Nirogacestat, a γ-Secretase Inhibitor for Desmoid Tumors.

BACKGROUND: Desmoid tumors are rare, locally aggressive, highly recurrent soft-tissue tumors without approved treatments. METHODS: We conducted a phase 3, international, double-blind, randomized, placebo-controlled trial of nirogacestat in adults with progressing desmoid tumors according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Patients were assigned in a 1:1 ratio to receive the oral γ-secretase inhibitor nirogacestat (150 mg) or placebo twice daily. The primary end point was progression-free survival. RESULTS: From May 2019 through August 2020, a total of 70 patients were assigned to receive nirogacestat and 72 to receive placebo. Nirogacestat had a significant progression-free survival benefit over placebo (hazard ratio for disease progression or death, 0.29; 95% confidence interval, 0.15 to 0.55; P<0.001); the likelihood of being event-free at 2 years was 76% with nirogacestat and 44% with placebo. Between-group differences in progression-free survival were consistent across prespecified subgroups. The percentage of patients who had an objective response was significantly higher with nirogacestat than with placebo (41% vs. 8%; P<0.001), with a median time to response of 5.6 months and 11.1 months, respectively; the percentage of patients with a complete response was 7% and 0%, respectively. Significant between-group differences in secondary patient-reported outcomes, including pain, symptom burden, physical or role functioning, and health-related quality of life, were observed (P≤0.01). Frequent adverse events with nirogacestat included diarrhea (in 84% of the patients), nausea (in 54%), fatigue (in 51%), hypophosphatemia (in 42%), and maculopapular rash (in 32%); 95% of adverse events were of grade 1 or 2. Among women of childbearing potential receiving nirogacestat, 27 of 36 (75%) had adverse events consistent with ovarian dysfunction, which resolved in 20 women (74%). CONCLUSIONS: Nirogacestat was associated with significant benefits with respect to progression-free survival, objective response, pain, symptom burden, physical functioning, role functioning, and health-related quality of life in adults with progressing desmoid tumors. Adverse events with nirogacestat were frequent but mostly low grade. (Funded by SpringWorks Therapeutics; DeFi ClinicalTrials.gov number, NCT03785964.).

SOX8 Knockdown Overcomes Enzalutamide Resistance in Castration-Resistant Prostate Cancer by Inhibiting the Notch Signaling Pathway.

Castration-resistant prostate cancer (CRPC) is still challenging to treat. Dissatisfaction with androgen signal-targeted therapy forces people to look for other treatment strategies. Therefore, this study is aimed at exploring the role of SOX8/Notch signaling in CRPC. The upregulation of SOX8, Notch4, and Hes5 indicated a poor progression-free survival (PFS) in CRPC patients. The expression of these proteins was also upregulated in enzalutamide-resistant LNCaP cells (Enza-R). Moreover, knocking down SOX8 inhibited malignant biological behaviors and decreased the activation of Notch signaling in Enza-R cells. Importantly, knocking down SOX8 obviously reversed the enzalutamide resistance in Enza-R cells, while RO0429097 (a γ secretase inhibitor inactivates Notch signaling) exerted similar effects. At last, we found that both SOX8 knockdown and/or RO0429097 suppressed tumor growth and bone metastasis in vivo. Altogether, our study indicated that the SOX8/Notch signaling is involved in CRPC and that these enzymes are possible targets to develop novel treatment for CRPC.

Enzyme-substrate interface targeting by imidazole-based γ-secretase modulators activates γ-secretase and stabilizes its interaction with APP.

Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.

Gamma Secretase Inhibitors as Potential Therapeutic Targets for Notch Signaling in Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with few effective therapeutics. The Notch signaling pathway is evolutionarily conserved with oncogenic properties, but it has not been well studied in uLMS. The purpose of our study was to determine expression of Notch family genes and proteins and to investigate the therapeutic effect of γ-secretase inhibitors (GSIs), indirect inhibitors of Notch signaling, in uLMS. We determined expression of Notch genes and proteins in benign uterine smooth muscle tissue, fibroids, and uLMS samples by immunostaining and in two uLMS cell lines, SK-UT-1B (uterine primary) and SK-LMS-1 (vulvar metastasis) by RT-PCR, Western blot and immunostaining. We exposed our cell lines to GSIs, DAPT and MK-0752, and measured expression of HES1, a downstream effector of Notch. Notch proteins were differentially expressed in uLMS. Expression of NOTCH3 and NOTCH4 was higher in uLMS samples than in benign uterine smooth muscle and fibroids. Expression of NOTCH4 was higher in SK-LMS-1 compared to SK-UT-1B. Exposure of SK-UT-1B and SK-LMS-1 to DAPT and MK-0752 decreased expression of HES1 and decreased uLMS cell viability in a dose- and time-dependent manner that was unique to each GSI. Our findings suggest that GSIs are potential therapeutics for uLMS, albeit with limited efficacy.

γ-secretase inhibitor disturbs the morphological development of differentiating neurons through affecting Notch/miR-342-5p.

During neurodevelopment, differentiation of neural stem/progenitor cells (NSPCs) into neurons are regulated by many factors including Notch signaling pathway. Herein, we report the effect of a Notch signaling blocker, i.e. γ -secretase inhibitor (GSI), on this differentiating process, especially on the morphological development. NSPCs were cultured and induced to differentiate with or without GSI. The neurite outgrowth was impeded by GSI application and the expression of a Notch signaling downstream effector miR-342-5p increased with the downregulated expression of Notch effectors Hes1 and Hes5. Upregulated expression of miR-342-5p in differentiating NSPCs could shorten the neurite length of progeny neurons, which was similar to the effect of GSI. To avoid the possible influence from astrocytes into neurons, we directly applied cultured neurons, on which GSI could shorten the processes and RBP-J knockdown could also reduce the neurite length. Similarly, transfection of miR-342-5p mimics or inhibitors into PC12 cells led to shorter or longer processes of cells compared with control ones. Furthermore, in differentiating NSPCs, GSI-induced shorter neurites could be partially rescued by miR-342-5p inhibitors, and STAT3 was one of the possible targets of miR-342-5p during this differentiating process as indicated by results of Western Blot test, luciferase reporter assay and GFP reporter assay. To further demonstrate the role of STAT3, it was introduced into GSI-treated neurons and the GSI-affected neurites could also be partially rescued. In conclusion, GSI could influence the morphological development of neurons and the possible mechanism involved Notch/miR-342-5p and STAT3. These results would be informative for future therapeutic research.

γ-Secretase inhibitors suppress IL-20-mediated osteoclastogenesis via Notch signalling and are affected by Notch2 in vitro.

Rheumatoid arthritis (RA) is a chronic immune disease involving the small joints, which often causes irreversible damage. In recent years, elevated interleukin 20 (IL-20) has been observed in synovial fluid, while IL-20 receptor overexpression has been observed in synovial cells. IL-20 is a pleiotropic cytokine that participates in various immune diseases. Further understanding of the relationship between IL-20 and RA can help to identify a potential clinical treatment for RA. This study demonstrated that IL-20 can regulate osteoclast differentiation and function in a dose-dependent manner, while influencing the expression of Notch signalling. Quantitative reverse transcription polymerase chain reaction and western blotting showed that γ-secretase-inhibiting drugs can reverse the effects of IL-20. The effects of Notch2 on IL-20-induced osteoclastogenesis were investigated by immunofluorescence and Notch2 gene silencing via transfection of small interfering RNA; the results showed that Notch2 obviously affected the expression levels of the key protein NFATc1 and downstream osteoclastic proteins. In conclusion, we found that IL-20 regulated the osteoclastogenesis in a dose-dependent manner via Notch signalling, primarily by means of Notch2 activity. This study may help to find new targets for RA treatment.

NOTCH1 PEST domain variants are responsive to standard of care treatments despite distinct transformative properties in a breast cancer model.

Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications.

Intrauterine growth restriction neonates present with increased angiogenesis through the Notch1 signaling pathway.

Intrauterine growth restriction (IUGR) is associated with increased perinatal mortality and morbidity, and plays an important role in the development of adult cardiovascular diseases. This study brings forward a hypothesis that Human umbilical vein endothelial cells (HUVECs) from IUGR newborns present dysfunctions and varying changes of signaling pathways as compared to the Control group. Similar pathways may also be present in pulmonary or systemic vasculatures. HUVECs were derived from newborns. There were three groups according to the different fetal origins: normal newborns (Control), IUGR from poor maternal nutrition (IUGR1), and pregnancy-induced hypertension (IUGR2). We found that IUGR-derived HUVECs showed a proliferative phenotype compared to those from normal subjects. Interestingly, two types IUGR could cause varying degrees of cellular dysfunction. Meanwhile, the Notch1 signaling pathway showed enhanced activation in the two IUGR-induced HUVECs, with subsequent activation of Akt or extracellular signal regulated protein kinases1/2 (ERK1/2). Pharmacological inhibition or gene silencing of Notch1 impeded the proliferative phenotype of IUGR-induced HUVECs and reduced the activation of ERK1/2 and AKT. In summary, elevated Notch1 levels might play a crucial role in IUGR-induced HUVECs disorders through the activation of ERK1/2 and AKT. These pathways could be potential therapeutic targets for prevention of the progression of IUGR associated diseases later in life.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

Shear stress-induced cellular senescence blunts liver regeneration through Notch-sirtuin 1-P21/P16 axis.

BACKGROUND AND AIMS: The mechanisms involved in liver regeneration after partial hepatectomy (pHx) are complicated. Cellular senescence, once linked to aging, plays a pivotal role in wound repair. However, the regulatory effects of cellular senescence on liver regeneration have not been fully elucidated. APPROACH AND RESULTS: Mice subjected to pHx were analyzed 14 days after surgery. The incomplete remodeling of liver sinusoids affected shear stress-induced endothelial nitric oxide synthase (eNOS) signaling on day 14, resulting in the accumulation of senescent LSECs. Removing macrophages to augment LSEC senescence led to a malfunction of the regenerating liver. A dynamic fluctuation in Notch activity accompanied senescent LSEC accumulation during liver regeneration. Endothelial Notch activation by using Cdh5-CreERT NICeCA mice triggered LSEC senescence and senescence-associated secretory phenotype, which disrupted liver regeneration. Blocking the Notch by γ-secretase inhibitor (GSI) diminished senescence and promoted LSEC expansion. Mechanically, Notch-hairy and enhancer of split 1 signaling inhibited sirtuin 1 (Sirt1) transcription by binding to its promoter region. Activation of Sirt1 by SRT1720 neutralized the up-regulation of P53, P21, and P16 caused by Notch activation and eliminated Notch-driven LSEC senescence. Finally, Sirt1 activator promoted liver regeneration by abrogating LSEC senescence and improving sinusoid remodeling. CONCLUSIONS: Shear stress-induced LSEC senescence driven by Notch interferes with liver regeneration after pHx. Sirt1 inhibition accelerates liver regeneration by abrogating Notch-driven senescence, providing a potential opportunity to target senescent cells and facilitate liver repair after injury.

A Local Desymmetrization Approach to Piperidinyl Acetic Acid γ-Secretase Modulators.

A desymmetrization-based approach for the synthesis of piperidinyl acetic acid γ-secretase modulators has been developed. The synthetic sequence features the use of N-tert-butanesulfinyl imine reduction and a diastereoselective lactam formation to set up the chiral centers. The synthetic utility is demonstrated by the concise asymmetric synthesis of γ-secretase modulator GSM-1.

Discovery of the Oxadiazine FRM-024: A Potent CNS-Penetrant Gamma Secretase Modulator.

The recent approval of aducanumab for Alzheimer's disease has heightened the interest in therapies targeting the amyloid hypothesis. Our research has focused on identification of novel compounds to improve amyloid processing by modulating gamma secretase activity, thereby addressing a significant biological deficit known to plague the familial form of the disease. Herein, we describe the design, synthesis, and optimization of new gamma secretase modulators (GSMs) based on previously reported oxadiazine 1. Potency improvements with a focus on predicted and measured properties afforded high-quality compounds further differentiated via robust Aß42 reductions in both rodents and nonhuman primates. Extensive preclinical profiling, efficacy studies, and safety studies resulted in the nomination of FRM-024, (+)-cis-5-(4-chlorophenyl)-6-cyclopropyl-3-(6-methoxy-5-(4-methyl-1H-imidazole-1-yl)pyridin-2-yl)-5,6-dihydro-4H-1,2,4-oxadiazine, as a GSM preclinical candidate for familial Alzheimer's disease.

γ-secretase inhibitors, DAPT and RO4929097, promote the migration of Human Glioma Cells via Smad5-downregulated E-cadherin Expression.

Malignant gliomas are a type of central nervous system cancer with extremely high mortality rates in humans. γ-secretase has been becoming a potential target for cancer therapy, including glioma, because of the involvement of its enzymatic activity in regulating the proliferation and metastasis of cancer cells. In this study, we attempted to determine whether γ-secretase activity regulates E-cadherin to affect glioma cell migration. The human glioma cell lines, including LN18 and LN229, and the γ-secretase inhibitors, including N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and RO4929097, were used in this study. It was shown that γ-secretase activity inhibition by DAPT and RO4929097 could promote LN18 and LN229 glioma cell migration via downregulating E-cadherin mRNA and protein expressions, but not via affecting E-cadherin protein processing. In addition, γ-secretase activity inhibition was regulated by bone morphogenetic proteins-independent Smad5 activation in glioma cells. Moreover, endogenous Smad1 in glioma cells was found to play an important role in regulating E-cadherin expression and subsequent cell migration but did not affect DAPT-stimulated effects. These results help further elucidate the molecular mechanisms of γ-secretase activity regulation involved in controlling glioma cell malignancy. Information about a potential role for Smad1/5 activity upregulation and subsequent E-cadherin downregulation during inhibition of γ-secretase activity in the development of gliomas is therefore relevant for future research.

Evaluation of Virtual Screening Strategies for the Identification of γ-Secretase Inhibitors and Modulators.

γ-Secretase is an intramembrane aspartyl protease that is important in regulating normal cell physiology via cleavage of over 100 transmembrane proteins, including Amyloid Precursor Protein (APP) and Notch family receptors. However, aberrant proteolysis of substrates has implications in the progression of disease pathologies, including Alzheimer's disease (AD), cancers, and skin disorders. While several γ-secretase inhibitors have been identified, there has been toxicity observed in clinical trials associated with non-selective enzyme inhibition. To address this, γ-secretase modulators have been identified and pursued as more selective agents. Recent structural evidence has provided an insight into how γ-secretase inhibitors and modulators are recognized by γ-secretase, providing a platform for rational drug design targeting this protease. In this study, docking- and pharmacophore-based screening approaches were evaluated for their ability to identify, from libraries of known inhibitors and modulators with decoys with similar physicochemical properties, γ-secretase inhibitors and modulators. Using these libraries, we defined strategies for identifying both γ-secretase inhibitors and modulators incorporating an initial pharmacophore-based screen followed by a docking-based screen, with each strategy employing distinct γ-secretase structures. Furthermore, known γ-secretase inhibitors and modulators were able to be identified from an external set of bioactive molecules following application of the derived screening strategies. The approaches described herein will inform the discovery of novel small molecules targeting γ-secretase.