Gamma-secretase Inhibitor Treatment Research Articles

Impact of Gamma-Secretase Inhibition on the Multiple Myeloma Immune Microenvironment

Background: Therapy with monoclonal antibodies, chimeric antigen receptor (CAR) T-cells, and bispecific T-cell engagers (BiTE) has significantly changed the treatment landscape for multiple myeloma (MM). B-cell maturation antigen (BCMA), a cell surface protein highly expressed in malignant plasma cells, has been established as an effective MM cell target, however, achieving long-term responses in relapsed or refractory patients remains a challenge. Resistance to BCMA targeting may result from the enzymatic cleavage of BCMA by surface membrane gamma-secretase resulting in reduced target antigen density on MM cells, and from the soluble BCMA ectodomain that may hinder tumor cell recognition through binding to circulating therapeutic antibodies or to CAR T-cells. Methods: We have previously demonstrated that gamma-secretase inhibitors (GSIs) abrogate enzymatic function and increase BCMA surface density on plasma cells with a concomitant decrease in soluble BCMA concentrations [Pont, MJ. et al. Blood, 134(2019)]. In a phase I, first-in-human clinical trial (n=18; relapsed/refractory MM) combining the GSI, crenigacestat, with anti-BCMA CAR T-cell therapy (FCARH143), we recently demonstrated that plasma cell BCMA antibody-binding capacity increased a median of 12-fold among 17/18 (94%) of participants after they received a 5-day GSI “run-in” (25 mg orally administered QOD for 3 doses) [Cowan AJ, et al. Lancet Oncology. 24(7) 2023]. Overall, 78% of participants experienced a very good partial response or better to GSI with FCAR143, but the duration of response was significantly longer in patients without prior anti-BCMA exposure. Here, we report results from an analysis of the bone marrow aspirates collected at enrollment and again within 12 hours of the third “run-in” dose. We leveraged single-cell RNA sequencing (scRNAseq) coupled with the single-cell assay for transposase-accessible chromatin sequencing (scATACseq) to profile the transcriptome and chromatin accessibility of individual cells within the bone marrow microenvironment. Results: Single-cell multi-omic analysis was performed on 32 bone marrow mononuclear cell samples from 16 patients collected before and within 12 hours after the third oral dose of GSI. Six patients had previously been treated with BCMA-directed therapy, including antibody-drug conjugates, CAR T-cells, or BiTEs. After quality filtering, including compensation for ambient RNA, 142,498 cells were analyzed (median 6,373 cells/sample, range 613-17,150 cells/sample). Cells were automatically classified into 92 immune cell types using Celltypist and a reference dataset trained on immune sub-populations from 20 tissues in 18 studies. Comparing samples pre- and post-GSI exposure revealed a significant decrease in the abundance of plasmablasts, non-classical monocytes, germinal center B cells, mid-erythroid cells, and central memory/naïve cytotoxic T cells after GSI treatment. Additionally, gene expression analysis detected increased expression of Notch signaling genes, NEURL1 and HES1, in non-classical monocytes following GSI treatment. Although RNA expression of TNFRSF17 ( BCMA) did not significantly change, chromatin accessibility was considerably higher in patients without a history of prior BCMA-directed therapy following GSI treatment. In non-plasma cells, we observed increased chromatin accessibility after GSI treatment involving genes encoding proteins known to be cleaved by gamma-secretase, such as ERBB4, NOTCH1, NOTCH2, and TNFRSF17. Accessibility of CD38, the target of daratumumab, was significantly increased in B cells, and SLAMF7, the target of elotuzumab, was significantly increased in plasma cells. Conclusions: BCMA cleavage from myeloma cells' surface is a putative resistance mechanism to BCMA-targeting immunotherapy. This study assessed the single-cell transcriptome and chromatin accessibility in the bone marrow environment of 16 patients given GSI monotherapy to ultimately enhance the efficacy of subsequent anti-BCMA CAR T-cell therapy. We found that prior BCMA-targeted therapy resulted in reduced chromatin accessibility within the BCMA epigenome. Surprisingly, GSI administration also modified the epigenome of other gamma-secretase sensitive genes. These data provide insight into the molecular consequences of prior BCMA-targeted therapy and GSI exposure that may inform future studies.

Loss of p53 enhances the tumor-initiating potential and drug resistance of clonogenic multiple myeloma cells.

Tumor relapse and drug resistance are major factors that limit the curability of multiple myeloma (MM). New regimens have improved overall MM survival rates, but patients with high-risk features continue to have inferior outcomes. Chromosome 17p13 deletion (del17p) that includes the loss of the TP53 gene is a high-risk cytogenetic abnormality and is associated with poor clinical outcomes owing to relatively short remissions and the development of pan-drug resistant disease. Increased relapse rates suggest that del17p enhances clonogenic growth, and we found that the loss of p53 increased both the frequency and drug resistance of tumor-initiating MM cells (TICs). Subsequent RNA sequencing (RNA-seq) studies demonstrated significant activation of the Notch signaling pathway and upregulation of inhibitor of DNA binding (ID1/ID2) genes in p53-knock out (p53-KO) cells. We found that the loss of ID1 or HES-1 expression or treatment with a gamma-secretase inhibitor (GSI) significantly decreased the clonogenic growth of p53-KO but not p53 wild-type cells. GSI treatment in a small set of MM specimens also reduced the clonogenic growth in del17p samples but not in non-del17p samples. This effect was specific as overexpression of the Notch intracellular domain (NICD) rescued the effects of GSI treatment. Our study demonstrates that the Notch signaling and ID1 expression are required for TIC expansion in p53-KO MM cells. These findings also suggest that GSI may be specifically active in patients with p53 mutant MM.

Abstract 3928: Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is hematologic tumor characterized by the diffuse infiltration of the bone marrow by malignant hematopoietic cells expressing immature T cell markers. Although T-ALL currently has better cure rates primarily due to multiagent or intensified chemotherapy, the prognosis for patients who are resistant or develop relapse to therapy remains very poor. Aberrant Notch homolog-1 (NOTCH1) signaling is a major driver of T-ALL pathogenesis as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. Gamma Secretase Inhibitors (GSIs) which effectively block the activation of oncogenic protein NOTCH1 are potential candidates for the treatment of T-ALL. However, the clinical application of GSIs is hampered by severe gastrointestinal toxicity due to the inhibition of NOTCH1 signaling in the gut. Here we demonstrate that combination therapy of GSIs and a small molecule inhibitor of the neddylation pathway circumvents the GSI-induced gut toxicity in vitro and in vivo. Genome-wide CRISPR loss-of-function screen in LS174T adenocarcinoma cells revealed neddylation pathway as a main regulator of massive goblet cell differentiation upon NOTCH1 inhibition. Genetic and pharmacologic inhibition of the neddylation pathway in LS174T cells rescued GSI-induced differentiation and cell death. Mechanistically, neddylation inhibition increases the protein stability of Hairy and enhancer of split-1 (HES1), a known regulator of absorptive and secretory cell fate decisions. Combination treatment of GSI and neddylation inhibitor in C57/BL6 mice showed a profound decrease in the number of goblet cells and maintained HES1 protein levels in the intestinal epithelium compared to GSI treatment alone. Remarkably, combined treatment of GSI and neddylation inhibitor in NOTCH1-induced T-ALL mice showed leukemia regression and improved overall survival without any associated gut toxicity. Overall, these results substantiate the potential of targeting NOTCH1 and neddylation pathway in the treatment of NOTCH1-induced T-ALL. Citation Format: Carla Bertulfo, Pablo Perez-Duran, Teresa Palomero, Adolfo Ferrando. Therapeutic targeting of NOTCH1 and neddylation pathway in T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3928.

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.

Notch signaling regulates function of human mucosal-associated invariant T (MAIT) cells.

Notch signaling plays an important role in the development of T lymphocytes and regulates their effector functions. The regulatory roles of Notch signaling on T cells have been intensely investigated, but whether it involves in effector functions of mucosal-associated invariant T (MAIT) cells has never been reported. To elucidate the expression profiles of Notch receptors/ligands and to investigate their roles in human MAIT cell function. Peripheral blood mononuclear cells (PBMCs) from health donors were stimulated with or without anti-CD3/ CD28-coupled beads, recombinant IL-12/IL-18 cytokines, riboflavin- or non-riboflavin-synthesizing bacterial cultured supernatant for 24 hours. The expression profiles of Notch receptors and ligands on MAIT cells were detected by flow cytometry. PBMCs were treated with a Notch signaling inhibitor, gamma secretase inhibitor (GSI), before stimulation to investigate the impact of interfering with Notch signaling on activation and function of MAIT cells. Resting MAIT cells predominantly expressed Notch2 receptor and the ligand, Jagged 2, on their surface. Upon stimulation, MAIT cells further upregulated Notch2 and also Notch1 with its cleaved form, indicating active Notch signaling. Cytokines and cytotoxic molecules which are secreted by activated MAIT cells, were suppressed by treatment with GSI. Moreover, both TCR-dependent MAIT cell activation by microbial-derived riboflavin intermediates and TCR-independent MAIT cell activation driven by IL-18 in synergy with IL-12, were blocked by GSI treatment. Notch signaling is operating in MAIT cells and is involved in their activation both in a TCR-independent and -dependent manners.

Inhibition of the Numb/Notch signaling pathway increases radiation sensitivity in human nasopharyngeal carcinoma cells.

Radiation therapy is the primary treatment for primary nasopharyngeal carcinoma (NPC). The aim of this study is to identify the effect of the Numb/Notch signaling pathway on radiation sensitivity in human NPC cells. NPC tissues and normal nasopharyngeal tissues were collected. To evaluate the regulatory effects of the Numb/Notch signaling pathway, NPC cells were subjected to radiotherapy and various doses of the Numb/Notch signaling pathway inhibitor gamma secretase inhibitor (GSI). Next, the expression of Notch and Numb proteins was determined in NPC tissues and normal nasopharyngeal tissues, and the correlation of Notch and Numb protein expression with the clinicopathological features of NPC tissues was analyzed. Then, the effect of radiotherapy on NPC cell survival rate, survival fraction, apoptosis rate, proliferation, migration, and invasion as well as Numb/Notch signaling pathway-related molecules was detected. The results demonstrated that the Numb/Notch signaling pathway was activated in NPC tissues. Following treatment with radiotherapy and GSI, the Numb/Notch signaling pathway was inhibited. In addition, the NPC cell survival rate, survival fraction, cell proliferation, migration, and invasion were decreased, whereas the colony number and apoptosis rate were increased. Following radiotherapy and GSI treatment, Numb expression was increased, whereas Notch1, Hes1, Jagged1, and c-Myc expression was decreased. However, the greatest difference was noted upon treatment with radiotherapy +15 μM GSI. The results reported in this study suggest that a high dose of the inhibitor of the Numb/Notch signaling pathway GSI increased the radiation sensitivity in human NPC cells.

NOTCH1 Represses MCL-1 Levels in GSI-resistant T-ALL, Making them Susceptible to ABT-263.

Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

307 Dermatologic toxicities associated with chronic gamma-secretase inhibitor treatment for desmoid tumor

307 Dermatologic toxicities associated with chronic gamma-secretase inhibitor treatment for desmoid tumor

Mutations at 3' Untranslated Region (3'UTR) of NOTCH1 Are Associated with Low CD20 Expression Levels in Chronic Lymphocytic Leukemia

Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein.Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels.Methods.NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI).Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3’UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3’UTR NOTCH1 mutations (3’UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3’UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3’UTR group according to the substantially higher mutational burden detected for the 3’UTR mutation. ii) NOTCH1 protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3’UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3’UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3’UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p<0.0001) and non-trisomy 12 CLL (mean MFI in 16 3’UTR NOTCH1-mut cases = 2033.50 vs. mean MFI in 468 NOTCH1-wt cases = 3294.07; p=0.0001), and comparable to coding NOTCH1-mut cases (trisomy 12 CLL, mean MFI in 34 coding NOTCH1-mut cases = 2570.73; p=0.8530; non-trisomy 12 CLL, mean MFI in 56 coding NOTCH1-mut cases = 2538.13; p=0.1500) (Fig. 1b). Similarly, in both trisomy 12 CLL and non-trisomy 12 CLL categories, MS4A1 transcript levels were lower in 3’UTR NOTCH1-mut cases than in NOTCH1-wt cases (p=0.0274 and p=0.0072, respectively), again with expression levels comparable with coding NOTCH1-mut cases (p=0.2874 and p=0.9610, respectively, Fig. 1c). iv) Susceptibility to anti-CD20 in vitro. In keeping with CD20 levels, 3’UTR NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab, in CDC assay, than NOTCH1-wt cells (7 3’UTR NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.67% vs. 25.56%; p=0.0349; mean relative lysis upon ofatumumab: 39.26% vs. 60.64%; p=0.0286). In the same manner, coding NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab than NOTCH1-wt cases (9 coding NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.53% vs. 25.56%; p=0.0339; mean relative lysis upon ofatumumab: 30.60% vs. 60.64%; p=0.0114; Fig. 1d). v) Modulation of CD20 expression by NOTCH1 inhibition. GSI treatment increased both CD20 protein and transcript levels in 3’UTR NOTCH1-mut cases, as previously reported for coding NOTCH1-mut cases (p=0.0376 and p=0.0326, respectively; Fig. 1e,Pozzo et al., Leukemia, 2016).Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract P3-06-15: Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer

Abstract Background: Breast cancer is the second leading cause of cancer-associated death in women. Triple negative breast cancer (TNBC) accounts for 10-15% of breast cancer, lacks expression of ER, PR, and HER2 gene amplification. Due to lack of targeted therapy, TNBC has the worst prognosis. TNBC is a heterogeneous disease with six distinct subtypes. Current treatment includes combination of surgery, radiation therapy and chemotherapy. Notch signaling is increased in TNBC and predicts for worst overall outcome. The goal of the study is to identify novel Notch-biomarkers that predict sensitivity of subtypes of TNBC to Notch inhibition in both bulk and cancer stem cells. Methods: Two subtypes of TNBC, Mesenchymal stem-like MDA-MB-231 and basal like MDA-MB-468 cells were used. Notch signaling was evaluated using both RNA-sequencing and quantitative real time PCR (q-RTPCR). Cancer stem cell markers were evaluated using Aldefluor assay, CD44 high/ CD24 low expression, and mammosphere forming assay. To study the effect of chemotherapy and Notch, MDA-MB-231 and MDA-MB-468 cells were treated with carboplatin or a pan Notch inhibitor- gamma secretase inhibitor (GSI) or combination of both and cell viability was measured using the trypan blue exclusion test. Results: The RNA-seq and RT-PCR results showed that Notch3 is differentially expressed in the two cell lines. Notch3 was knocked down using siRNA and its effect on cell viability was assessed after GSI or carboplatin or combination treatment. MDA-MB-468 cells had higher Notch receptors and activity, most notably Notch3. MDA-MB-468 cells had high levels of Aldefluor whereas MDA-MB-231 cells showed higher levels of CD44 High/CD24 Low expression. MDA-MB-468 cells had higher mammosphere forming efficiency (MFE) compared to MDA-MB-231. Notch inhibition decreased MFE of MDA-MB-468 whereas it increased MFE of MDA-MB-231 cells. MDA-MB-468 cells have lower IC50 for carboplatin or GSI compared to MDA-MB-231 cells, as measured by cell viability assay. However, combination treatment lowered IC50 for GSI in MDA-MB-231 cells as compared to MDA-MB-468 cells. Since Notch3 levels were strikingly different between the two cell lines, we hypothesized that Notch3 levels are necessary for GSI sensitivity. Carboplatin treatment increased Notch3 in MDA-MB-231 cells and the increase in Notch3 could be the reason for lower IC50 of GSI during combination treatment as increased Notch3 would provide the substrate for gamma-secretase. In MDA-MB-468 cells, Notch3 knockdown significantly reduced cell viability and was similar to GSI, suggesting that GSI acts through Notch3. Furthermore, Notch3 knockdown and carboplatin treatment reduced viability comparable to carboplatin and GSI treatment, reinforcing that the GSI acts through Notch3 in TNBC. Using the Kaplan-Meier Plotter, high Notch3 predicted poor recurrence free survival post chemotherapy in patients with ER-, HER2-, basal breast cancer. Conclusions: GSI acts through Notch3 in two TNBC subtypes and combination of chemotherapy with Notch inhibition results in a better outcome as compared to either drug alone. Future experiments would elucidate the role of Notch3 inhibition in targeting cancer stem cells post chemotherapy treatment in different subtypes of TNBC. Citation Format: Shah D, Osipo C. Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-15.

Notch Signaling in Inflammation-Induced Preterm Labor.

Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.

Notch3-specific inhibition using siRNA knockdown or GSI sensitizes paclitaxel-resistant ovarian cancer cells.

Notch signaling plays an important role in ovarian cancer chemoresistance, which is responsible for recurrence. Gamma-secretase inhibitor (GSI) is a broad-spectrum Notch inhibitor, but it has serious side effects. The efficacy of Notch3-specific inhibition in paclitaxel-resistant ovarian cancers was assessed in this study, which has not yet been evaluated relative to GSI. To analyze the effect of Notch3-specific inhibition on paclitaxel-resistant ovarian cancers, we compared cell viability, apoptosis, cell migration, angiogenesis, cell cycle, and spheroid formation after treatment with either Notch3 siRNA or GSI in paclitaxel-resistant SKpac cells and parental SKOV3 cells. Expression levels of survival, cell cycle, and apoptosis-related proteins were measured and compared between groups. Notch3 was significantly overexpressed in chemoresistant cancer tissues and cell lines relative to chemosensitive group. In paclitaxel-resistant cancer cells, Notch inhibition significantly reduced viability, migration, and angiogenesis and increased apoptosis, thereby boosting sensitivity to paclitaxel. Spheroid formation was also significantly reduced. Both Notch3 siRNA-treated cells and GSI-treated cells arrested in the G2/M phase of the cell cycle. Proteins of cell survival, cyclin D1 and cyclin D3 were reduced, whereas p21 and p27 were elevated. Both GSI and Notch3 siRNA treatment reduced expression of anti-apoptotic proteins (BCL-W, BCL2, and BCL-XL) and increased expression of pro-apoptotic proteins (Bad, Bak, Bim, Bid, and Bax). These results indicate that Notch3-specific inhibition sensitizes paclitaxel-resistant cancer cells to paclitaxel treatment, with an efficacy comparable to that of GSI. This approach would be likely to avoid the side effects of broad-spectrum GSI treatment. © 2015 Wiley Periodicals, Inc.

Inhibition of the Notch1 Pathway in Chronic Lymphocytic Leukemia Promotes Apoptosis and Inhibits Proliferation

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008).Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A).Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037).Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells.In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PR02: Targeting NOTCH1 and C-MYC in humanized models of relapsed and induction failure pediatric T-ALL

Abstract Although prognosis has improved for children with T cell acute lymphoblastic leukemia (T-ALL), 20-30% of patients undergo induction failure (IF) or relapse. Leukemia-initiating cells (L-ICs) are hypothesized to be resistant to chemotherapy and to mediate relapse. We and others have shown that Notch1 directly regulates c-Myc and c-Myc is a known regulator of quiescence in stem and progenitor populations, leading us to examine whether c-Myc inhibition results in efficient targeting of T-ALL-initiating cells. We demonstrate that c-Myc suppression by shRNA or pharmacological approaches prevents leukemia initiation in mice by reducing the L-IC population. Consistent with its anti-L-IC activity in mice, treatment with the BET bromodomain BRD4 inhibitor JQ1 reduces C-MYC expression and inhibits the growth of relapsed and induction failure (IF) pediatric T-ALL samples in vitro. Although treatment of relapsed T-ALL cells with gamma secretase inhibitors (GSI) or JQ1 reduces, it does not abrogate C-MYC mRNA levels, indicating a role for dual NOTCH and MYC inhibition in relapsed disease. To test this possibility, we engrafted immunodeficient NSG mice with relapsed pediatric T-ALL cells and treated them with vehicle, GSI, JQ1 or with GSI and JQ1 and monitored survival. Although JQ1 or GSI treatment alone significantly prolonged survival, the combination therapy was more effective at extending survival. These findings reveal a requirement for MYC in mouse and human L-IC maintenance and provide evidence that NOTCH1 and MYC inhibition may be an effective therapy for relapsed/IF T-ALL patients. This abstract is also presented as Poster A48. Citation Format: Justine E. Roderick, Jessica Tesell, Alejandro Gutierrez, Thomas Look, Jun Qi, James E. Bradner, Michelle A. Kelliher. Targeting NOTCH1 and C-MYC in humanized models of relapsed and induction failure pediatric T-ALL. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR02.

Abstract 3099: Dual targeting of mTOR and Notch disrupts growth and promotes apoptosis in diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine gliomass (DIPGss) account for 10% of pediatric brain tumors and have a universally poor prognosis. Our laboratory has shown that targeting the NOTCH pathway in glioblastoma (GBM) with gamma secretase inhibitors (GSI), inhibits the growth and viability of tumor neurospheres. Primary DIPG as well as DIPG cell lines express have very high levels of NOTCH activity as measured by western blot and qPCR assays. AKT amplification is observed in primary DIPG, and TORC1/TORC2 activation occurs in DIPG cell lines as measured by western blot. The mTOR and NOTCH pathways are known to interact and alter cancer stem cell growth, stemness, and metabolism. We hypothesized that dual targeting of NOTCH and mTOR in DIPG would lead to decreased proliferation and increased apoptosis compared to targeting either pathway in isolation. In established DIPG cell lines, we observed that GSI treatment decreases NOTCH activity in a dose dependent manner as evidenced by decreased expression of NOTCH targets expression of Hes1 and Hey1 via as measured by western blot and qPCR assays. We also observed a dose-dependent reduction in DIPG proliferation and growth after gamma-secretase inhibition as measured by BrdU (Day 7, DMSO to 5 uM MRK003 had p value &amp;lt;0.05) and MTT assays (Day 5 and Day 7, DMSO to 2 uM and 5 uM MRK003 p value &amp;lt;0.05). However, inhibition of growth and increased apoptosis were not complete. We observed a similar decrease in proliferation and increase in apoptosis using the dual TORC1/TORC2 inhibitor PPT242. Combination therapy with MRK003 and PP242 led to significantly increased apoptosis compared to either treatment alone (cell cycle Sub-G1: DMSO 13.2% vs 1000nM PP242-5uM M003 28.9%). This data suggest that dual targeting of the mTOR and NOTCH pathways with GSI will decrease DIPG cell growth and proliferation, increase cell death, and reduce tumorigenicity. Citation Format: Isabella C. Taylor, Marianne Hutt-Cabezas, Melanie Weingart, Kathy Warren, Howard Chang, Javad Nazarian, Charles G. Eberhart, Eric H. Raabe. Dual targeting of mTOR and Notch disrupts growth and promotes apoptosis in diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3099. doi:10.1158/1538-7445.AM2014-3099

Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phospho-proteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle arrest. Taken together, these results indicate that Notch signaling positively regulates phosphorylation of Akt, possibly via phosphorylation of RGS19, and inhibition of both molecules affects the cell survival and cell cycle of macrophages upon LPS stimulation.

T-ALL Leukemia Cells Instructively Modulate Leukemia Niche To Suppress Normal Hematopoiesis

IntroductionAcute leukemias are often associated with the suppression of hematopoiesis. This condition is generally believed to reflect the “crowding-out” effect by the rapidly proliferating leukemia cells. Emerging data, however, has indicated that leukemia cells can instructively modulate the marrow microenvironment that is essential for the maintenance of hematopoietic homeostasis. One of the underlying mechanisms likely implicates leukemia-associated disruption of the marrow stem cell niche; however, the mechanisms by which acute leukemia employs to modulate niche architectural alterations are not well understood. A better understanding of the leukemia niche elements and the implicated molecular pathways involved in the niche regulation will facilitate the development of therapies to accelerate hematopoietic recovery and reconstitution in leukemia patients. MethodsWe used AML human NSG xenografts and the murine T-ALL model induced by the retroviral transfection of marrow progenitors with activated form of Notch1 (ICN1). In the murine T-ALL model, the transformed progenitors were co-transplanted with the wild type (WT) marrow cells into irradiated immune-sufficient C57BL/6 mice. The activated Notch1 drives leukemia transformation characterized by the expansion of an immature CD4+/CD8+ population of T progenitors. We tracked the niche location of leukemia using multi-photon intravital microscopy. The niche cell compartment and the reconstitution of co-transplanted marrow cells were characterized by flow cytometry, RT-PCR and in vitro differentiation assays. ResultsIn NSG mice receiving primary human AML cells, we observed that leukemia development was accompanied by a suppression of the host hematopoietic stem cell (HSC) compartment. A similar hematopoietic suppression was observed in the C57BL/6 mice receiving ICN1-expressing progenitors but not in the mice receiving control cells in a disease stage-dependent manner, characterized by a loss of the HSC population at the later disease stage and a prominent suppression of B lymphopoiesis in the companion non-transformed marrow compartment. Co-culturing with leukemia cells only modestly affected HSPC differentiation, indicating elements of leukemia microenvironment are also important to mediate the hematopoietic suppression.To understand how leukemia cells modulate niche, we longitudinally tracked the niche localization and the proliferation of pre-leukemia cells in lethally irradiated host mice. We found that ICN1-expressing individual pre-leukemia cells can be identified on day1 at the peri-vascular niche after adoptive transfer, and appeared as clusters around day8 followed by a rapid expansion phase 2-3 weeks after transplantation. We then demonstrated that the CD8+ leukemia cells, which are enriched for leukemia initiating cells, but not the CD8- cells, homed to the peri-vascular niche in the secondary leukemia transplanted hosts. Further, co-transplantation of leukemia cells with WT HSCs decreased HSC homing to the marrow and displaced them from the peri-vascular niche. In addition, leukemia mice displayed a marked loss of endosteal-lining osteoblasts, accompanied with a down-regulation of the transcription factors essential for the osteoblast differentiation in both osteoblastic progenitors and the multi-potent stromal cells (MSCs), and leukemia MSCs displayed a suppressed osteoblast formation when compared to control MSCs. In contrast, expressions of Notch signaling targets as well as CXCL12 were up-regulated in the leukemia stromal cells. The requirement of Notch in the alteration of leukemia niche function is supported by the observation that gamma-secretase inhibitor (GSI) treatment of mice receiving GSI-insensitive ICN1 cells attenuated the HSC suppression. ConclusionIn summary, we found a potentially common mechanism employed by different types of acute leukemia to instructively modulate niche components essential for normal hematopoiesis. Specifically, we identified that T-ALL leukemia cells driven by ICN1 induces a hematopoietic suppression through competitive displacement of the normal HSCs from the proliferating peri-vascular niche and an ablation of the endosteal osteoblasts. Our future studies will target to elucidate the mechanistic co-regulation of normal and malignant hematopoiesis by leukemia microenvironment. Disclosures:No relevant conflicts of interest to declare.

Abstract C40: Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells

Abstract In previous studies we targeted hypoxic Non-Small Cell Lung Cancer (NSCLC) tumor tissue using gamma-secretase inhibitors (GSI) in a preclinical model of advanced NSCLC. GSI treatment of mice suggested reactivation of mTORC-1 in otherwise quiescent hypoxic NSCLC tissue. Methods included immunohistochemistry and cytochemistry, immunoblotting, immunoprecipitation, siRNA-mediated gene knockdown, nucleotide and amino acid analogs incorporation, FACS analysis, various imaging techniques, Q-PCR, gene transfection, cap binding assays, RNA and antibodies arrays. GSI treatment of mice showed a re-induced phosphorylation of 4E-BP1 at T37/46 in hypoxic tumor masses. In vitro studies indicated a role for APP in this phenomenon. APP downregulation caused a rearrangement in 4E-BP1's phosphorylation pattern at residues T37/46, S65 and T70, reversed by overexpression of the APP C-terminal domain. Increased recruitment of eIF4A on the mRNA cap was observed. These events coincided with a drastic increase in the rate of global protein synthesis, an event that appeared to be mTOR-independent. Meanwhile, NSCLC cells with depleted APP were arrested in G0/G1 through a cyclin C-mediated mechanism. Cells arrested in G0/G1 with augmented rate of global protein synthesis (both cap- and IRES-dependent) increased their size and underwent a substantial necrotic cell death due to cell membrane permeabilization. These results indicate a novel role for APP in synchronizing the rate of protein synthesis and cell cycle progression, raising questions on how these processes interact in malignant transformation. The data presented here suggest that cells of different origin, including NSCLC cells, have developed some mechanisms to couple cell growth with cell cycle progression independent of overlapping signals which derive, among others, from the PI3-K pathway. APP seems to play a central role in this synchronization, and its loss of function causes cell size abnormalities and death. Citation Format: Anna Sobol, Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan Weber, Maurizio Bocchetta. Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C40.

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.

Gamma-secretase inhibition attenuates oxaliplatin-induced apoptosis through increased Mcl-1 and/or Bcl-xL in human colon cancer cells

The Notch signaling pathway plays a significant role in differentiation, proliferation, apoptosis, and stem cell processes. It is essential for maintenance of the normal colon crypt and has been implicated in colorectal cancer oncogenesis. Downregulation of the Notch pathway through gamma-secretase inhibitors (GSIs) has been shown to induce apoptosis and enhance response to chemotherapy in a variety of malignancies. In this study, we analyzed the effect of MRK-003 (Merck), a potent inhibitor of gamma-secretase, on oxaliplatin-induced apoptosis in colon cancer. Unexpectedly, gamma-secretase inhibition reduced oxaliplatin-induced apoptosis while GSI treatment alone was shown to have no effect on growth or apoptosis. We determined that the underlying mechanism of action involved an increase in protein levels of the anti-apoptotic Bcl-2 family members Mcl-1 and/or Bcl-xL which resulted in reduced Bax and Bak activation. Blocking of Mcl-1 and/or Bcl-xL through siRNA or the small molecule inhibitor obatoclax restored the apoptotic potential of cells treated with both oxaliplatin and MRK-003. Moreover, obatoclax synergized with MRK-003 alone to induce apoptosis. Our findings warrant caution when treating colon cancer with the combination of GSIs and chemotherapy, whereas other drug combinations, such as GSIs plus obatoclax, should be explored.