pan-Notch Inhibitor Research Articles

The bHLH transcription factor ASCL1 promotes differentiation of endocrine cells in the stomach and is regulated by Notch signaling.

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.

Abstract 2484: The role of the PI3K/Akt signaling pathway on LIN28B-mediated colorectal cancer metastasis

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death worldwide. The 5-year survival rate is 90% in patients with localized tumors, but the survival rate drastically decreases to 14% in patients with distant metastasis. Therefore, it is imperative to elucidate the molecular mechanisms underlying CRC metastasis. The RNA-binding protein LIN28B is overexpressed in over 30% of patients with CRC and is associated with poor prognosis. Indeed, our previous work revealed that LIN28B promotes colorectal polyps and tumors in a genetic mouse model (PMID: 24142874) and metastasis in a subcutaneous xenograft model of CRC (PMID: 21512136). In the present study, we investigated the mechanism by which LIN28B promotes colorectal tumor progression and metastasis. To assess LIN28B-mediated biological changes, we established CRC cells (DLD-1 and LoVo) with overexpression of LIN28B. LIN28B overexpression increased the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in CRC cells. Knockdown of claudin-1 (CLDN1) or NOTCH3 expression in LIN28B-overexpresed CRC cells, or treatment of the cells with the pan-Notch inhibitor DAPT (γ-secretase inhibitor), revealed that the PI3K/Akt pathway works downstream of the tight junction protein CLDN1 and the transmembrane receptor NOTCH3. Furthermore, cell-cell contact of CRC cells was required for the activation of the NOTCH3 and PI3K/Akt signaling pathways, suggesting for an important role of CLDN1 as a tight junction protein upstream of NOTCH3 and PI3K. Inhibition of PI3K by either the pan-PI3K inhibitor LY294002 or PI3K-α-specific inhibitor Alpelisib (BYL719) suppressed LIN28B-mediated CRC cell migration and wound healing rate. Using a mouse model of metastatic CRC, we show that Alpelisib treatment significantly reduced LIN28B-induced liver metastases formation. Interestingly, the inhibitory effect of Alpelisib was even more pronounced when Alpelisib was given in combination with the pan-Notch inhibitor DAPT, resulting in complete inhibition of visible liver metastases formation. Immunohistochemical staining revealed that Alpelisib did not affect proliferation, apoptosis, or angiogenesis of the liver metastases, whereas DAPT may inhibit angiogenesis. Taken together, our results indicate that LIN28B promotes cell migration and liver metastasis formation through CLDN1 and NOTCH3-mediated activation of PI3K signaling. Development of new therapies that target the PI3K/Akt pathway may provide an effective strategy for preventing metastases in CRC patients. This work is supported by NIH 9R01CA277795-22, NIH 5P30CA0113696 and the American Cancer Society. The authors declare no conflicts of interest. Citation Format: Alice E. Shin, Kensuke Sugiura, Kensuke Suzuki, Yasunori Masuike, Christopher J. Lengner, Anil K. Rustgi. The role of the PI3K/Akt signaling pathway on LIN28B-mediated colorectal cancer metastasis [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 2484.

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

1148P - ACCURACY a phase (P) II trial of AL101, a pan-Notch inhibitor, in recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC) patients (pts) with Notch activating mutations (Notch act mut): Preliminary safety and efficacy data

BackgroundNotch signaling plays a key role in tumorigenesis. AL101 is a γ-secretase inhibitor that potently inhibits signaling through Notch receptors 1-4. AL101 has robust antitumor activity in ACC patient-derived xenograft models with Notchact mut (AACR ‘19, Abstr 4885). In P1 testing, AL101 was well tolerated, with manageable AEs and a recommended P2 dose of 4mg IV QW (ASCO ‘18, Abstr 2515). One of the 4 responders in P1 had ACC with Notchact mut. ACC is a rare chemotherapy-refractory cancer of the secretory glands. Notchact mut are found in ∼20% of ACC pts, characterized by a particularly aggressive disease and poor prognosis. There is no proven active treatment for R/M ACC. MethodsACCURACY (NCT03691207) is an open-label, single-arm, multicenter study of AL101 (4mg IV QW) in R/M ACC pts (bone-exclusive disease allowed) with known Notch1-4act mut (ASCO ‘19, Abstr TPS6098). Pts with disease progression ≤6 months of enrollment or newly diagnosed metastatic disease are allowed. Primary endpoint: ORR by RECIST v1.1 (or modified MD Anderson bone criteria), by independent review committee (IRC). Secondary endpoints: ORR by investigator review (IR), duration of response by IRC and IR, PFS by IRC, OS, and safety. Per Simon optimal design, 12 pts are enrolled in stage 1; if≥2 pts respond, 24 additional pts are enrolled in stage 2. If≥6 pts of 36 respond, the trial is deemed positive. This design yields 5% type I error rate and 80% power, if ORR is 25%. ResultsIn stage 1, 12 pts are being treated (median of 1.5 cycles, as of May ‘19). Most pts are males, with ECOG PS of 0, and with Notch mutations in the PEST domain (Table).Table: 1148PDisposition and Baseline characteristics of patients treated with the investigational new drug AL101Table: 1148PScreened/Enrolled (signed consent), n18Screen failures, n6Treated, n12Median number of cycles, n1.5Gender, nMale Female8 4Median age, years56.5Race, nWhite Black Asian Other Not Reported N/A6 1 0 1 2 2ECOG performance status, n0 1 N/A6 4 2Disease status, nWith nodal or visceral metastases With bone-exclusive metastases10 2Type of mutations, nIn the NRR region In the PEST domain In the NRR and PEST regions3 6 3N/A=not available ConclusionsIn the stage 1 of the ACCURACY trial, 12 pts with Notch1-4act mut are being treated with the pan-Notch inhibitor AL101. The trial will advance to stage 2 if≥2 pts respond. Efficacy/safety data on these first 12 pts will be presented at the meeting. Accrual to stage 2 is ongoing. Clinical trial identificationNCT03691207. Editorial acknowledgementFrancesca Balordi, PhD, of The Lockwood Group (Stamford, CT, USA), in accordance with Good Publication Practice (GPP3) guidelines, funded by Ayala Pharmaceuticals, Inc. Legal entity responsible for the studyAyala Pharmaceuticals, Inc. FundingAyala Pharmaceuticals, Inc. DisclosureR. Ferrarotto: Honoraria (self), Advisory / Consultancy: Ayala Pharmaceuticals; Honoraria (self): Regeneron. A. Ho: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Ayala Pharmaceuticals; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Merck; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Eisai; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): AstraZeneca; Advisory / Consultancy, Research grant / Funding (self): Genentech/Roche; Research grant / Funding (self): Celldex Therapeutics; Research grant / Funding (self): Bayer; Research grant / Funding (self): Eli Lilly and Company; Research grant / Funding (self): Astellas Pharma; Research grant / Funding (self): Daiichi Sankyo; Research grant / Funding (self): Allos Therapeutics; Research grant / Funding (self): Pfizer; Honoraria (self), Advisory / Consultancy: Sanofi Genzyme; Honoraria (self), Advisory / Consultancy: Sun Pharma; Honoraria (self), Advisory / Consultancy: Regeneron; Honoraria (self), Advisory / Consultancy: TRM Oncology; Research grant / Funding (self), Travel / Accommodation / Expenses: Kura Oncology; Travel / Accommodation / Expenses: Ignyta. L.J. Wirth: Honoraria (self), Advisory / Consultancy: Ayala Pharmaceuticals; Honoraria (self), Advisory / Consultancy: Eisai. C. Rodriguez: Honoraria (self), Advisory / Consultancy: Cue Biopharma; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Merck. E. Dekel: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ayala Pharmaceuticals. R.M. Walker: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ayala Pharmaceuticals. C. Nadri-Shay: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ayala Pharmaceuticals. A. Vergara-Silva: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ayala Pharmaceuticals. All other authors have declared no conflicts of interest.

Abstract 2695: Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors

Abstract NOTCH signalling is a key development pathway whose aberrant activation plays an onco-driver role in human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss-of-function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumors harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumors. On the other hand, GSIs act as pan-NOTCH inhibitors and thereby are able to target human tumors positive for NOTCH1-NOTCH4 receptors. However, clinical development of these pan-NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in intestine, where NOTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effect on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, cMYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre-requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1st and 2nd generation NOTCH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan-NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan-NOTCH inhibitors that blocks signalling by directly targeting the NOTCH transcription complex. Here we present further in vivo characterization of the development candidate CB-103. A comprehensive in vitro study demonstrated that CB-103 acts as a pan-NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB-103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB-103 fully engages the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia. Citation Format: Rajwinder Lehal, Jelena Zaric, Michele Vigolo, Charlotte Urech, Maximilien Murone, Freddy Radtke. Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2695.

Abstract 4837: Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy

Abstract Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and currently no targeted therapy is available for MEC patients. MEC is characterized by a unique chromosomal translocation t(11;19)(q14-21;p12-13) that generates the CRTC1-MAML2 fusion. Our understanding of CRTC1-MAML2-mediated tumorigenesis and maintenance remains limited. MEC is considered a disease of stem/progenitor cells; however, the regulation of human MEC stem/progenitor cells has not yet been characterized. In this study, we focused on Notch signaling, a pathway important in regulating normal and cancerous stem cells, and investigated its regulatory role in human MEC cancer stem/progenitor cells as well as its potential therapeutic potential. First, we determined the status of Notch signaling activation in MEC cells. Western blotting showed the presence of cleaved active NOTCH1 and the expression of Notch downstream target gene HES1 in human CRTC1-MAML2 fusion-positive MEC cells, indicative of active Notch signaling in MEC. Second, we utilized two approaches of blocking endogenous Notch signaling in human MEC cells in vitro and in vivo, i.e. dnMAML1, a pan-Notch inhibitor that blocks the formation of the Notch transcriptional activation complex, and dibenzazepine (DBZ), a γ-secretase inhibitor that interferes with Notch receptor processing. We observed that Notch signaling inhibition had no effect on the proliferation of bulk MEC cells, but significantly reduced oncosphere forming capacity and ALDH-positive population in human MEC in vitro, suggesting that Notch signaling contributes to the maintenance of MEC stem/progenitor cells. Further, Notch signaling inhibition significantly reduced the growth of human MEC xenografts, indicating an essential role of Notch signaling in MEC growth in vivo. Finally, since we previously showed that the CRTC1-MAML2-induced autocrine AREG-EGFR signaling was required for the growth and survival of human MEC cells, we reasoned that simultaneous targeting of Notch and EGFR signaling will eliminate cancer stem and non-stem cell populations, likely improving therapeutic efficacy. Indeed, concurrent inhibition of Notch and EGFR signaling via respective DBZ and Erlotinib synergistically blocked the colony and oncosphere formation capacity of human MEC cells in vitro and in vivo. Collectively, our data demonstrate that Notch signaling is a critical regulator for maintaining MEC cancer-initiating cells and that the combination strategy targeting Notch and EGFR signaling is a potentially effective approach for MEC treatment. Citation Format: Wei Ni, Zirong Chen, Xin Zhou, Rongqiang Yang, Frederic Kaye, Lizi Wu. Role of Notch signaling in human mucoepidermoid carcinoma and combined targeting of Notch and EGFR signaling as an anti-cancer strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4837.

ACCURACY: phase (P) 2 trial of AL101, a pan-Notch inhibitor, in patients (pts) with recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC) with Notch activating mutations (Notchact mut).

TPS6098 Background: Notch signaling plays a key role in tumorigenesis. Notch cleavage by γ-secretase frees the Notch intracellular domain, which promotes the expression of target genes involved in cancer. AL101, a small molecule, is a γ-secretase inhibitor that potently inhibits Notch1-4, resulting in robust antitumor activity in vivo (PMID 26005526), including ACC xenograft models with Notchact mut (Ferrarotto, AACR 2019, Abstr 4885). Three P1 trials tested AL101 as monotherapy or in combination regimens in > 200 solid/hematologic cancer pts. In the P1 trial of AL101 monotherapy, conducted in 94 pts with advanced/metastatic solid tumors refractory to standard therapies (Tx), AL101 was generally well tolerated, with manageable AEs, and the recommended P2 dose was 4 mg IV once weekly (QW; El-Khoueiry, ASCO 2018, Abstr 2515). 4 pts had objective responses, 2 of those had Notchmut (1 of which had ACC). ACC, a rare cancer that most commonly develops in the major salivary glands, but can also arise in minor salivary glands in the trachea, lacrimal gland, and other sites, is refractory to chemotherapy, with a high recurrence rate. Notchact mut are found in a subset of ACC pts (11%–22%), with particularly aggressive disease and poor prognosis. There is no proven active treatment for R/M ACC pts (PMID 27870570). Methods: ACCURACY (NCT03691207) is an open-label, single-arm, multicenter study of AL101 (4 mg IV QW) in pts with R/M ACC (bone-exclusive disease included) with known Notch1-4act mut. Pts with disease progression within 6 months of enrollment or newly diagnosed metastatic pts are allowed; pts who received > 3 prior systemic Tx are excluded. Primary endpoint: ORR by RECIST v1.1 (or modified MDA bone criteria), by independent review committee (IRC). Secondary endpoints: ORR by investigator review (IR), duration of response by IRC and IR, PFS by IRC, OS, and safety. Per the Simon optimal design, 12 pts are enrolled in stage 1; if ≥2 pts respond, 24 additional pts are enrolled in stage 2. If ≥4 pts in stage 2 respond, the trial is deemed positive. This design yields 5% type I error rate and 80% power, if ORR is 25%. 4 of planned 36 pts have been enrolled as of 2/12/19. Clinical trial information: NCT03691207.

Abstract P4-07-05: A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant

Abstract The Notch signaling pathway plays a central role in cellular differentiation, growth and stem cell maintenance. Expression and activation of Notch pathway receptors and ligands have differential outcomes depending on the tissue, localization and cell type. When Notch pathway is aberrantly activated by genetic lesions, it can be a major driver for Notch-dependent cancers and can cause resistance to standard of care treatment. We and others have shown that in Estrogen Receptor (ER)-positive breast cancers, estrogen deprivation caused by endocrine therapy results in Notch1 and Notch4 activation. In turn, Notch1 stimulated ER-dependent transcription in the absence of estrogen, causing endocrine resistance. Combinations of Notch inhibitors and endocrine therapy are effective in preclinical models of ER-positive breast cancer and have shown promising signals in early clinical trials. Cellestia's lead development candidate CB-103 is a small molecule, first-in-class, oral pan-Notch inhibitor. CB-103 selectively blocks Notch pathway activation-related gene transcription through binding to a Notch specific protein in the transcription factor complex. The blockade occurs by protein-protein interaction inhibition with a binding site critical for the assembly of the Notch transcription complex. This is a unique mode of action, which allows blocking Notch signaling regardless of the genetic lesions which have activated the pathway. We have performed mammosphere assays to test the potency and efficacy of this compound on stem cell ability to form sphere. We used two different doses of CB-103; either alone or in combination with a fixed dose of Fulvestrant (30nM), a SERD, in our mammosphere assays. Two different ER+ luminal,endocrine resistant cell lines were tested and compared with their parental controls. From our data, it's apparent that there is a synergistic effect when using CB-103 in combination with Fulvestrant. It's also evident that the efficacy of CB-103 is maximal maximizes at the lowest concentration tested in our assays. The combination was effective in 3 out of 4 models. However, the effect of CB-103 on MCF7-TAMR either as a single agent or in combination was not statistically significant. Cellestia has received regulatory approval to start clinical development with CB-103 in a first-in-human study Phase l – lla study investigating safety (Ph l) and preliminary single agent efficacy (Ph lla) of CB-103 in patients with advanced solid cancers and haematological malignancies. Our data support the notion of testing this agent in ER-positive breast cancer in combination with SERDs. Citation Format: Majumder S, Crabtree J, Hossain F, Murone M, Lehal R, Miele L. A novel, first in class Notch transcriptional inhibitor, CB-103 has activity on luminal breast cancer stem cells in combination with fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-05.

Ets1 Enhances Context-Dependent Notch1 Activity in T-Cell Leukemia

The discovery of NOTCH1 as the most frequently mutated oncogene in T-ALL patients raised hopes for targeted therapy in this cancer. Unfortunately, in clinical trials, the pan-Notch inhibitor GSI caused excessive GI toxicity. Mice treated continuously with GSI die from intestinal stem cell loss and severe intestinal secretory cell metaplasia. Intermittent dosing of GSI is tolerable, but has weak anti-cancer effects. Thus, the challenge has been to find ways to selectively disable Notch in T-ALL. Our idea to meet this challenge stems from work by others showing that Notch cannot activate enhancers by itself. Notch requires a favorable “chromatin context” at its enhancers that is created by cooperating transcription factors. In theory, one could target cell-specific factors at these enhancers in order to avoid the intolerable effects of pan-Notch inhibition. In support of this, others showed that ubiquitous deletion of the T-cell specific Notch-dependent Myc enhancer in mice impairs T-ALL proliferation and thymopoiesis, but has no effect on other tissues. We previously showed that the transcriptional coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell Myc enhancer. However, it was unclear what other factors promote context-dependent Notch activity.Ets1 is an attractive candidate. It can bind nucleosome-occupied regions in T-cell precursors and most Notch response elements in T-ALL cells, including the T-cell MYC enhancer. To investigate its importance, we generated conditional Ets1 knockout mice. Deletion of Ets1 in hematopoietic cells using the VavCre transgene caused a 21-fold loss of thymocytes starting at the earliest stage. This was 4-fold more severe than the loss of thymocytes in Notch-deficient mice. Deletion of Ets1 using a ubiquitous tamoxifen-inducible Cre caused a Notch loss-of-function phenotype in the intestine with a 1.4 to 2.3-fold increase in goblet cells. This was milder than the effects of GSI (3.3 to 4.2-fold increase). ~64% of the Ets1-deleted mice died from unclear causes. In vivo deletion of Ets1 in Notch1-induced murine T-ALLs reduced blast counts by 30-fold and prolonged survival. In a panel of human T-ALL cell lines, on average, knockdown with two different shEts1 reduced proliferation by 2 and 9-fold respectively over ~1.5 weeks of culture. This was superior to the effects of GSI (up to 2-fold inhibition). A small molecule inhibitor of Usp9x, the deubiquitinase of Ets1, induced Ets1 protein degradation and impaired T-ALL cell proliferation with submicromolar GI50. In PDX models, shEts1 reduced circulating blasts by 44-fold and prolonged survival.To identify the mechanism by which Ets1 promotes T-ALL, we performed endogenous co-IP assays, which showed that Ets1 interacts with Notch1 and its cofactor Zmiz1. Further, Ets1 binding by ChIP correlated with Zmiz1 binding (R2=0.93). Knockdown of Ets1 reduced Zmiz1, Ets1, and Notch1 binding to enhancers of major T-ALL oncogenes, MYC and IL7R. RNA-Seq showed that Ets1 co-regulates the expression of ~30% of Notch1 target genes. Multiple MSigDB enrichment analyses of both Ets1 and Notch-regulated genes showed that the MYC and MTORC pathways were the #1 or #2 most enriched list. Enforced expression of Myc partially rescued the proliferation of human T-ALL cell lines deprived of Ets1. Based on these data, we predicted that Ets1 inhibition would sensitize enhancers to Notch inhibition. Accordingly, Ets1 withdrawal promoted the effects of GSI in repressing Myc expression and cell proliferation. Further, in our mouse model of Notch-induced T-ALL, Ets1 deletion in combination with intermittent doses of GSI reduced blast counts and prolonged survival more effectively than either treatment alone.Our data support an emerging model in which cofactors like Ets1 create a favorable chromatin context for Notch1 to activate a subset of response elements. The context dependence of Ets1 action, which promotes certain oncogenic signals of Notch1 in T cells, might be clinically relevant. Ets1 deprivation inhibited thymopoiesis and leukemic proliferation more effectively and with less intestinal toxicity than Notch deprivation. Our data suggest that inhibiting Ets1, possibly through targeted protein degradation, would combat important drivers of the Notch pathway with reduced adverse effects linked to pan-Notch inhibition. DisclosuresNo relevant conflicts of interest to declare.

PO-107 Notch signalling pathway is required for proliferation, repair and differentiation of primary human lung stem cells upon irradiation

IntroductionLung cancer is the leading cause of cancer death in western countries. While significant progress has been made in terms of treatment, radiotherapy is limited by dose-limiting side-effects. Reducing side-effects may improve tumour control by dose-escalation and treatment-time. The NOTCH signalling pathway plays an important role in differentiation of the airway epithelium and its deregulation is associated with lung cancer. However, the mechanism by which NOTCH inhibition integrates with airway repair is unknown. What is currently lacking are primary human lung tissue models that enable robust evaluation of the effects of treatment which can guide the selection of combination treatments for lung cancer.Material and methodsWe therefore investigated the effects of inhibiting NOTCH in human basal stem cells from normal primary bronchial epithelial cells isolated from lung cancer patients undergoing lobectomy. Basal stem cells isolated from 3 different patients were treated with the pan NOTCH inhibitor (DBZ) and irradiated with 2–4 Gy. Incucyte experiment, Edu/PI staining and clonogenic assay were used to evaluate proliferation and clonal capacity. To evaluate DNA damage response 53 bp1 staining was performed. To study Notch inhibition in basal cells differentiation the tracheal epithelium of 2–5 Gy irradiated mice was seeded in ALI system and confocal stainings were performed.Results and discussionsIncucyte experiment revealed a reduced proliferation of stem cells upon NOTCH inhibition which is further reduced upon irradiation. NOTCH inhibition induces accumulation of the cells in GO-G1 phase and a significant reduction in S phase which was exacerbated upon irradiation. The reduction in proliferation also led to a reduction in long term survival. We observed activation of the DNA damage response pathway by expression of pCHK2, pATM upon NOTCH inhibition which was enhanced by radiation. 53 BP1 foci 24 hours after RT was higher when NOTCH was inhibited demonstrating that NOTCH is required for the DNA damage response. In cultured primary stem cells from irradiated murine tracheas NOTCH inhibition induced a fate change by blocking mucous differentiation and inducing cillilary differentiation and enhancing progenitor p63 +cells. However, overall proliferation was reduced.ConclusionNOTCH pathway is needed for stem cell survival and DNA damage response upon irradiation of undifferentiated progenitors. These studies underscore the importance of studying the effect of targeted anti-cancer drugs for normal tissue effects.

A phase I study of AL101, a pan-NOTCH inhibitor, in patients (pts) with locally advanced or metastatic solid tumors.

2515Background: Notch signaling can be deregulated in human cancer. AL101 (formerly BMS-906024), a gamma secretase inhibitor that potently inhibits all Notch receptors, was evaluated. The primary o...

66P - Pharmacological activity of CB-103: An oral pan-NOTCH inhibitor targeting the NOTCH transcription complex

66P - Pharmacological activity of CB-103: An oral pan-NOTCH inhibitor targeting the NOTCH transcription complex

Abstract B061: Targeting lymphomas with the novel first-in-class pan-NOTCH transcription inhibitor CB-103

Abstract Background: NOTCH pathway is recurrently activated in human cancers, including lymphomas, and overactivation of the pathway has been correlated to a worse prognosis in patients with solid tumors, leukemias, and lymphomas, making it an attractive target for treatment (Rossi et al, JEM 2012; Rossi et al, Blood 2012; Kridel et al, Blood 2012). However, so far, pharmacologic approaches to inhibit its activity are limited to the use of nonspecific gamma secretase inhibitors and blocking antibodies with limited application. None of these therapeutic agents have been approved yet for cancer treatment. CB-103 is a novel first-in-class pan-NOTCH inhibitor that blocks signaling by directly and specifically targeting the NOTCH transcription complex, and it has shown preclinical activity in a variety of solid tumor and leukemia models (Lehal et al, AACR 2017). Here, we present the very first results with CB-103 in a large panel of B and T cell lymphoma cell lines. Methods: 58 human lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 9 mantle cell lymphomas; 6 marginal zone lymphomas; 9 T-cell lymphomas; 7 others], 2 murine and 1 canine, were exposed to increasing doses of CB-103 for 72h. Cell proliferation was measured by using the MTT assay. Transcriptome analysis was performed using the Illumina HumanHT 12 Expression BeadChips and with the HTG EdgeSeq Oncology Biomarker Panel, a targeted RNA-Seq investigating over 2500 genes, followed by a limma moderate t-test, and GSEA. Results: CB-103 presented a median IC50 above 20 μM across the whole panel of 61 lymphoma cell lines (range from 400 nM to > 20 μM), without significant differences among lymphoma subtypes. Importantly, 13/58 cell lines had an IC50 <10 μM and were defined as sensitive. These cell lines were derived from ABC DLBCL (3/7, 43%), GCB DLBCL (5/20, 25%), MCL (3/9, 33%), marginal zone lymphoma (1/6, 17%), and canine DLBCL (1/1, 100%). The sensitive cell lines, when compared with those presenting IC50 >10 μM, presented a gene expression signature significantly enriched with genes involved in the epithelial-mesenchymal transition (FDR 0.0014), a Notch-related process. Indeed, the transcripts higher in the sensitive cell lines included, among others, DLL1 (P 0.010; Log Fold Change 2.39), BAMBI (P 0.046; Log Fold Change 0.92), MCM7 (P 0.015; Log Fold Change 1), and ULK1 (P 0.045; Log Fold Change 2.39), all connected to NOTCH pathway. Finally, 10 of the CB-103 sensitive human cell lines were also exposed to another NOTCH inhibitor, the gamma secretase inhibitor LY-3039187, which resulted active only in one MCL cell line, while it had IC50 >20 μM in the other cell lines tested. Conclusions: CB-103 presented in vitro antilymphoma activity in a restricted number of B cell lymphoma cell lines, which were characterized by a NOTCH-related gene expression signature. Further studies with the compound are ongoing and a phase I is planned to start in Q3 2017 (EUDRACT Number 2017-001491-35). Citation Format: Filippo Spriano, Chiara Tarantelli, Alberto Arribas, Eugenio Gaudio, Luciano Cascione, Luca Aresu, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Maximilien Murone, Dirk Weber, Rajwinder Lehal, Freddy Radtke, Francesco Bertoni. Targeting lymphomas with the novel first-in-class pan-NOTCH transcription inhibitor CB-103 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B061.

411P - Pharmacological activity of CB-103: An oral pan-NOTCH inhibitor with a novel mode of action

Reference EPFL-CONF-232358View record in Web of Science Record created on 2017-11-08, modified on 2017-11-20

T Cell-Restricted Notch Signaling Contributes to Pulmonary Th1 and Th2 Immunity during Cryptococcus neoformans Infection.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans Inhibition of T cell-restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ-, IL-5-, and IL-13-producing CD4+ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8+ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.

Abstract 4851: NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851

Notch Signaling Mediated By Dll1/4 Notch Ligands Controls the Pathogenesis of Both Multi-Organ System Non-Sclerodermatous and Sclerodermatous Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT) with limited success of current therapeutic options. We have shown that Notch inhibition in donor T cells or systemic neutralization of the Notch ligands Delta-like1 and Delta-like4 (Dll1/4) prevents acute GVHD in multiple mouse allo-HCT models (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., JCI 2013). However, the role of Notch signaling in cGVHD remains unknown. To address this question, we used genetic and antibody-mediated strategies of Notch inhibition in two established and pathophysiologically distinct mouse models of cGVHD characterized by dominance of sclerodermatous changes (Scl) or non-Scl multi-organ system disease that includes bronchiolitis obliterans (BO), respectively.In the B10.D2→BALB/c MHC-matched, minor antigen mismatched model of Scl-cGVHD, transgenic expression of the pan-Notch inhibitor DNMAML in T cells induced high level protection from cGVHD as assessed using clinical and pathological scoring criteria. Systemic antibody-mediated blockade of Dll1/4 Notch ligands prevented cGVHD when given early after allo-HCT, with dominant effects of Dll4 inhibition alone. However, Dll4 blockade provided no therapeutic benefits if delayed by 48 hours after allo-HCT or when targeting fully established Scl-cGVHD, suggesting that Dll4-mediated Notch signals are critical in this model when delivered to T cells early after allo-HCT. To understand the impact of Notch inhibition during priming of alloantigen-specific T cells, we studied immunodominant Vβ3+CD4+ T cells that expand in response to a host superantigen encoded by Mtv6 in BALB/c recipients. Both control and Notch-inhibited Vβ3+CD4+ T cells upregulated activation markers and expanded to comprise >80% of donor T cells in lymphoid and target organs by day 6. However, Notch inhibition markedly decreased IFN-γ, TNF-α, and IL-17 production in these cells, while expanding Vβ3+CD4+FoxP3+ regulatory T cells. Thus, Notch inhibition preserved in vivo T cell proliferation and expansion, but tilted the balance in favor of alloantigen-specific regulatory T cells over highly inflammatory effector T cells. In the B6→B10.BR MHC-mismatched model of allo-HCT, characterized by prominent germinal center (GC) responses and BO-cGVHD, genetic pan-Notch inhibition in T cells with DNMAML blocked GC formation and provided long-lasting high level protection from BO, as assessed using pulmonary function tests to measure resistance, elastance and compliance, as well as pathological examination (day 56 after allo-HCT). Flow cytometric evaluation showed that follicular helper T cells (CD4+PD-1hiCXCR5+) and cells with a GC B cell phenotype (CD19+GL7+CD95hi) were markedly decreased in numbers. Genetic deletion of Notch1 or Notch2 in donor T cells also prevented the immunological manifestations of cGVHD. Antibody-mediated blockade of the Notch ligands Dll1, Dll4, or both in the peri-transplant period protected recipients from BO-cGVHD. In contrast to our observations in Scl-cGVHD, delayed Dll1/4 blockade starting at day 28 after allo-HCT preserved pulmonary function, decreased GC formation and alloantibody deposition in target tissues, and ameliorated BO-cGVHD. Thus, Dll1/4 inhibition could provide therapeutic benefits even in established BO-cGVHD.Altogether, we identified a key role for early Notch signals in alloantigen-specific T cells that define subsequent cGVHD pathogenesis. Interference with Notch signaling early after allo-HCT provided long-lasting protection from cGVHD. In addition, our observations in the multi-organ system BO-cGVHD model suggest a therapeutic potential for delayed inhibition of Dll1 or Dll4 Notch ligands in an active disease setting driven by alloantibody formation. Our preclinical data suggest that Notch signaling should be explored as a novel druggable target to prevent or treat different forms of cGVHD. DisclosuresYan:Genentech, Inc.: Employment, Equity Ownership. Siebel:Genentech Inc.: Employment, Equity Ownership.

High selective pressure for Notch1 mutations that induce Myc in T-cell acute lymphoblastic leukemia

AbstractActivating NOTCH1 mutations are frequent in human T-cell acute lymphoblastic leukemia (T-ALL) and Notch inhibitors (γ-secretase inhibitors [GSIs]) have produced responses in patients with relapsed, refractory disease. However, sustained responses, although reported, are uncommon, suggesting that other pathways can substitute for Notch in T-ALL. To address this possibility, we first generated KrasG12D transgenic mice with T-cell–specific expression of the pan-Notch inhibitor, dominant-negative Mastermind (DNMAML). These mice developed leukemia, but instead of accessing alternative oncogenic pathways, the tumor cells acquired Notch1 mutations and subsequently deleted DNMAML, reinforcing the notion that activated Notch1 is particularly transforming within the context of T-cell progenitors. We next took a candidate approach to identify oncogenic pathways downstream of Notch, focusing on Myc and Akt, which are Notch targets in T-cell progenitors. KrasG12D mice transduced with Myc developed T-ALLs that were GSI-insensitive and lacked Notch1 mutations. In contrast, KrasG12D mice transduced with myristoylated AKT developed GSI-sensitive T-ALLs that acquired Notch1 mutations. Thus, Myc can substitute for Notch1 in leukemogenesis, whereas Akt cannot. These findings in primary tumors extend recent work using human T-ALL cell lines and xenografts and suggest that the Notch/Myc signaling axis is of predominant importance in understanding both the selective pressure for Notch mutations in T-ALL and response and resistance of T-ALL to Notch pathway inhibitors.

A Practical Approach for Enantio- and Diastereocontrol in the Synthesis of 2,3-Disubstituted Succinic Acid Esters: Synthesis of the pan-Notch Inhibitor BMS-906024

An oxidative intermolecular enolate heterocoupling reaction was employed for the synthesis of anti-2,3-disubstituted succinic acid mono- and differentially protected diesters. Tactical approaches to access all the diastereomers are discussed. The method was applied to the synthesis of a potent anticancer agent, BMS-906024.

Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis

ObjectiveWe tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue.DesignMice were treated with the pan-Notch...