doses. The safety profile was consistent with the GSI class of drugs. Tumor response, volume reduction and T2 signal reduction were observed earlier in the 1.2 mg QD group, with deeper and maintained treatment responses. This dose was selected for study in RINGSIDE Phase 3, which is currently enrolling in multiple countries. Clinical trial information: NCT04871282 .

Population pharmacokinetic-pharmacodynamic model of nirogacestat effects on B-cell maturation antigen in healthy subjects.

8027 Background: B-cell maturation antigen (BCMA) is expressed on the membrane of normal plasma cells (PCs) and multiple myeloma (MM) cells and is the target of several investigational agents and approved products for MM treatment. Mechanistically, BCMA is cleaved from the cell surface by the enzyme gamma secretase, resulting in reduced levels of membrane-bound BCMA (mbBCMA) and release of soluble BCMA. Gamma secretase inhibitors (GSIs) have been shown to increase BCMA receptor density and potentiate the activity of several BCMA-targeted therapies in both clinical trials and in vitro studies. Although the effect of GSIs on BCMA receptor density has been well characterized in vitro, their effect on BCMA dynamics is incompletely understood in humans. This study sought to evaluate the pharmacodynamics (PD) of the GSI nirogacestat on mbBCMA in healthy subjects. Methods: A total of 23 healthy subjects were administered single doses of 50, 150, or 300 mg nirogacestat or multiple doses of 100 mg every 12 hours. Density of mbBCMA on PCs isolated from whole blood and bone marrow was measured by flow cytometry at baseline and 1, 2, 4, 8, 24, and 48 hours after single doses and 24 and 48 hours after the first dose in subjects receiving multiple doses. Nirogacestat serum concentrations were also measured at multiple time points to fully characterize the pharmacokinetics (PK). A population PK-PD model of the effect of nirogacestat on BCMA dynamics in humans was developed and used to simulate various administration dose levels and schedules to find an optimal nirogacestat regimen that continually increases mbBCMA over the dosing interval. Results: A 2-compartment model with linear absorption and elimination described serum nirogacestat in healthy subjects. A sequential PK-PD model that included an effect compartment and drug effect on mbBCMA cleavage rate described mbBCMA values. Simulations indicated that a 100-mg dose administered twice daily sufficiently maintained elevated and clinically relevant mbBCMA levels. Exposure and efficacy benchmarks from in vitro experiments and published clinical studies were used to contextualize simulations and hypothesize various dose and schedule scenarios to minimize dose of the BCMA-targeting therapy while maximizing BCMA potentiation. Conclusions: These results suggest that nirogacestat dose and schedule may have direct implications on the potentiation of BCMA-targeting therapies. Simulations suggest that the 100-mg, twice-daily nirogacestat dose results in elevated mbBCMA levels that could even allow for use of lower doses of BCMA-targeted agents while maintaining or improving efficacy. Study results along with this PK-PD model will be used to support nirogacestat dose and schedule in ongoing and planned studies in patients with MM.

Tumor volume and T2 hyperintensity changes from DeFi: A phase 3, randomized, controlled trial of nirogacestat in patients with desmoid tumors.

11514 Background: MRI tumor volume or T2 signal intensity changes may represent novel imaging techniques that could have a prognostic or predictive value for assessing response in desmoid tumors (DT). For example, hyperintense areas on T2-weighted images have been associated with active fibroblastic proliferation and ≥90% hyperintensity is associated with DT disease progression, whereas iso- or hypo-intense areas are associated with inactive sites of disease. In DeFi (NCT03785964), the novel gamma secretase inhibitor nirogacestat (n=70 patients) significantly improved the primary endpoint of PFS compared with placebo (n=72 patients; hazard ratio, 0.29 [95% CI, 0.15, 0.55; P&lt;0.001]) and significantly improved the secondary endpoint of ORR per blinded, independent central review (41% vs 8%; P&lt;0.001). Complete responses were achieved in 7% with nirogacestat and 0% with placebo. Here, we present an exploratory analysis of MRI tumor volume and T2 signal intensity changes in DeFi. Methods: Eligible adults had histologically confirmed DT that had progressed ≥20% per RECIST v1.1 within 12 months of screening. Patients were randomized 1:1 to receive nirogacestat 150 mg or placebo twice daily taken continuously in 28-day cycles. Volumetric MRI and T2 hyperintensity of the largest target tumor were evaluated at screening and every 6 cycles during the double-blind phase. MRI T2 signal intensity is represented as the ratio of hyperintensity in total tumor volume to muscle background. A blinded, independent, central radiologist reviewed all MRI scans. Results: Nirogacestat treatment led to more substantial reductions versus placebo in tumor volume and T2 hyperintensity ratio (Table). At baseline, similar proportions of patients in each arm had a T2 hyperintensity ratio ≥90% (95% vs 97%). This ratio changed from ≥90% at baseline to &lt;90% at any time after baseline in 34% of patients in the nirogacestat arm and 15% of those on placebo. Conclusions: In DeFi, nirogacestat demonstrated substantial reduction of tumor volume and T2 hyperintensity of the largest target tumors by MRI compared with placebo in adults with DT. These results are consistent with the significant improvement in ORR achieved with nirogacestat. These data suggest that volumetric MRI and T2 hyperintensity might provide additional information in the evaluation of treatment response in DT. The prognostic or predictive value of these imaging techniques in DT should be further studied. Clinical trial information: NCT03785964 . [Table: see text]