Gamma-secretase Inhibitor Research Articles

NEPHROPROTECTIVE EFFECT OF GAMMA-SECRETASE INHIBITOR ON SEPSIS- INDUCED RENAL INJURY IN MOUSE MODEL OF CLP.

The aim: This study was set out to assess the potential protective impact of MK0752 (a gamma secretase inhibitor) on sepsis-induced renal injury through modulation of inflammatory and oxidative stress pathways. Materials and methods: Twenty-four Swiss-albino mice aged between eight and twelve week and weighted twenty to thirty-seven grams were randomly allocated into four groups (n=6 in each group). Sham group (laparotomy without cecal ligation and puncture (CLP), sepsis group (laparotomy with CLP), vehicle-treated group (equivalent volume of DMSO before the CLP), MK0752 treated group (5 mg/kg) single daily dose for three days before the CLP. Blood samples were used to assess the serum levels of urea and creatinine. The kidneys were used to assess tissue levels of the TNF-α, IL-10, IL-6, TNFR1, VEGF, notch1, jagged1 and tissue damage by histopathological analysis. Results: The current study shows that pretreatment with MK0752 ameliorates the renal damage by significantly reducing the proinflammatory cytokines and notch1 signaling. Conclusions: Taken together, these results suggest that MK0752 could be protective against the renal injury induced by sepsis through its ameliorative impact on renal architecture and modulating cytokines and Notch1 singling pathway. Further studies regarding the role of Notch signaling pathways would be worthwhile.

Low-Dose Metformin Treatment Reduces In Vitro Growth of the LL/2 Non-small Cell Lung Cancer Cell Line.

Lung cancer maintains a relatively small survival rate (~19%) over a 5-year period and up to 80-85% of all lung cancer diagnoses are Non-Small Cell Lung Cancer (NSCLC). To determine whether metformin reduces non-small cell lung cancer (NSCLC) LL/2 cell growth, cells were grown in vitro and treated with metformin for 48 h. qPCR was used to assess genes related to cell cycle regulation and pro-apoptotic markers, namely Cyclin D, CDK4, p27, p21, and HES1. Treatment with 10 mM metformin significantly reduced HES1 expression (p = 0.011). Furthermore, 10 mM metformin treatment significantly decreased REDD1 (p = 0.0082) and increased p-mTOR Ser2448 (p = 0.003) protein expression. Control cells showed significant reductions in phosphorylated p53 protein expression (p = 0.0367), whereas metformin treated cells exhibited reduced total p53 protein expression (p = 0.0078). There were no significant reductions in AMPK, PKB/AKT, or STAT3. In addition, NSCLC cells were treated for 48 h. with 10 mM metformin, 4 µM gamma-secretase inhibitor (GSI), or the combination of metformin (10 mM) and GSI (4 µM) to determine the contribution of respective signaling pathways. Metformin treatment significantly reduced total nucleus expression of the proliferation maker Ki-67 with an above 65% reduction in Ki-67 expression between control and metformin-treated cells (p = 0.0021). GSI (4 µM) treatment significantly reduced Ki-67 expression by ~20% over 48 h (p = 0.0028). Combination treatment (10 mM metformin and 4 µM GSI) significantly reduced Ki-67 expression by more than 50% over 48 h (p = 0.0245). As such, direct administration of metformin (10 mM for 48 h) proved to be an effective pharmaceutical agent in reducing the proliferation of cultured non-small cell cancer cells. These intriguing in vitro results, therefore, support the further study of metformin in appropriate in vivo models as an anti-oncogenic agent and/or an adjunctive therapy.

NOTCH1 mutation in chronic lymphocytic leukaemia is associated with an enhanced cell cycle G1/S transition and specific cyclin overexpression: Preclinical ground for targeted inhibition.

Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G0 -G1 cell cycle phase, due to the expression of dysregulated cyclin genes. Of note, the activating nature of the NOTCH1 mutation in T lymphoblastic leukaemia also drives the dysregulation of cell cycle genes. Our goal was to comprehensively revisit the cell cycle in NOTCH1-mutated CLL (NOTCH1MUT ) to test for potential therapeutic targets. Among 378 NGS-annotated CLL cases, NOTCH1MUT cells displayed a unique transcriptome profile of G0 -G1 cell cycle components, with an overexpression of early-phase effectors, reaching a 38-, 27- and ninefold change increase for the complex elements CCND3, CDK4 and CDK6, respectively. This NOTCH1MUT cells' profile was related to more cells traversing through the cell cycle. In-vitro targeted inhibition of NOTCH1 gamma-secretase and CDK4/6 reversed the distribution of cells through the cycle phases and enhanced the killing of NOTCH1MUT CLL cells, suggesting new therapeutic approaches.

In silico identification of potential γ-secretase inhibitor of marine-algal origin: an anticancer intervention

ABSTRACT Gamma secretase (GS) activates notch signalling pathway (NSP) by liberating the truncated notch intracellular domain (NICD). The NSP is associated with the cancer development and progression, which makes GS a potential therapeutic target. Now day’s marine compounds emerged as a major source of bioactive entity. The NSP inhibition potential of marine-algal compounds has not yet been studied. Thus, in the present study, we have used molecular docking, molecular dynamics (MD) simulations, principal component analysis (PCA) and free energy and binding energy calculations to identify the potential GS inhibitors of marine-algal origin. Laminarin showed better docking score (−12.72) compared to the known GS inhibitor DAPT (−9.2). Laminarin formed H-Bond interaction with the Asp257 and Asp385 required for the catalytic cleavage activity of gamma-secretase. It potentially stabilised the structural parameters (RMSD, RMSF, Rg and SASA) of GS catalytic subunit compared to DAPT during the MD simulation. The PCA and free energy calculation revealed conformationally and energetically stable Laminarin–GS complex formation. Laminarin showed lower binding energy (−44.75 kcal/mol) with GS catalytic subunit than DAPT (−20.92 kcal/mol). In conclusion, the present study provides a marine-algal compound as a novel potential GS inhibitor, which requires further validation in experimental model.

Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma.

Vasculogenic mimicry (VM) is the process where cancer cells adopt endothelial characteristics by forming tube-like structures and perfusing channels. This phenomenon has been demonstrated in several types of solid tumors and associated with the growth and survival of tumor cells. In this study, we investigated the presence of VM formation in human pancreatic ductal adenocarcinoma (PDAC) and elucidated the molecular mechanisms underlying the VM process. In human PDAC tissues, CD31-negative, periodic acid-Schiff (PAS)-positive channels were predominantly found in desmoplastic areas, which are generally also hypovascularized. We found a positive correlation of VM capacity to tumor size and NOTCH1 expression and nuclear localization with statistical significance, implicating that Notch activity is involved with VM formation. Additionally, our data showed that the presence of growth or angiogenic factors significantly increased Notch activity in PDAC cell lines and upregulated several mesenchymal marker genes, such as TWIST1 and SNAI1, which can be inhibited by a gamma-secretase inhibitor. Our data showed that Notch signaling plays an important role in inducing VM formation in PDAC by promoting the epithelial-to-mesenchymal transition process.

Pharmacodynamic Effects of Nirogacestat, a Gamma Secretase Inhibitor, on B-Cell Maturation Antigen in Healthy Participants

Purpose: B-cell maturation antigen (BCMA) is expressed on the cell membrane of normal plasma cells (PCs) and multiple myeloma (MM) cells and is the target of several investigational agents and approved products for the treatment of MM. Low BCMA receptor density may be associated with lower response rates, less durable responses, or resistance to BCMA therapies. Mechanistically, BCMA is cleaved from the cell surface by the enzyme gamma secretase (GS), which results in reduced levels of membrane bound BCMA (mbBCMA) and generation of soluble BCMA (sBCMA). GS inhibitors (GSIs) have been shown to increase mbBCMA, and potentiation of the activity of several BCMA-targeted therapies has been demonstrated in vitro and in clinical studies in combination with GSIs. While the effect of GS inhibition on mbBCMA has been reproducibly characterized in vitro, the effect on BCMA dynamics has yet to be adequately characterized in humans. Therefore, this study sought to evaluate the pharmacodynamics of the GSI nirogacestat on mbBCMA on PCs in healthy participants. Methods: An adaptive design clinical study in healthy participants used quantitative flow cytometry to evaluate mbBCMA receptor density on PCs collected from bone marrow (BM) and whole blood (WB) following administration of nirogacestat. In the first cohort, 9 participants received a single dose of nirogacestat 150 mg, and mbBCMA density on PCs isolated from BM and WB was measured at baseline and 4, 8, 24, or 48 hours post-dose. Changes in the percentage of mbBCMA+ PCs, serum concentrations of sBCMA, and serum concentrations of nirogacestat were also determined. Based on the results of the first cohort, participants were enrolled in a second cohort in which different dose levels, treatment schedules, and post-dose collection time points were included to refine the exposure-response relationship and to better understand the dynamics of mbBCMA. Results: Nirogacestat dosing in healthy participants resulted in a rapid increase in mbBCMA density on PCs isolated from BM and WB. Maximal increases in mbBCMA (8- to 12-fold) were observed from 4 to 8 hours post-dose and returned to baseline levels by 24 to 48 hours. These changes in mbBCMA density correlated with serum concentrations of nirogacestat. The responses in BM and WB were generally similar, but there were some differences in mbBCMA dynamics. The serum nirogacestat concentrations and mbBCMA density values were used to develop a pharmacokinetic/pharmacodynamic (PK/PD) model that describes mbBCMA dynamics relative to GSI concentrations, including receptor density on PCs in both BM and WB. Results of the final PK/PD model will be presented. Conclusions: Administration of a single dose of nirogacestat in healthy participants resulted in a robust but temporary increase in mbBCMA receptor density on PCs in both BM and WB that correlated with nirogacestat serum concentrations, indicating that the turnover of mbBCMA on PCs is rapid and its abundance on the cell surface is directly regulated by GS activity. PK/PD modeling showed that sustained serum concentrations of nirogacestat are necessary for maintaining elevated levels of mbBCMA receptor density, thereby suggesting that the schedule of nirogacestat dosing may have direct implications on the potentiation of BCMA-targeting therapies. This PK/PD study supports the use of nirogacestat to increase mbBCMA levels on myeloma cells in patients receiving BCMA-directed therapy. Ongoing and planned studies are evaluating mbBCMA dynamics with nirogacestat combined with BCMA-targeted treatments in patients with MM.

Magnetismm-4: An Open Label, Phase 1B/2 Umbrella Study of Elranatamab in Combination with Other Anti-Cancer Treatments for Patients with Multiple Myeloma

Background Elranatamab (PF-06863135) is a humanized bispecific molecule that targets multiple myeloma (MM) cells via B-cell maturation antigen (BCMA) and T cells via CD3 to induce T cell-mediated cytotoxicity. In MagnetisMM-1 (NCT03269136), an ongoing, phase 1 study in patients with relapsed or refractory MM (RRMM), subcutaneous (SC) elranatamab yielded an overall response rate (ORR) of 64% and had a manageable safety profile (Jakubowiak et al, J Clin Oncol, 2022). In the phase 2 MagnetisMM-3 (NCT04649359) study in patients with heavily pretreated RRMM, SC elranatamab was well tolerated and yielded an ORR of 61% at the recommended phase 2 dose (Lesokhin et al, J Clin Oncol, 2022). Study Design and Methods MagnetisMM-4 (NCT05090566) is a phase 1B/2, open label, non-randomized, multicenter, umbrella study of elranatamab in combination with other anti-cancer treatments for patients with MM. The estimated enrollment is 105 patients. The study is split into 2 sub-studies (Figure 1). In each sub study, a 2 step-up priming dose of elranatamab will be employed to mitigate the risk of cytokine release syndrome and a Bayesian logistic regression model will be utilized for dose escalation. Sub-study A (SSA) is comprised of 2 phases in which patients will receive elranatamab + the gamma-secretase inhibitor (GSI) nirogacestat. Studies have shown that GSIs block BCMA cleavage and can potentially enhance BCMA-directed therapy (Eastman et al, Blood, 2019; Pont et al, Blood, 2019; Cowan et al, Blood, 2019). Phase 1 of SSA is designed to evaluate the safety, tolerability and select a recommended dose and regimen for the combination of elranatamab and nirogacestat. The primary endpoint for SSA Phase 1 is dose limiting toxicity (DLT) up to 35 days. Phase 2 of SSA will further evaluate the efficacy and safety of the combination of elranatamab and nirogacestat. The primary endpoint for SSA phase 2 is objective response rate (ORR) assessed every 4 weeks for ~2 years. A key secondary endpoint in both phases of SSA is the incidence of treatment-emergent adverse events (TEAEs). In sub-study B (SSB), patients will receive elranatamab + lenalidomide + dexamethasone. The dose escalation part of SSB is designed to assess the safety and tolerability of elranatamab in combination with lenalidomide and dexamethasone. The primary outcome of the dose escalation part is DLT up to 42 days. The dose expansion part of SSB is designed to evaluate the overall safety profile of elranatamab in combination with lenalidomide and dexamethasone. The primary endpoints of the dose expansion part of SSB are incidence of TEAEs and laboratory abnormalities across the duration of the study. A key secondary endpoint in SSB is ORR. Additional secondary endpoints in both SSA and SSB are time to response, duration of response, complete response (CR) rate, duration of CR, progression-free survival, overall survival, minimal residual disease negativity rate, immunogenicity to elranatamab, and elranatamab pharmacokinetics. Inclusion criteria in SSA and SSB include age ≥18 years, RRMM with ≥3 prior lines of therapy, refractory to at least one immunomodulatory drug, one proteasome inhibitor, and one anti-CD38 antibody, measurable disease by serum M-protein ≥0.5g/dL, urinary M-protein excretion ≥200mg/24h, or serum immunoglobulin free light chain (FLC) ≥10mg/dL and abnormal serum immunoglobulin kappa to lambda FLC ratio, and Eastern Cooperative Oncology Group performance status ≤1 (Table 1). Exclusion criteria include active plasma cell leukemia, amyloidosis, stem cell transplant within 12 weeks prior to enrolment or active graft-versus-host disease, POEMS syndrome, any active uncontrolled infection, impaired cardiovascular function or clinically significant cardiovascular disease within 6 months prior to enrolment, and previous administration with an investigational drug within 30 days or 5 half-lives preceding the first dose of study treatment. In SSA, patients who received prior treatment with a BCMA-bispecific antibody are excluded, and in SSB, patients who received prior treatment with BCMA-directed therapy are excluded. The study is enrolling the Phase 1 portions and is being opened at multiple sites in the United States and Canada. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Modulation of CD21 Antigen Density Enhances Chimeric Antigen Receptor (CAR-T) Cell Function

Introduction Antigen density has emerged as an important factor regulating CAR-T cell activity with low antigen density being associated with reduced potency in pre-clinical models across several targets. In clinical trials of CD22 CAR-T cells, the low and heterogeneous CD22 expression in B-ALL enabled CD22lo blasts to escape CAR-T cell detection leading to antigen dim relapses (Nat Med. 2018, 24:20-28). We have previously reported on the development of novel CD21 directed CAR-T cells (CAR21) for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) (Blood. 2021, 138:902). Donor derived CAR21 T cells targeting membrane proximal epitopes and utilizing a Fab-CAR architecture were capable of specific cytotoxicity and cytokine secretion against CD21 positive target cells in vitro and were active in vivo. However, in co-culture assays, CAR21 cells secreted less IL-2 against cell lines and primary tumors expressing CD21 at very low antigen density (<1000 copies/cell). We have shown that CD21 is expressed in ~60% of primary T-ALL samples (n=58) although at low antigen density (mean 1262 copies/cell) compared to more established targets such as CD19. We wanted to explore the impact of this low antigen density on CD21 CAR-T function and whether it was possible to upregulate CD21 density by pharmacological means. Results We have previously shown that CD21 is a direct transcriptional target of the NOTCH-MYC pathway and that PI3K/mTOR inhibition of T-ALL results in activation of this pathway and consequent CD21 up-regulation (Leukaemia. 2013, 27:650). We therefore incubated six T-ALL cell lines with four inhibitors of the PI3K/mTOR/AKT pathway (AZD5363, GDC0941, BEZ235 and rapamycin) for 48 and 72hrs and showed surface CD21 up-regulation by flow cytometry of > two-fold at both time points for all inhibitors, with BEZ235 showing the greatest fold increases across all cell lines. Incubation with the gamma-secretase inhibitor L685 resulted in significantly down regulated CD21 expression across all cell lines in keeping with the known NOTCH regulation of CD21 expression. We were next interested in the persistence of this enhanced CD21 expression after drug withdrawal. In two T-ALL cell lines, PF382 and RPMI8402, the impact of BEZ235, rapamycin and L685 was sustained at 48hrs after washing off. PI3K/ mTOR mediated CD21 manipulation did not occur in a CD21 negative T-ALL cell line, normal donor B and T cells or B-ALL patient samples suggesting the effect was specific to T-ALL. When clinically approved PI3K inhibitors (Idelalisib, Duvelisib, Copanlisib, Everolimus) were used, an increase in CD21 expression was again seen across 5 T-ALL cell lines tested. Greatest fold increases (> 2-fold) in CD21 expression were seen with the dual PI3Kα and PI3Kδ inhibitor copanlisib and the mTOR inhibitor rapamycin. No consistent up regulation of CD7 or CCR9 by flow was seen after 48-hour copanlisib incubation suggesting that the effect was specific to CD21. PI3K inhibition was additionally able to mediate 2-fold increases in CD21 expression in a CD21 low patient-derived xenograft T-ALL sample. Further, in a MOLT-4 murine xenograft model of T-ALL, 2 doses of copanlisib were able to produce a 2-fold increase in CD21 expression. These PI3K-mediated increases in CD21 antigen density translated into improved in vitro CAR21 functionality. Across 3 T-ALL cell lines - MOLT-4, CUTLL-1 and RPMI-8402, 48hr pre-incubation with both rapamycin and copanlisib prior to co-culture with CAR21 or CAR19 cells, resulted in improved CD21 specific cytotoxicity, IFN-G and IL-2 release. However, in the same co-culture assay, when copanlisib was added directly to the co-culture rather than via pre-incubation, there was a significant attenuation of IFN-G and IL-2 production by CAR21 suggesting a direct inhibitory effect on CAR-T effector functions in keeping with the known immunosuppressive effects of PI3K inhibitors. Conclusion In summary, we have shown that CD21 expression in T-ALL can be increased through PI3K pathway inhibition and that enhanced CD21 antigen density through pharmacological priming results in improved in vitro CAR21 efficacy. In vivo models analyzing the impact of copanlisib tumor priming on CAR-T efficacy and persistence are ongoing. Target antigen modulation is a promising strategy for enhancing CAR efficacy and reducing risk of antigen negative relapse in low antigen density targets.

Antibody drug conjugates for the treatment of multiple myeloma.

The treatment landscape of multiple myeloma (MM) has evolved substantially, but it remains largely incurable so new treatment options are required. Antibody drug conjugates (ADCs) are an emerging therapeutic class used in Cancer to deliver targeted therapy. ADCs are composed of three components, an antibody, a chemical linker and a payload which must be chosen carefully to be effective and safe. This alternative mechanism of action to standard treatments makes ADCs an attractive class for further development. However, several ADCs have been investigated but many have not moved further than phase 1 trials, highlighting the challenges in designing an effective and tolerable ADC. Belantamab Mafodotin is currently the only ADC licensed for MM although others are currently under evaluation. Belantamab Mafodotin demonstrated efficacy as monotherapy in triple class exposed patients and combinations are under development which maintain safety with encouraging efficacy particularly at earlier lines of therapy. Retaining an acceptable adverse event profile for ADCs remains vital for their success. Strategies to mitigate ocular events for Belantamab Mafodotin involve lower and less frequent dosing as well as the use of gamma secretase inhibitors. The optimal sequencing of ADCs within the treatment pathway including novel immunotherapies is now under evaluation.

Gamma-secretase inhibitor does not induce cytotoxicity in adult T-cell leukemia cell lines despite NOTCH1 expression

BackgroundActivated mutations in NOTCH1 are drivers of T-cell type acute lymphoblastic leukemia/lymphoma. The γ-secretase inhibitor (GSI), which suppresses the function of NOTCH1, is expected to be a molecular-targeted agent. NOTCH1 is also expressed in other malignant neoplasms. We aimed to determine the function of NOTCH1 expression and the effects of GSI on adult T-cell leukemia/lymphoma (ATL) caused by long-term human T-cell leukemia virus type I (HTLV-1) infection.MethodsWe analyzed the expression of NOTCH1 in six ATL- and HTLV-1-infected cell lines and investigated the influence of activated NOTCH1 (i.e., the cleaved form of NOTCH1) together with GSI on cell proliferation.ResultsActivated NOTCH1 found in ATL- and HTLV-1-infected cell lines was undetectable after incubation with GSI, regardless of Tax expression (HTLV-1-coded protein). Whole-exome sequencing revealed that activated NOTCH1 mutations were undetectable in six ATL- and HTLV-1-infected cell lines, regardless of abundant NOTCH1 expression. Moreover, GSI did not suppress the growth of ATL cell lines.ConclusionsThese findings suggested that NOTCH1 protein is constitutively activated but is likely a passenger during NOTCH1-mutation-negative ATL cell proliferation.

Hesperidin potentially interacts with the catalytic site of gamma-secretase and modifies notch sensitive genes and cancer stemness marker expression in colon cancer cells and colonosphere

Gamma secretase (GS) produces Notch Intracellular Domain (NICD) by trans-membrane cleavage of notch receptor. The NICD enters the nucleus and activates the notch signaling pathway (NSP) by activating notch-responsive gene transcription. Hyperactivation of NSP is related to cancer aggressiveness, therapy resistance, and poor therapy outcome, and decreased overall disease-free survival in patients. Till date, none of the GS inhibitors (GSI) has been clinically approved due to their toxicity in patients. Thus in the present study, we explored the GS catalytic site binding potential of hesperidin (natural flavone glycoside) and its effect on notch responsive gene expression in HCT-116 cells. Molecular docking, MM-GBSA binding energy calculations, and molecular dynamics (MD) simulation experiments were performed to study the GS catalytic site binding potential of hesperidin. The compound showed better GS catalytic site binding potential at the active site compared to experimentally validated GSI, N-N-(3, 5-Difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester (DAPT) in molecular docking and MM-GBSA experiments. MD simulation results showed that hesperidin forms stable and energetically favorable complex with gamma secretase in comparison to standard inhibitor (DAPT)-GS complex. Further, in vitro experiments showed that hesperidin inhibited cell growth and sphere formation potential in HCT-116 cells. Further, hesperidin treatment altered notch responsive genes (Hes1, Hey1, and E-cad) and cancer stemness/self-renewal markers expression at transcription levels. In conclusion, hesperidin produces toxicity in HCT-116 cells and decreases colonosphere formation by inhibiting transcription of notch signaling pathway target genes and stemness markers. Communicated by Ramaswamy H. Sarma

Rutin Potentially Binds the Gamma Secretase Catalytic Site, Down Regulates the Notch Signaling Pathway and Reduces Sphere Formation in Colonospheres.

Rutin, a natural flavonol, can modulate molecular signaling pathways and has considerable potential in cancer treatment. However, little is known about the effect of rutin on the notch signaling pathway (NSP) in cancer and cancer stem-like cells. In this study, we explored the effect of rutin on gamma secretase (GS, a putative notch signaling target) inhibition mediated NICD (Notch Intracellular Domain) production in colon cancer cells. Molecular docking, MM-GBSA, and Molecular dynamics (MD) simulation experiments were performed to check rutin’s GS catalytic site binding potential. The HCT-116 colon cancer and cancer stem-like cells (colonospheres) were utilized to validate the in silico findings. The NICD production, notch promoter assay, expression of notch target genes, and cancer stemness/self-renewal markers were studied at molecular levels. The results were compared with the Notch-1 siRNA transfected test cells. The in silico study revealed GS catalytic site binding potential in rutin. The in vitro results showed a decreased NICD formation, an altered notch target gene (E-cad, Hes-1, and Hey-1) expression, and a reduction in stemness/self-renewal markers (CD44, c-Myc, Nanog, and Sox2) in test cells in a time and dose-dependent manner. In conclusion, rutin inhibits the notch signaling pathway and reduces the stemness/self-renewal property in colon cancer cells and the colonospheres by targeting gamma secretase. The clinical efficacy of rutin in combination therapy in colon cancer may be studied in the future.

1488MO Initial results of phase II/III trial of AL102 for treatment of desmoid tumors (DT)

AL102 is a potent, orally available, selective gamma secretase inhibitor (GSI) under investigation as an antineoplastic agent. GSIs have antitumor activity against DT, including AL101 (2 confirmed PRs >3.5 years and >1 year; 1 stable disease for 1 year). There remains an unmet need for effective systemic therapy in DT, associated with durable tolerability, tumor regression, and symptom improvement.

LBA2 DeFi: A phase III, randomized controlled trial of nirogacestat versus placebo for progressing desmoid tumors (DT)

DT are rare, locally aggressive soft-tissue tumors without approved systemic therapy. Nirogacestat (niro), a novel oral gamma secretase inhibitor (GSI) has shown antitumor activity in patients with DT.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Gamma secretase inhibitors as potential therapeutic targets for the notch signaling pathway in uterine leiomyosarcoma (199)

Gamma secretase inhibitors as potential therapeutic targets for the notch signaling pathway in uterine leiomyosarcoma (199)

Notch signaling as a novel target for uterine leiomyosarcoma (259)

<b>Objectives:</b> The Notch signaling pathway is evolutionarily conserved and has been implicated as a potential therapeutic target in several cancers. Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with poor survival, higher prevalence in Black women than White women, and few effective adjuvant therapies. The objective of this study was to determine the expression of Notch in uLMS as a foundation for investigating the Notch signaling pathway as a potential therapeutic target in uLMS. <b>Methods:</b> We analyzed previously published mRNA sequencing (seq) datasets on LMS, including uLMS and other sarcoma patient tissue samples for expression of Notch pathway genes. We performed immunostaining for <i>NOTCH1</i> and <i>NOTCH3</i> on uterine tissue from benign hysterectomies (BH, <i>n</i>=3) and uLMS samples (<i>n</i>=30) from a multiple organ LMS tissue array (US Biomax, Inc). <i>NOTCH1</i> expression was measured as a percent positive stain per area, and <i>NOTCH3</i> expression was quantified with an H-Score. We analyzed Notch intracellular domain (ICD) protein expression in human uterine smooth muscle cell lines (hUT-SMC) and uLMS cell lines, SK-UT-1B and SK-LMS-1, by Western blot, and expression of Notch effectors, <i>HES1, HEY1,</i> and <i>NRARP,</i> by qPCR and immunofluorescence. Numerical data were reported as mean ± SEM or median ± interquartile range and compared using unpaired t-test or Mann-Whitney U, with p≤0.05 being statistically significant. <b>Results:</b> mRNA seq analysis revealed similar levels of expression for <i>NOTCH1, 2,</i> and <i>3</i> and <i>HES1</i> in LMS compared to other sarcomas. <i>NOTCH4</i> was not included in the original mRNA seq data and thus could not be evaluated for expression levels. Immunostaining revealed that median H-score of <i>NOTCH3</i> in uLMS was higher than in BH (211.4, IQR: 165.6-239.3 vs 100.3, IQR: 100.0-100.5, p≤0.05), whereas mean expression of <i>NOTCH1</i> was similar (3.7 ± 1.6% vs 1.9 ± 0.5%, p>0.05). Western blot analysis demonstrated that ICD for <i>NOTCH1-4</i> was expressed in hUT-SMC, N1ICD and N2ICD were expressed in SK-UT-1B, and N1ICD, N2ICD, and N4ICD were expressed in SK-LMS-1. mRNA expression of <i>HES1, HEY1,</i> and <i>NRARP</i> and nuclear expression of <i>HES1</i> indicate active Notch signaling in SK-LMS-1 and SK-UT-1B cells. <b>Conclusions:</b> Notch is expressed in uLMS patient tissues and uLMS cell lines. Notch signaling and distinct expression of Notch proteins in uLMS cell lines support the investigation of Notch inhibition as a potential target for treatment options in uLMS. Further studies are warranted to determine the efficacy of targeting Notch inhibition using therapies, such as gamma-secretase inhibitors alone or in combination with a cytotoxic agent.

The Role of Pharmacotherapeutic Agents in Children with Desmoid Tumors.

Desmoid tumors (DT) are rare fibroblastic, soft-tissue tumors that do not metastasize but can aggressively infiltrate tissues causing significant chronic discomfort and/or functional impairment. In the pediatric population, the incidence of DT is greatest during infancy and adolescence but can occur at any age. Dysregulated β-catenin, most commonly resulting from mutations in either CTNNB1 or germline APC (adenomatous polyposis coli) drives DT. Most cases are sporadic but some are associated with predisposition syndromes such as familial adenomatous polyposis (FAP). Historically, treatment has been surgery. However, the recurrence rate after surgery can be high. Various systemic cytotoxic chemotherapy regimens used in other soft-tissue sarcomas have been applied to DT with differing results. Given the chronic and rarely life-threatening nature of this disease and the potential short- and long-term toxicity of these regimens, especially in children, alternative non-cytotoxic interventions have been investigated. Molecularly targeted agents such as tyrosine kinase and gamma secretase inhibitors have shown activity against DT. Innovative local control therapies are being employed as alternatives to surgery and radiation. Periods of prolonged stability and spontaneous regression in the absence of therapy in some patients has prompted wider adoption of an upfront active surveillance approach in the appropriate setting. This review will briefly summarize the epidemiology, pathophysiology, and clinical presentation of DT in children, then focus on historical, current, and future pharmacotherapeutic management and finally, propose areas for future study.

Abstract 6074: Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines

Abstract Introduction: Uterine leiomyosarcoma (uLMS) is a rare tumor with poor survival and few therapeutic options. The Notch pathway is evolutionarily conserved, is active in uterine processes, has oncogenic properties in many cancers, but has not been well studied in uLMS. Cancer stem cells (CSC) are subpopulations of cancer cells that are treatment resistant. In certain tumors, activation of canonical Notch signaling, via gamma secretase, supports CSC survival and the acquisition of chemoresistance. Our objective is to identify and characterize the subpopulation of uLMS cells that are viable after treatment with the half maximal inhibitory concentration (IC50) of MK-0752, a gamma secretase inhibitor (GSI) in phase I trials. We will determine Notch signaling activity and expression of markers associated with stem-like potential in GSI resistant uLMS cells. Methods: MTT assays were performed on SK-LMS-1 and SK-UT-1B, two uLMS cell lines, to identify the IC50 for MK-0752. Cells were exposed to MK-0752 at the IC50 or DMSO (control) for 24 hours and viable cells were collected. First, expression of stemness markers, CD133, cMYC and SOX2, in uLMS cells was compared to human uterine smooth muscle cells (hUT-SMC) at baseline. After treatment with MK-0752, the remaining subpopulations of resistant uLMS cells were evaluated for Notch signaling activity by expression of HES1. Stemness marker expression was also assessed. Gene expression was measured with qRT-PCR. Mean fold change (FC) gene expression was determined by FC experimental divided by FC control, relative to expression of 18S rRNA. P≤0.05 was considered significant. Results: In untreated uLMS cells, expression of CD133 and c-MYC was significantly higher in SK-LMS-1 (fibroblast morphology) vs hUT-SMC, while expression of CD133 and SOX2 was significantly higher in SK-UT-1B (epithelial morphology) vs hUT-SMC. After treatment with MK-0752 at IC50 (427.4 µM for SK-LMS-1 or 128.4µM for SK-UT-1B) expression of HES1 was decreased in SK-LMS-1 (0.35x, p≤0.05) and increased in SK-UT-1B (2.38x, p≤0.01) in treatment resistant, viable cells as compared to untreated cells. Expression of c-MYC was decreased (0.38x, p≤0.01) and CD133 was similar (1.64x, p&amp;gt;0.05) in GSI resistant SK-LMS-1 cells, while expression of CD133 and SOX2 was similar (1.43x and 1.30x, respectively, p&amp;gt;0.05) in GSI resistant SK-UT-1B cells compared to untreated cells. Conclusions: Cellular morphology, Notch signaling activity and expression of stemness markers differs in GSI resistant SK-LMS-1 compared to GSI resistant SK-UT-1B cells. The subpopulation of MK-0752 resistant SK-LMS-1 cells have reduced Notch activity and reduced expression of stemness marker, c-MYC, while the resistant SK-UT-1B cells have increased Notch activity. Further studies are required to identify additional factors associated with uLMS resistance to GSIs and the importance of this heterogeneity of uLMS in vivo. Citation Format: Yasmin Abedin, Emily Alpert, Erica Rego, Karla Larios, Emma Cheung, Sofia Gabrilovich, Qingshi Zhao, Mark H. Einstein, Nataki Douglas. Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6074.